SEMESTER III PAPER IX - IMMUNOLOGY & MMUNOTECHNOLOGY
UNIT - II
Antigens - properties, Epitopes, haptens, adjuvant, cross reactivity. Antibodies - properties,
structure (primary & secondary) and isotypes. Diversity and specificity. Anti antibodies.
UNIT - III
Serology - Introduction and classification of antigens and antibody reactions - Agglutination and
precipitation reaction. Strength of antigen and antibody bindings - affinity & avidity.
Complement pathway and complement fixation reaction. Immunofluorscence RIA, RAST,
ELISA and Flowcytometry. Monoclonal antibodies & its applications. (Hybridoma technique)
UNIT - IV
MHC antigens - types and functions. Regulation and response of immune system. Response of
B Cell to antigens. T cell products. Immunity to infectious diseases - Viral, bacterial and
protozoan . Hyper sensitivity reactions .
UNIT V
Transplantation immunology - Tissue transplantation and grafting . Mechanism of graft
acceptance and rejection. HLA typing Tumor immunology. Immunodeficiency diseases and auto
immunity. Vaccines - Types and vaccination methods.
UNIT I
Historical background and scope of immunology, Immunohaematology -ABO and Rh factor.
Cells and organs of immune system. Non immunological defence mechanism - Barriers,
Phagocytosis, inflammation, fever . Types of immunity - HI and CMI.
1. Explain the historical background and scope of immunology.

1718 - Lady Mary Wortley Montagu, the wife of the British ambassador to
Constantinople, observed the positive effects of variolation on the native population and
had the technique performed on her own children.

1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner)

1837 - First description of the role of microbes in putrefaction and fermentation
(Theodore Schwann)

1838 - Confirmation of the role of yeast in fermentation of sugar to alcohol (Charles
Cagniard-Latour)

1840 - First "modern" proposal of the germ theory of disease (Jakob Henle)

1850 - Demonstration of the contagious nature of puerperal fever (childbed fever) (Ignaz
Semmelweis)

1857-1870 - Confirmation of the role of microbes in fermentation (Louis Pasteur)

1862 - phagocytosis (Ernst Haeckel)

1867 - First aseptic practice in surgery using carbolic acid (Joseph Lister)

1876 - First demonstration that microbes can cause disease-anthrax (Robert Koch)

1877 - Mast cells (Paul Ehrlich)

1878 - Confirmation and popularization of the germ theory of disease (Louis Pasteur)

1880 - 1881 -Theory that bacterial virulence could be attenuated by culture in vitro and
used as vaccines. Proposed that live attenuated microbes produced immunity by depleting
host of vital trace nutrients. Used to make chicken cholera and anthrax "vaccines" (Louis
Pasteur)

1883 - 1905 - Cellular theory of immunity via phagocytosis by macrophages and
microphages (polymorhonuclear leukocytes) (Elie Metchnikoff)

1885 - Introduction of concept of a "therapeutic vaccination". First report of a live
"attenuated" vaccine for rabies (Louis Pasteur).

1888 - Identification of bacterial toxins (diphtheria bacillus) (Pierre Roux and Alexandre
Yersin)

1888 - Bactericidal action of blood (George Nuttall)

1890 - Demonstration of antibody activity against diphtheria and tetanus toxins.
Beginning of humoral theory of immunity. (Emil von Behring) and (Shibasaburo
Kitasato)

1891 - Demonstration of cutaneous (delayed type) hypersensitivity (Robert Koch)

1893 - Use of live bacteria and bacterial lysates to treat tumors-"Coley's Toxins"
(William B. Coley)

1894 - Bacteriolysis (Richard Pfeiffer)

1896 - An antibacterial, heat-labile serum component (complement) is described (Jules
Bordet)

1900 - Antibody formation theory (Paul Ehrlich)

1901 - blood groups (Karl Landsteiner)

1902 - Immediate hypersensitivity anaphylaxis (Paul Portier) and (Charles Richet)

1903 - Intermediate hypersensitivity, the "Arthus reaction" (Maurice Arthus)

1903 - Opsonization

1905 - "Serum sickness" allergy (Clemens von Pirquet and (Bela Schick)

1909 - Paul Ehrlich proposes "immune surveillance" hypothesis of tumor recognition and
eradication

1911 - 2nd demonstration of filterable agent that caused tumors (Peyton Rous)

1917 - hapten (Karl Landsteiner)

1921 - Cutaneous allergic reactions (Otto Prausnitz and Heinz Küstner)

1924 - Reticuloendothelial system

1938 - Antigen-Antibody binding hypothesis (John Marrack)

1940 - Identification of the Rh antigens (Karl Landsteiner and Alexander Weiner)

1942 - Anaphylaxis (Karl Landsteiner and Merill Chase)

1942 - Adjuvants (Jules Freund and Katherine McDermott)

1944 - hypothesis of allograft rejection

1945 - Coombs Test aka antiglobulin test (AGT)

1946 - identification of mouse MHC (H2) by George Snell and Peter A. Gorer

1948 - antibody production in plasma B cells

1949 - growth of polio virus in tissue culture, neutralization with immune sera, and
demonstration of attenuation of neurovirulence with repetitive passage (John Enders) and
(Thomas Weller) and (Frederick Robbins)

1949 - immunological tolerance hypothesis

1951 - vaccine against yellow fever

1953 - Graft-versus-host disease

1953 - immunological tolerance hypothesis

1957 - Clonal selection theory (Frank Macfarlane Burnet)

1957 - Discovery of interferon by Alick Isaacs and Jean Lindenmann

1958-1962 - Discovery of human leukocyte antigens (Jean Dausset and others)

1959-1962 - Discovery of antibody structure (independently elucidated by Gerald
Edelman and Rodney Porter)

1959 - Discovery of lymphocyte circulation (James Gowans)

1960 - Discovery of lymphocyte "blastogenic transformation" and proliferation in
response to mitogenic lectins-phytohemagglutinin (PHA) (Peter Nowell)

1961-1962 Discovery of thymus involvement in cellular immunity (Jacques Miller)

1961- Demonstration that glucocorticoids inhibit PHA-induced lymphocyte proliferation
(Peter Nowell)

1963 - Development of the plaque assay for the enumeration of antibody-forming cells in
vitro (Niels Jerne) (Albert Nordin)

1964-1968 T and B cell cooperation in immune response

1965 - Discovery of the first lymphocyte mitogenic activity, "blastogenic factor" (Shinpei
Kamakura) and (Louis Lowenstein) (J. Gordon) and (L.D. MacLean)

1965 - Discovery of "immune interferon" (gamma interferon) (E.F. Wheelock)

1965 - Secretory immunoglobulins

1967 - Identification of IgE as the reaginic antibody (Kimishige Ishizaka)

1968 - Passenger leukocytes identified as significant immunogens in allograft rejection
(William L. Elkins and Ronald D. Guttmann)

1969 - The lymphocyte cytolysis Cr51 release assay (Theodore Brunner) and (JeanCharles Cerottini)

1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA

1972 - Structure of the antibody molecule

1973 - Dendritic Cells first described by Ralph M. Steinman

1974 - T-cell restriction to major histocompatibility complex (Rolf Zinkernagel and
(Peter C. Doherty)

1975 - Generation of the first monoclonal antibodies (Georges Köhler) and (César
Milstein)

1976 - Identification of somatic recombination of immunoglobulin genes (Susumu
Tonegawa)

1979 - Generation of the first monoclonal T cells (Kendall A. Smith)

1980-1983 - Discovery and characterization of the first interleukins, 1 and 2 IL-1 IL-2
(Kendall A. Smith)

1981 - Discovery of the IL-2 receptor IL2R (Kendall A. Smith)

1983 - Discovery of the T cell antigen receptor TCR (Ellis Reinherz) (Philippa Marrack)
and (John Kappler) (James Allison)

1983 - Discovery of HIV (Luc Montagnier)

1984 - The first single cell analysis of lymphocyte proliferation (Doreen Cantrell) and
(Kendall A. Smith)

1985-1987 - Identification of genes for the T cell receptor

1986 - Hepatitis B vaccine produced by genetic engineering

1986 - Th1 vs Th2 model of T helper cell function (Timothy Mosmann)

1988 - Discovery of biochemical initiators of T-cell activation: CD4- and CD8-p56lck
complexes (Christopher E. Rudd)

1990 - Gene therapy for SCID

1991 - Role of peptide for MHC Class II structure ([Scheherazade Sadegh-Nasseri] &
[Ronald N. Germain])

1992- Discovery of transitional B cells (David Allman & Michael Cancro)

1994 - 'Danger' model of immunological tolerance (Polly Matzinger)

1995 - Regulatory T cells (Shimon Sakaguchi)

1995 - First dendritic cell vaccine trial reported by Mukherji et al.

1996-1998 - Identification of Toll-like receptors

2001 - Discovery of FOXP3 - the gene directing regulatory T cell development

2005 - Development of human papillomavirus vaccine (Ian Frazer)

Scope of immunology


Of the four major causes of death – injury, infection, degenerative

disease and cancer – onl y the fi rst two regularl y kill their victims

before child -bearing age, which means that they are a potential source

of lost genes. Therefore any mechanism that reduces their effects has

tremendous survival value, and we see this in the processes of,
respectivel y,

healing and immunity.

Immunit y is concerned with the recognition and disposal of foreign

or ‘non-self’ material that enters the body (represented by red arrows

in the fi gure), usually in the form of life -threatening infectious
microorganisms

but sometimes, unfortunatel y, in the shape of a life -saving

kidney graft. Resistance to infection may b e ‘innate’ (i.e. inborn and

unchanging) or ‘acquired’ as the result of an adaptive immune

response (centre).

Immunology is the study of the organs, cells and molecules responsible

for this recognition and disposal (the ‘immune system’), of how

they respond and interact, of the consequences – desirable (top) or

otherwise (bottom) – of their activit y, and of the ways in which they

can be advantageously increased or reduced.

By far the most important t ype of foreign material that needs to be

recognized and disposed of is the microorganisms capable of causing

infectious disease and, strictl y speaking, immunit y begins at the point

when they enter the body. But it must be remembered that the fi rst

line of defence is to keep them out, and a variet y of external defences
have evolved for this purpose. Whether these are part of the immune
s ystem is a purel y semantic question, but an immunologist is certainl y
expected to know about them.

Non-self A widel y used term in immunology, covering everything that

is detectabl y different from an animal’s own constituents. Infectious

microorganisms, together with cells, organs or other materials from

another animal, are the most important non -self substances from an

immunological viewpoint, but drugs and even normal foods, whic h

are, of course, non -self too, can sometimes give rise to immunit y.

Detection of non -self material is carried out by a range of receptor

molecules (see Figs 11 –15).

Infection Parasitic viruses, bacteria, protozoa, worms or fungi that

attempt to gain acces s to the body or its surfaces are probabl y the chief
raison d’être of the immune system. Higher animals with a damaged or
defi cient immune system frequentl y succumb to infections that normal
animals overcome.


External defences The presence of intact skin on the outside and

mucous membranes lining the hollow viscera is in itself a powerful

barrier against entry of potentiall y infectious organisms. In addition,

there are numerous antimicrobial (mainl y antibacterial) secretions in

the skin and mucous surface s; these include l ysoz yme (also found in

tears), lactoferrin, defensins and peroxidases. More specialized

defences include the extreme acidit y of the stomach (about pH 2), the

mucus and upwardl y beating cilia of the bronchial tree, and specialized
surfactant proteins that recognize and clump bacteria that reach the lung
alveoli. Successful microorganisms usuall y have cunning ways of
breaching or evading these defences.

Innate resistance Organisms that enter the body (shown in the fi gure as
dots or rods) are often eliminated within minutes or hours by inborn, ever present mechanisms, while others (the rods in the fi gure) can avoid this
and survive, and may cause disease unless they are dealt with by adaptive
immunit y (see below). These mechanisms have evolv ed to dispose of
pathogens (e.g. bacteria, viruses) that if

unchecked can cause disease. Harmless microorganisms are usuall y

ignored by the innate immune system. Innate immunit y also plays a

vital role in initiating the adaptive immune response.

Adaptive immune response The development or augmentation of

defence mechanisms in response to a particular (‘specifi c’) stimulus,

e.g. an infectious organism. It can result in elimination of the

microorganism and recovery from disease, and often leaves th e host

with specifi c memory, enabling it to respond more effectivel y on

reinfection with the same microorganism, a condition called acquired

resistance. Since the body has no prior way of knowing which
microorganisms are and which are not harmless, all fo reign material is
usuall y responded to as if it were harmful, including relativel y
inoffensive pollens, etc.

Vaccination A method of stimulating the adaptive immune response

and generating memory and acquired resistance without suffering the

full effects o f the disease. The name comes from vaccinia, or cowpox,

used by Jenner to protect against smallpox.

Grafting Cells or organs from another individual usuall y survive

innate resistance mechanisms but are attacked by the adaptive immune
response, leading to r ejection.

Autoimmunity The body’s own (‘self’) cells and molecules do not

normall y stimulate its adaptive immune responses because of a variet y of
special mechanisms that ensure a state of self -tolerance, but in certain
circumstances they do stimulate a re sponse and the body’s own structures
are attacked as if they were foreign, a condition called autoimmunit y or
autoimmune disease.

Hypersensitivity Sometimes the result of specifi c memory is that re exposure to the same stimulus, as well as or instead of e liminating

the stimulus, has unpleasant or damaging effects on the body’s own

tissues. This is called hypersensitivit y; examples are allergies such as hay
fever and some forms of kidney disease.

Immunosuppression Autoimmunit y, hypersensitivit y and, above a ll,

graft rejection sometimes necessitate the suppression of adaptive

immune responses by drugs or other means.

Immunohaematology -ABO and Rh factor.
Immunohematology, more commonly known as blood banking is a branch of hematology
which studies antigen-antibody reactions and analogous phenomena as they relate to the
pathogenesis and clinical manifestations of blood disorders. A person employed in this field is
referred to as an immunohematologist. Their day to day duties include blood typing, crossmatching and antibody identification
CELLS OF THE IMMUNE RESPONSE
Immune responsive cells can be divided into five groups based on i) the presence of specific
surface components and ii) function: B-cells (B lymphocytes), T-cells (T lymphocytes),
Accessory cells (Macrophages and other antigen-presenting cells), Killer cells (NK and K cells),
and Mast cells. Some of the properties of each group are listed below.
Cell group
Surface components

Surface immunoglobulin
(Ag recognition)



Immunoglobulin Fc
receptor
B-lymphocytes
Function
Direct antigen
recognition

Differentiation into
Class II Major
antibody-producing
Histocompatability
plasma cells
Complex (MHC)

molecule (Ag
Antigen presentation
within Class II MHC
presentation)
T-lymphocytes

CD3 molecule


T-cell receptor (TCR,
humoral and cell-
Ag recognition)
mediated responses

Involved in both
Recognizes antigen
presented within Class
II MHC

Helper T-cells (TH)

CD4 molecule

Promotes
differentiation of Bcells and cytotoxic Tcells

Suppressor T-cells (TS)

CD8 molecule

Activates macrophages

Downregulates the
activities of other cells

Recognizes antigen
presented within Class I

Cytotoxic T-cells (CTL)

CD8 molecule
MHC

Kills cells expressing
appropriate antigen
Accessory cells

Variable

Phagocytosis and cell
killing

Bind Fc portion of
immunoglobulin
(enhances
phagocytosis)


component C3b
Immunoglobulin Fc
(enhances
receptor

Macrophages

Complement component
C3b receptor

Class II MHC molecule
Bind complement
phagocytosis)

Antigen presentation
within Class II MHC

Secrete IL-1
(macrokine) promoting
T-cell differentiation
and proliferation

Can be "activated" by
T-cell lymphokines

Dendritic cells

Polymorphonuclear
cells (PMNs)

Class II MHC molecule

Immunoglobulin Fc


Antigen presentation
within Class II MHC

Bind Fc portion of
receptor
immunoglobulin
Complement component
(enhances
C3b receptor
phagocytosis)

Bind complement
component C3b
(enhances
phagocytosis)

Killer cells


NK cells
Variable

Direct cell killing

Kills variety of target
cells (e.g. tumor cells,
Unknown
virus-infected cells,
transplanted cells)

Bind Fc portion of
immunoglobulin


K cells
Immunoglobulin Fc

Kills antibody-coated
target cells (antibody-
receptor
dependent cellmediated cytotoxicity,
ADCC)

Mast cells
High affinity IgE Fc

receptors
Bind IgE and initiate
allergic responses by
release of histamine
LYMPHOID TISSUES
Primary
Secondary
(Responsible for maturation
of Ag-reactive cells)
Thymus
Bone
(T-cell
marrow
(Sites for Ag contact and response)
Lymph nodes
Spleen
maturation)
(Expansion of lymphatic system,
(T-cell
(B-cell
maturation)
maturation)
separate from blood circulation. Deep
cortex harbors mostly T-cells,
superficial cortex harbors mostly Bcells)
(Similar to lymph nodes
but part of blood
circulation. Collects
blood-borne Ags)
. NON IMMUNOLOGICAL DEFENSE MECHANISM
Body Defenses
•
Physical barriers: skin & epithelial linings & cilia
•
Chemical: acids, mucous & lysozymes
•
Immune defenses – internal
•
•
Innate, non-specific, immediate response (min/hrs)
•
Acquired – attack a specific pathogen (antigen)
Steps in Immune defense
•
Detect invader/foreign cells
•
Communicate alarm & recruit immune cells
•
Suppress or destroy invader
•
Microbial killing by phagocytes:
•
Phagocytosis involves two steps namely attachment and ingestion. Following attachment
of the organism,
•
invagination of the phagocyte results in the formation of a phagosome. Some capsulated
bacteria don’t attach to the
•
phagocyte, but they can still be phagocytosed if they are coated with opsonins such as
IgG and complement
•
component (C3b). The engulfed bacteria are held inside a vacuole called phagosome. The
formation of phagosome
•
triggers respiratory bursts and fusion of lysosome with phagosome to form
phagolysosome
•
The phagocytes appear to kill engulfed bacteria by two pathways, oxygen independent pathway
and oxygen dependent pathway. The microbicidal mechanisms of the respiratory burst are
termed oxygen dependent and phagolysosome formations are termed oxygen
independent.Oxygen dependent mechanism involves catalytic conversion of molecular oxygen to
oxyhalide free radicals, which are highly reactive oxidizing agents. The phagocyte oxidase
present in the plasma membrane and phagolysosome reduce oxygen into reactive oxygen
intermediates such as superoxide radicals. Superoxide is converted to H2O2,which is used by
enzyme myeloperoxidase to convert unreactive halide ions to reactive hypohalous acids that are
toxic to bacteria.Oxygen independent mechanism involves release of lysosomal contents into
phagolysosomes. The content of lysosome includes lactoferrin, cathepsin
G, lysozyme and defensins etc.In addition to the phagocyte oxidase system, macrophages have
free-radical generating system, namely inducible nitric oxide synthase. This cytosolic enzyme is
absent in resting macrophages but can be induced in response to bacterial lipopolysaccharides
and IFN-γ. This enzyme catalyses the conversion of arginine to citrulline, and in the process
releases nitric oxide gas. Nitric oxide may then combine with H2O2 or superoxide to form highly
reactive peroxynitrite radicals that kill the microbes.
Dendritic cells:
These cells are derived from myeloid progenitor in the bone marrow and are morphologically
identified by spiny
membranous projection on their surfaces. Immature dendritic cells are located in epithelia of
skin, gastrointestinal
tract and respiratory tract and are called langerhan cells. They express low levels of MHC
proteins on their surface
and their main function is to capture and transport protein antigen to the draining lymph node.
During their migration
to the lymph node, dendritic cells mature into excellent antigen presenting cells (APC). Mature
dendritic cells reside
in the T cell area (paracortex) of the lymph node. Here, they are referred as interdigitating
dendritic cells. These
cells are distinct from the dendritic cells that occur in the germinal centers of lymphoid follicles
(follicular dendritic
cells) in lymph node, spleen and MALT. The follicular dendritic cells are not derived from the
bone marrow and their
role is to present antigen-antibody complex and complement products to B cell.
Lymphoid system:
Lymphoid organs are stationed throughout the body and are concerned with the growth,
development and
deployment of lymphocytes. These structurally and functionally diverse lymphoid organs and
tissues are
interconnected by the blood vessels and lymphatic vessels through which lymphocytes circulate.
The organs
involved in specific as well as non-specific immunity are classified as primary (central)
lymphoid organs and
secondary (peripheral) lymphoid organs. The blood and lymphatic vessels that carry
lymphocytes to and from the
other structures can also be considered lymphoid organs. Recently, it has become accepted that
the liver is also a
hematopoietic organ, giving rise to all leukocyte lineages.
PRIMARY LYMPHOID ORGANS:
Also called central lymphoid organs, these are responsible for synthesis and maturation of
immunocompetant cells.
These include the bone marrow and the thymus.
BONE MARROW:
All the cells of the immune system are initially derived from the bone marrow through a process
called
hematopoiesis. During foetal development hematopoiesis occurs initially in yolk sac and paraaortic mesenchyme
and later in the liver and spleen. This function is taken over gradually by the bone marrow.
During hematopoiesis,
bone marrow-derived stem cells differentiate into either mature cells or into precursors of cells
that migrate out of
the bone marrow to continue their maturation in thymus.
The bone marrow produces B cells, natural killer cells, granulocytes and immature thymocytes,
in addition to red
blood cells and platelets. It is both a primary and secondary lymphoid organ. The proliferation
and maturation of
precursor cells in the bone marrow are stimulated by cytokines, many of which are called colony
stimulating factors
(CSFs). The bone marrow also contains antibody secreting plasma cells, which have migrated
from the peripheral
lymphoid tissue.
THYMUS:
The thymus is a gland located in the anterior mediastinum just above the heart, which reaches its
greatest size just
prior to birth, then atrophies with age. This lymphoepithelial organ develops from ectoderm
derived from the third
branchial cleft and endoderm of the third branchial pouch.
Immature lymphocytes begin to accumulate in the thymus of human embryos at about 90-100
days after
fertilization. Initially most of these immature lymphocytes have come from the yolk sac and fetal
liver rather than the
bone marrow. Cells from the bone marrow, later migrate to the thymus as precursors and develop
into mature
peripheral T cells. Once the immature lymphocytes have passed the blood-thymus barrier they
are called
thymocytes. Mature T cells migrate from the thymus to secondary lymphoid organs such as
lymph node, Peyer's
patches and spleen.
Ultimately the thymus becomes an encapsulated and consists of many lobes, each divided into an
outer cortical
region and an inner medulla. The cortex contains mostly immature thymocytes, some of which
mature and migrate
to the medulla, where they learn to discriminate between self and non-self during foetal
development and for a short
time after birth. T cells leave the medulla to enter the peripheral blood circulation, through which
they are
transported to the secondary lymphoid organs. About 98% of all T cells die in the thymus.
The greatest rate of T cell production occurs before puberty. After puberty, the thymus shrinks
and the production of
new T cells in the adult thymus drops away. Children with no development of thymus suffer
from DiGeorge
syndrome that is characterized by deficiency in T cell development but normal numbers of B
cells.
PERIPHERAL LYMPHOID ORGANS:
While primary lymphoid organs are concerned with production and maturation of lymphoid
cells, the secondary or
peripheral lymphoid organs are sites where the lymphocytes localise, recognise foreign antigen
and mount
response against it. These include the lymph nodes, spleen, tonsils, adenoids, appendix, and
clumps of lymphoid
tissue in the small intestine known as Peyer's patches. They trap and concentrate foreign
substances, and they are
the main sites of production of antibodies.
Some lymphoid organs are capsulated such as lymph node and spleen while others are noncapsulated, which
include mostly mucosa-associated lymphoid tissue (MALT).
LYMPH NODE:
Clusters of lymph nodes are strategically placed in the neck, axillae, groin, mediastinum and
abdominal cavity,
where they filter antigens from the interstitial tissue fluid and the lymph during its passage from
the periphery to the
thoracic duct. The key lymph nodes are the axillary lymph nodes, the inguinal lymph nodes, the
mesenteric lymph
nodes and the cervical lymph nodes. Lymph nodes that protect the skin are termed somatic
nodes, while deep
lymph nodes protecting the respiratory, digestive and genitourinary tracts are termed visceral
nodes.
Each lymph node is surrounded by a fibrous capsule that is pierced by numerous afferent
lymphatics that drain
lymph into marginal sinus. The lymph flows through the medullary sinus and leaves through
efferent lymphatics.
Each lymph node is divided into an outer cortex, inner medulla and intervening paracortical
region. The cortex is
also referred as B cell area, which mainly consists of B cells. The cortex is a high traffic zone
where recirculating Tand
B lymphocytes enter from the blood. Aggregates of cells called follicles are present in the cortex,
which in turn
may have central areas called germinal centers. Follicles without germinal centers are called
primary follicles and
those with germinal centers are called secondary follicles. Primary follicles are rich in mature but
resting B cells.
Germinal centers develop in response to antigenic stimulation and consist of follicular dendritic
cells and reactive B
cells. The medulla contains a mixture of B cells, T cells, plasma cells and macrophages. The
medulla consists of
medullary cords that lead to the medullary sinus. The cords are populated by plasma cells and
macrophages.
Between these two zones, lie the paracotex (T cell area) that contains T lymphocytes, dendritic
cells and
mononuclear phagocytes. Most of the T cells (70%) located there are CD4+ helper cells.
SPLEEN:
Situated in the left upper quadrant of the abdomen and weighing about 150 grams, spleen is the
largest single
lymphoid organ in the body. It has a dense fibrous capsule with muscular trabeculae extending
inward to subdivide
the spleen into lobules. It filters blood and is the major organ in which antibodies are synthesized
and released into
circulation. In addition to capturing foreign antigens from the blood that passes through the
spleen, migratory
macrophages and dendritic cells also bring antigens to the spleen via the bloodstream. Persons
lacking spleen (eg.
splenectomy) are highly susceptible to infections with capsulated bacteria such as pneumococci
and meningococci.
Spleen is the major site for phagocytosis of antibody coated bacteria and destruction of aged
RBCs. It is supplied by splenic artery, which pierces the capsule at hilum and divides into
smaller branches that are surrounded by fibrous trabeculae. The spleen is composed of two types
of tissue, the red pulp and the white pulp. The red pulp contains vascular sinusoids, large number
of erythrocytes, resident macrophages, dendritic cells, granulocytes, few plasma cells and
lymphocytes. It is the site where aged platelets and erythrocytes are destroyed. The white pulp
contains the lymphoid tissue clustered around small arterioles and is known as a periarteriolar
lymphoid sheath (PALS). PALS contain mainly T lymphocytes, about 75% of which are CD4+
helper T cells. Attached to this are lymphoid follicles, some of which contain germinal centers.
Follicles and germinal center predominantly contain B cells. The PALS and follicles are
surrounded by rim of lymphocytes and macrophages,called marginal zone. Marginal zone is
composed of macrophages, B cells, and CD4+ helper T cells. The arterioles end in vascular
sinusoids in the red pulp, which in turn end in venules that drain into splenic vein. Antigens and
lymphocytes enter the spleen through vascular sinusoids. Activation of B cells occurs at the
juncton between follicle and PALS. Activated B cells then migrate to the germinal centers or into
the red pulp.
MUCOSA ASSOCIATED LYMPHOID TISSUE (MALT):
Approximately >50% of lymphoid tissue in the body is found associated with the mucosal
system. MALT is
composed of gut-associated lymphoid tissues (GALT) lining the intestinal tract, bronchusassociated lymphoid
tissue (BALT) lining the respiratory tract, and lymphoid tissue lining the genitourinary tract. The
respiratory,
alimentary and genitourinary tracts are guarded by subepithelial accumulations of lymphoid
tissue that are not
covered by connective tissue capsule. They may occur as diffuse collections of lymphocytes,
plasma cells and
phagocytes throughout the lung and lamina propria of intestine or as clearly organised tissue with
well-formed
lymphoid follicles. The well-formed follicles include the tonsils (lingual, palatine and
pharyngeal), Peyer’s patches in
the intestine and appendix. The major function of these organs is to provide local immunity by
way of sIgA (also
IgE) production. Diffuse accumulations of lymphoid tissue are seen in the lamina propria of the
intestinal wall. The
intestinal epithelium overlying the Peyer's patches is specialized to allow the transport of
antigens into the lymphoid
tissue. This function is carried out by cuboidal absorptive epithelial cells termed "M" cells, so
called because they
have numerous microfolds on their luminal surface. M cells endocytose, transport and present
antigens to
subepithelial lymphoid cells.
Majority of intra-epithelial lymphocytes are T cells, and most often CD8+ lymphocytes. The
intestinal lamina propria
contains CD4+ lymphocytes, large number of B cells, plasma cells, macrophages, dendritic cells,
eosinophils and
mast cells. Peyer’s patches contain both B cells and CD4+ T cells.
LYMPHOCYTES:
Lymphocytes are stem cells derived cells that mature either in the bone marrow or thymus.
Together, the thymus
and marrow bone marrow produce approximately 109 mature lymphocytes each day and the
adult human body
contains approximately 1012 lymphocytes. Lymphocytes comprise 20-40% (1000 - 4000
cells/μl) of all leukocytes.
The lymphocytes are distributed to blood, lymph and lymphoid organs.
Typically, lymphocyte is small, round, cell with diameter of 5-10μm, spherical nucleus, densely
compacted nuclear
chromatin and scanty cytoplasm. Though the cytoplasm contains mitochondria and ribosomes,
other organelles are
not detectable. Such mature but resting lymphocytes are known as naïve cells. They are
mitotically inactive but
when stimulated can undergo cell division. Naïve lymphocytes have a short life span and die in
few days after
leaving bone marrow or thymus unless they are stimulated. Once the lymphocyte is activated
(stimulated), they
become large (10-12μm), have more cytoplasm and more organelles. Activated lymphocytes
may undergo several
successive rounds of cell division over a period of several days. Some of the progeny cells revert
to the resting
stage and become memory cells, but can survive for several years in the absence of any antigenic
stimulus.
There are three major types of lymphocyte, B lymphocyte, T lymphocyte and NK cells. Different
lymphocytes are
identified by certain protein markers on their surface called "cluster of differentiation" or "CD"
system. One marker
that all leukocytes have in common is CD45. The presence of the markers can be detected using
specific
monoclonal antibodies.
Distribution of lymphocytes
Approximate %
Tissue T-Cells B-Cells NK Cells
Peripheral blood 70-80 10-15 10-15
Bone marrow 5-10 80-90 5-10
Thymus 99 <1 <1
Lymph node 70-80 20-30 <1
Spleen 30-40 50-60 1-5
B LYMPHOCYTE:
Also called B-cells, they are so called because in birds they were found to mature in bursa of
fabricius. Humans
don’t have an anatomical equivalent to bursa, but the development and maturation of these cells
occur in bone
marrow.
© Sridhar Rao P.N (www.microrao.com)
Ontogeny:
In mammals, the early stages of B cell maturation occur in the fetal liver and bone marrow. B
cell development
begins in the fetal liver and continues in the bone marrow throughout life.
The stages in B cell development in the bone marrow are:
Stem cell > pro-B cell > pre-B cell > small pre-B cell > immature B cell > mature B cell.
Distribution:
They account for 5-15% of lymphocytes (250 cells/μl) in circulation and 80-90% in bone
marrow, 20-30% in lymph
node and 50-60% in spleen.
Surface markers:
The most important surface marker on the surface of mature B cell is the surface
immunoglobulin. The surface
immunoglobulins are of IgM and IgD type. A B cell will have approximately 109
immunoglubulins of single specificity
on its surface. Markers/Receptors on B cells are Surface Immunoglobulin (IgM and IgD), CD40,
B7, ICAM-1, LFA-1,
MHC II, CD32 (Ig Fc receptor), CD35 (Receptor for complement component) and additional
markers that distinguish
B cells such as CD19, CD20, CD21 and CD22.
Demonstration of B cells:
EAC (Erythrocyte Amboceptor Complement) Rosettes: When sheep RBCs coated with antibody
and treated with
complement and B cells, a rosette is formed due to the presence of complement receptor on B
cells. B cells can be
demonstrated by immunofluorescence with fluorescent-labelled monoclonal antibodies against
surface markers
such as surface immunoglobulin.
On stimulation by pokeweed mitogen, they undergo blast transformation.
Functions of B-cells:
Direct antigen recognition and Antigen presentation
B cells may differentiate into plasma cells (which secrete large amounts of antibodies) or into
memory B cells.
Memory cells can survive 20 years or more.
Plasma cells:
These are the effector cells of the B-cell lineage and are specialised in secreting
immunoglobulins. When activated
B cells divide, some of its progeny become memory cells and the reminder become
immunoglobulin-secreting
plasma cells. Plasma cells are oval or egg shaped, have eccentrically placed nuclei, have
abundant cytoplasm
containing dense rough endoplasmic reticulum (the site of antibody production), perinuclear
Golgi body (where
immunoglobulins are converted to final form and packaged). Unlike B cells, immunoglobulins
are not present on the
surface of plasma cells. They have a short life span of few days to few weeks.
© Sridhar Rao P.N (www.microrao.com)
T LYMPHOCYTE:
Ontogeny:
The name "T-cell" is an abbreviation of "thymus dependent lymphocyte". T lymphocytes arise in
the bone marrow
as T-cell precursors, then migrate to and mature in the thymus. After entry into the thymus T-cell
precursors are
also referred to as "thymocytes".
In the thymus there are rearrangements at gene segments coding for the variable part of the TCR
(T Cell Receptor)
resulting in generation of diversity. T Cell Receptors are then expressed on the surface, which is
followed by
expression of either CD8 or CD4 surface molecules. Those cells expressing receptors that can
interact with self
MHC molecules are positively selected while those cells that express receptors that recognize
peptides derived
from self protein in association with self MHC are negatively selected. Such cells undergo clonal
deletion or anergy.
Distribution:
T cell accounts for 70-80% (1500 cells/μl) lymphocytes in peripheral blood, 5-10% in bone
marrow, 70-80% in
lymph node and 30-40% in spleen.
Surface markers:
The most important surface receptor is TCR. TCR are polypeptides that belong to the
immunoglobulin superfamily.
There are two kinds of TCR, one composed of a α-β heterodimer (TCR2) and the other
composed of a γ-δ
heterodimer (TCR1). An individual T cell can express either α-β or γ-δ as its receptor but never
both. 95% of T cells
express the α-β heterodimer. The other markers/receptors present on the surface are IL-2R, IL1R, CD2, CD3,
CD4/CD8, CD28, ICAM-1 and LFA-1. Nearly all the mature T lymphocytes express both CD2
and CD3 on their
surface. CD3, which is always found closely associated with TCR, is necessary for signal
transduction following
antigen recognition by the TCR.
Subsets of T Cells:
There are two major types of T cells, Helper (CD4) and Cytotoxic/Suppressor (CD8) T cells.
CD4 cells account for 45% (900/μl) of lymphocytes while CD8 cells account for 30% (600/μl).
� Helper T cells (TH) secrete cytokines that promote the proliferation and differentiation of
cytotoxic T cells, B
cells and macrophages and activation of inflammatory leukocytes. TH cells are identified by the
presence of
the CD4 marker. They recognize antigen when presented along with Class II MHC molecules.
TH cells are
further subdivided into the TH1 and TH2 subsets on the basis of the kinds of cytokines they
produce. TH1 cells
produce interleukin-2 (IL-2), interferon-gamma (IFNγ), and tumour necrosis factor-beta (TNF-β)
while TH2
cells produce IL-4, IL-5, IL-6, IL-10 and TGF-β.
� Cytotoxic T cells (TC) lyse cells with foreign antigens, e.g. tumour cells, virus-infected
cells, and foreign
tissue grafts. TC cells are identified by the presence of the CD8 marker. They recognize antigen
presented
when presented along with Class I MHC molecules. The suppressor T cells have a role in
downregulation of
immune response.
Demonstration of T cells:
�
-labelled monoclonal
antibodies
against TCR or other surface markers.
�
-Rosette/ SRBC rosette: T cells bind to sheep RBCs at 37oC forming rosettes.
�
(PHA) or
Concanavalin A.
© Sridhar Rao P.N (www.microrao.com)
Functions of Helper T-cells (TH):
Promotes differentiation of B-cells and cytotoxic T-cells
Activates macrophages
Functions of Cytotoxic/Suppressor T-cells (CTL):
Kills cells expressing appropriate antigen
Downregulates the activities of other cells
NK CELLS (LARGE GRANULAR LYMPHOCYTES):
Also called Large Granular Lymphocytes (LGLs), these are large lymphocytes containing
azurophilic granules in the
cytoplasm. NK cells derive form bone marrow but don't require thymus for development. NK
cells are so called
because they kill variety of target cells (such as tumour cells, virus-infected cells, transplanted
cells) without the
participation of MHC molecules. They can kill target cell without a need for activation unlike
cytotoxic T
lymphocytes. Hence they mediate a form of natural (innate) immunity.
Distribution:
They account for 10-15% of blood lymphocytes. They are rare in lymph nodes and don't
circulate through lymph.
Surface markers:
NK cells lack any surface immunoglobulins, TCR or CD4 makers; instead they have CD16
(Immunoglobulin Fc
receptor) and CD56. Approximately 50% of human NK cells express only one form of CD8.
Other receptors include
IL-2R, CD2, ICAM-1 and LFA-1.
Functions:
NK cells are activated by recognition of antibody-coated cells, virus infected cell, cell infected
with intracellular
bacteria and cells lacking MHC I proteins. Activation of NK cell results in cytolysis of target and
cytokine secretion
but no clonal expansion. Interestingly, NK cells are inhibited on contact with MHC I proteins.
NK cells can kill antibody-coated target cells, which is mediated through Fc receptor present on
its surface. This is
called antibody-dependent cell cytotoxicity (ADCC).
NK cells also participate
in Graft vs Host reaction in
recipient of bone marrow
transplants. NK cells can
be activated by IL-2 so
that their cytotoxic
capacity is enhanced.
Such cells are called
Lymphokine Activated
Killer cells (LAK) and have
been used clinically to treat tumours. LAK cells have enhanced cytolytic activity and are
effective against wide
range of tumour cells. Activated NK cells produce cytokines such as IFN-γ, TNFα, GM-CSF and
CSF-1 all of which
are immunomodulators.
LYMPHOCTE RECIRCULATION:
The movement of lymphocytes
via the blood stream and
lymphatics from peripheral tissue
to another is called lymphocyte
recirculation. Lymphocytes are
migratory cells; mature
lymphocytes continually migrate
in and out of all peripheral
lymphoid tissue. At an average
each cell changes location once
or twice each day. At any given
point of time 1-2% of
lymphocytes will be in transit. In
most lymphoid organs, they
enter through blood and exit
through lymphatics, but in spleen they enter and leave directly through blood. As lymphocytes
migrate, they can
survey the body for foci of infection or presence of foreign antigens. Such a movement also
helps to maintain a
balance in distribution of lymphocytes in the body.
Cell-mediated immunity
The second arm of the immune response is refered to as CellMediated Immunity (CMIR). As the
name implies, the functional "effectors" of this response are various immune cells. Cell mediated immunity is
produced
by
the
sensetised
involve antibodies or complement but
lymphocytes
rather
.
involves
It
the
is
an immune
activation
of
response that
does
macrophages, natural
not
killer
cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an
antigen and this results in the lysis of the microbial antigens.
Historically, the immune system was separated into two branches: humoral immunity, for which the
protective function of immunization could be found in the humor (cell-free bodily fluid or serum) and cellular
immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T
cells provide protection against different pathogens.
If an individual is exposed to a particularantigan for the second time, immune response occurs more
quickly and more abundantly than during the first exposure. This is known as the secondary response. Both
humoral and cell mediated immunity are associated with immunologic memory that is the immune system is
able to retain the memory of the first antigenic exposure and there by during the secons similar exposure it
produces a quick response.
Cellular immunity protects the body by:

apoptosis in body cells displaying epitopes of foreign antigen on their
surface, such as virus-infected cells, cells with intracellular bacteria, and
cancer cells displaying tumor antigens;

activating macrophages and natural killer cells, enabling them to destroy
intracellular pathogens

stimulating cells to secrete a variety of cytokines that influence the
function of other cells involved in adaptive immune responses and innate
immune responses.
Cell-mediated immunity is directed primarily at microbes that survivein
phagocytes and microbes that infect non-phagocytic cells. It is most effective
in removing virus-infected cells, but also participates in defending
against fungi, protozoans, cancers, and intracellular bacteria. It also plays a
major role in transplant rejection.
These functions include:
Phagocytosis and killing of intracellular pathogens
Direct cell killing by NK and K cells
Direct cell killing by cytotoxic T cells
These responses are especially important for destroying intracellular bacteria, eliminating viral infections
and destroying tumor cells.
CELLS OF IMMUNE SYSTEM
The immune system operates by producing cells.The important cells as follows
LYMPHOCYTES
Lymphocytes are the mononucleate, nongranular leukocytes of lymphoid tissue, participating in
immunity. They are found in blood, lymph and lymphoid tissues such as spleen, lymph nodes, tonsils, peyers
patches etc. They are spherical or ovoid in shape. They have a diameter of 7 to 12 microns. They have a large
nucleus and cytoplasm.
The lymphocyes are of three types. They are B lymphocytes, Tlymphocytes and null cells.T he null cells
constitute about 3%, the B cells about 27% and the T cells about 70% of the total T lymphocytes.
The B lymphocytes mature in bursa of Fabricus in birds or in bone marrow of mammals. The B
lymphocytes produce antibodies and hence they are responsible for humoral immunity. The B cells kill
bactreria, virus etc
The T lyphocytes mature in the thymus under the influence of thymic hormones. The T lymphocytes bring
about the cell mediated immunity. The T cells are responsible for the killing of cancer cells,killing viral infected
cells, allograft etc.
The null cells form a third population of lymphocytes. They are intermediate between T and B cells. They
have cytotoxic activity.
B LYMPHOCYTES
The lymphocytes that mature in bursa of fabricius or bone marrow and that brings about humoral
immunity is called B lymphocytes. The B lymphocytes have a large nucleus. They are found in the blood ad
lymph. But they are highly concentrated in lymph nodes and spleen. They contain surface immunoglobulins.
The B lymphocytes have the following markers.

Ia(immune associated) protein which binds with the Ia receptor of the T
lymphocytes.
IgM.

Fc protein to bind with the Fc fragment of the immunoglobulin

CRI and CR3 receptors of complement system.

Surface immunoglobulins
The immature B cell changes in to mature B cell which has IgD molecules on the surface in addition to
PLASMA CELLS
These are the end cells of the B lymphocytes. They secrete immunoglobulins. They are very rarely seen in
the plasma of blood but are but are found mainly in the lymph nodes and spleenThe cytoplasm is completely
filled with rough endoplasmic reticulam. This is a site of protein synthesis namely synthesis of
immunoglobulins.
MACROPHAGE ACTIVATION
Macrophages are the large mononuclear phagocytic cells derived from monocytes. They are components
of the reticuloendothelial system. They are distributed through out the body but concentrated in lymph nodes,
spleen, bone marrow and liver. They are large lymphoid cells. They have large nucleus. They contain large
number of lysosomes.
While the production of antibody through the humoral immune response can
effectively lead to the elimination of a variety of pathogens, bacteria that have
evolved to invade and multiply within phagocytic cells of the immune response
pose a different threat. The following graphics illustrate this dilemma:
Non-encapsulated microorganisms are easily phagocytosed and killed within macrophages.
Encapsulated microorganisms require the production of antibody in order to be effectively phagocytosed. Once engul
however, they are easily killed.
cellular microorganisms
Intracellular microorganisms elicit the production of antibody, which allows effective phagocytosis. Once engul
however, they survive within the phagocyte and eventually kill it.
IFN
TNF
Intracellular microorganisms also activate specific T-cells, which then release lymphokines (e.g. IFN, TNF) that ca
macrophage activation. Activated ("killer") macrophages are then very effective at destroying the intracellular pathogens
This process can be further illustrated by considering the following experiment
known as "Koch's phenomenon". Inoculation of an unimmunized guinea pig with
a lethal dose of the intracellular pathogen Mycobacterium tuberculosis (MT)
results in death of the animal. Inoculation with a sub-lethal dose induces
immunity.Inoculation of an MT-immunized guinea pig with a lethal dose of MT
causes a local reaction ("delayed hypersensitivity") one to two days later.
Inoculation of an MT-immunized guinea pig with a lethal dose of a different
intracellular pathogen, Listeria monocytogenes (LM) again results in death of
the animal.Inoculation of an MT-immunized guinea pig with a lethal dose of
LM and MT causes a delayed hypersensitivity reaction.These results
demonstrate the specific (T-cell mediated) and non-specific (macrophage
mediated) aspects of this type of cell mediated immunity.
T LYMPHOCYTES
The mononucleated non granular leukocyte that matures in thymus and that
bringa about cell mediated immunity is called T lymphocyte. They vare highly
concentrated in blood and spleen. The T cell markers as follows.

Erythocyte receptor- It recognizes the sheep erythrocytes
T cell antigen receptor – It recognizes MHC antigens
The Ia protein receptor etc
1. T helper cells
They are sub population of T lymphocytes that help B cells and T cells in
immune responses They are regulator cells. They help the B and T cells in
many ways . The t helper cells contain glycoprotein molecules called CD4 molecules on the
urface. The HIV infects mainly these cells. The TH cells are activated by very small quantities
of antigen which can not activate other cells. They secrete lymphokines.
2. T suppressor cells
These cells are a sub population of T cells that suppresses the activity of B cells and other T
Cells. They are the regulatory T cells. They inhibit antibody production by B cells. They
suppress the functions of the T killer cells and T helper cells. They are responsible for
immune tolerance by limiting the ability of the immune system to attack a persons own
bodt tissue.
CELL MEDIATED CYTOTOXICITY
The second half of the cell-mediated immune response is involved in rejection of
foreign grafts and the elimination of tumors and virus-infected cells. The
effector cells involved in these processes are cytotoxic T-lymphocytes (CTLs), NKcells and K-cells. Each of these effector cells recognizes their target by different
means, described below.
Cytotoxic T-lymphocytes
CTLs, like other T-cells are both antigen and MHC-restricted. That is, CTLs require i) recognition of a
specific antigenic determinant and ii) recognition of "self" MHC (Click here to review these requirements).
Briefly, CTLs recognize antigen via their T-cell receptor. This receptor makes specific contacts with the
antigenic determinant and the target cell's class I MHC molecule. CTLs also express CD8, which may assist the
antigen recognition process. Once recognition is successful, the CTL "programs" the target cell for selfdestruction. This process is thought to occur in one of several possible ways. First, CTLs may release a
substance known as perforin in the space between the CTL and its target. In the presence of calcium ions, the
perforin polymerizes, forming channels in the target cell's membrane. These channels may cause the target cell
to lyse. Second, the CTL may also release various enzymes that pass through the polyperforin channels, causing
target cell damage. Third, the CTL may release lymphokines and/or cytokines that interact with specific
receptors on the target cell surface, causing internal responses that lead to destruction of the target cell. CTLs
principally act to eliminate endogenous antigens.
NULL CELLS
These are lymphocytes with cytotoxic properties. They are neither B cells or T cells. They are
intermediate between these. They form less than 3%. Two types namely natural killer cells and killer cells.
NK-CELLS
NK cells are part of a group know as the "large granular lymphocytes". These cells are generally
non-specific, MHC-unrestricted cells involved primarily in the elimination of neoplastic or
tumor cells.
The precise mechanism by which they
recognize their target cells is not clear. Probably, there is some type of NK-determinant
expressed by the target cells that is recognized by an NK-receptor on the NK cell surface.
Once the target cell is recognized, killing occurs in a manner similar to that produced by
the CTL.
K-CELLS
K-cells are probably not a separate cell type but rather a separate function of the NK group. K-cells contain
immunoglobulin Fc receptors on their surface and are involved in a process known as Antibody-dependent Cellmediated Cytotoxicity (ADCC). ADCC occurs as a consequence of antibody being bound to a target cell
surface via specific antigenic determinants expressed by the target cell. Once bound, the Fc portion of the
immunoglobulin can be recognized by the K-cell. Killing then ensues by a mechanism similar to that employed
by CTLs. This type of CMIR can also result in Type II hypersensitivities.
UNIT-II
Antibody
Antibodies (also known as immunoglobulins[1], abbreviated Ig) are gamma globulin proteins
that are found in blood or other bodily fluids of vertebrates, and are used by the immune system
to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made
of basic structural units—each with two large heavy chains and two small light chains—to form,
for example, monomers with one unit, dimers with two units or pentamers with five units.
Antibodies are produced by a kind of white blood cell called a plasma cell. There are several
different types of antibody heavy chains, and several different kinds of antibodies, which are
grouped into different isotypes based on which heavy chain they possess. Five different antibody
isotypes are known in mammals, which perform different roles, and help direct the appropriate
immune response for each different type of foreign object they encounter.[2]
Though the general structure of all antibodies is very similar, a small region at the tip of the
protein is extremely variable, allowing millions of antibodies with slightly different tip
structures, or antigen binding sites, to exist. This region is known as the hypervariable region.
Each of these variants can bind to a different target, known as an antigen.[3] This huge diversity
of antibodies allows the immune system to recognize an equally wide variety of antigens. The
unique part of the antigen recognized by an antibody is called the epitope. These epitopes bind
with their antibody in a highly specific interaction, called induced fit, that allows antibodies to
identify and bind only their unique antigen in the midst of the millions of different molecules that
make up an organism. Recognition of an antigen by an antibody tags it for attack by other parts
of the immune system. Antibodies can also neutralize targets directly by, for example, binding to
a part of a pathogen that it needs to cause an infection.[4]
The large and diverse population of antibodies is generated by random combinations of a set of
gene segments that encode different antigen binding sites (or paratopes), followed by random
mutations in this area of the antibody gene, which create further diversity.[2][5] Antibody genes
also re-organize in a process called class switching that changes the base of the heavy chain to
another, creating a different isotype of the antibody that retains the antigen specific variable
region. This allows a single antibody to be used by several different parts of the immune system.
Production of antibodies is the main function of the humoral immune system.

Forms
Surface immunoglobulin (Ig) is attached to the membrane of the effector B cells by its
transmembrane region, while antibodies are the secreted form of Ig and lack the trans membrane
region so that antibodies can be secreted into the bloodstream and body cavities. As a result,
surface Ig and antibodies are identical except for the transmembrane regions. Therefore, they are
considered two forms of antibodies: soluble form or membrane-bound form (Parham 21-22).
The membrane-bound form of an antibody may be called a surface immunoglobulin (sIg) or a
membrane immunoglobulin (mIg). It is part of the B cell receptor (BCR), which allows a B cell
to detect when a specific antigen is present in the body and triggers B cell activation.[7] The BCR
is composed of surface-bound IgD or IgM antibodies and associated Ig-α and Ig-β heterodimers,
which are capable of signal transduction.[8] A typical human B cell will have 50,000 to 100,000
antibodies bound to its surface.[8] Upon antigen binding, they cluster in large patches, which can
exceed 1 micrometer in diameter, on lipid rafts that isolate the BCRs from most other cell
signaling receptors.[8] These patches may improve the efficiency of the cellular immune
response.[9] In humans, the cell surface is bare around the B cell receptors for several thousand
ångstroms,[8] which further isolates the BCRs from competing influences.
Immunoglobulin A
Isotypes
Name
Types
IgA
2
IgD
1
Antibody Complexes
IgE
1
IgG
4
IgM
1
Antibodies can come in different varieties known as isotypes or classes. In placental mammals
there are five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. They are each named
with an "Ig" prefix that stands for immunoglobulin, another name for antibody, and differ in their
biological properties, functional locations and ability to deal with different antigens, as depicted
in the table.[13]
The antibody isotype of a B cell changes during cell development and activation. Immature B
cells, which have never been exposed to an antigen, are known as naïve B cells and express only
the IgM isotype in a cell surface bound form. B cells begin to express both IgM and IgD when
they reach maturity—the co-expression of both these immunoglobulin isotypes renders the B cell
'mature' and ready to respond to antigen.[14] B cell activation follows engagement of the cell
bound antibody molecule with an antigen, causing the cell to divide and differentiate into an
antibody producing cell called a plasma cell. In this activated form, the B cell starts to produce
antibody in a secreted form rather than a membrane-bound form. Some daughter cells of the
activated B cells undergo isotype switching, a mechanism that causes the production of
antibodies to change from IgM or IgD to the other antibody isotypes, IgE, IgA or IgG, that have
defined roles in the immune system.
Structure
Antibodies are heavy (~150 kDa) globular plasma proteins. They have sugar chains added to
some of their amino acid residues.[15] In other words, antibodies are glycoproteins. The basic
functional unit of each antibody is an immunoglobulin (Ig) monomer (containing only one Ig
unit); secreted antibodies can also be dimeric with two Ig units as with IgA, tetrameric with four
Ig units like teleost fish IgM, or pentameric with five Ig units, like mammalian IgM.[16]
Several immunoglobulin domains make up the two heavy chains (red and blue) and the two light chains
(green and yellow) of an antibody. The immunoglobulin domains are composed of between 7 (for
constant domains) and 9 (for variable domains) β-strands. See also:
The variable parts of an antibody are its V regions, and the constant part is its C region.
Immunoglobulin domains
The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical
heavy chains and two identical light chains connected by disulfide bonds.[13] Each chain is
composed of structural domains called immunoglobulin domains. These domains contain about
70-110 amino acids and are classified into different categories (for example, variable or IgV, and
constant or IgC) according to their size and function.[17] They have a characteristic
immunoglobulin fold in which two beta sheets create a “sandwich” shape, held together by
interactions between conserved cysteines and other charged amino acids.
Heavy chain
There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and
μ.[3] The type of heavy chain present defines the class of antibody; these chains are found in IgA,
IgD, IgE, IgG, and IgM antibodies, respectively.[4] Distinct heavy chains differ in size and
composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately
550 amino acids.[3]
1. Fab region
2. Fc region
3. Heavy chain (blue) with one variable (VH) domain followed by a constant domain (CH1), a hinge region,
and two more constant (CH2 and CH3) domains.
4. Light chain (green) with one variable (VL) and one constant (CL) domain
5. Antigen binding site (paratope)
6. Hinge regions.
In birds, the major serum antibody, also found in yolk, is called IgY. It is quite different from
mammalian IgG. However, in some older literature and even on some commercial life sciences
product websites it is still called "IgG", which is incorrect and can be confusing.
Each heavy chain has two regions, the constant region and the variable region. The constant
region is identical in all antibodies of the same isotype, but differs in antibodies of different
isotypes. Heavy chains γ, α and δ have a constant region composed of three tandem (in a line) Ig
domains, and a hinge region for added flexibility;[13] heavy chains μ and ε have a constant region
composed of four immunoglobulin domains.[3] The variable region of the heavy chain differs in
antibodies produced by different B cells, but is the same for all antibodies produced by a single B
cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids
long and is composed of a single Ig domain.
Light chain
.
In mammals there are two types of immunoglobulin light chain, which are called lambda (λ) and
kappa (κ).[3] A light chain has two successive domains: one constant domain and one variable
domain. The approximate length of a light chain is 211 to 217 amino acids.[3] Each antibody
contains two light chains that are always identical; only one type of light chain, κ or λ, is present
per antibody in mammals. Other types of light chains, such as the iota (ι) chain, are found in
lower vertebrates like Chondrichthyes and Teleostei.
CDRs, Fv, Fab and Fc Regions
Some parts of an antibody have unique functions. The arms of the Y, for example, contain the
sites that can bind two antigens (in general identical) and, therefore, recognize specific foreign
objects. This region of the antibody is called the Fab (fragment, antigen binding) region. It is
composed of one constant and one variable domain from each heavy and light chain of the
antibody.[18] The paratope is shaped at the amino terminal end of the antibody monomer by the
variable domains from the heavy and light chains. The variable domain is also referred to as the
FV region and is the most important region for binding to antigens. More specifically, variable
loops of β-strands, three each on the light (VL) and heavy (VH) chains are responsible for binding
to the antigen. These loops are referred to as the complementarity determining regions (CDRs).
In the framework of the immune network theory, CDRs are also called idiotypes. According to
immune network theory, the adaptive immune system is regulated by interactions between
idiotypes.
The base of the Y plays a role in modulating immune cell activity. This region is called the Fc
(Fragment, crystallizable) region, and is composed of two heavy chains that contribute two or
three constant domains depending on the class of the antibody.[3] Thus, the Fc region ensures that
each antibody generates an appropriate immune response for a given antigen, by binding to a
specific class of Fc receptors, and other immune molecules, such as complement proteins. By
doing this, it mediates different physiological effects including opsonization, cell lysis, and
degranulation of mast cells, basophils and eosinophils.[13][19]
Function
Activated B cells differentiate into either antibody-producing cells called plasma cells that
secrete soluble antibody or memory cells that survive in the body for years afterward in order to
allow the immune system to remember an antigen and respond faster upon future exposures.[20]
At the prenatal and neonatal stages of life, the presence of antibodies is provided by passive
immunization from the mother. Early endogenous antibody production varies for different kinds
of antibodies, and usually appear within the first years of life. Since antibodies exist freely in the
bloodstream, they are said to be part of the humoral immune system. Circulating antibodies are
produced by clonal B cells that specifically respond to only one antigen (an example is a virus
capsid protein fragment). Antibodies contribute to immunity in three ways: they prevent
pathogens from entering or damaging cells by binding to them; they stimulate removal of
pathogens by macrophages and other cells by coating the pathogen; and they trigger destruction
of pathogens by stimulating other immune responses such as the complement pathway.[21]
The secreted mammalian IgM has five Ig units. Each Ig unit (labeled 1) has two epitope binding Fab
regions, so IgM is capable of binding up to 10 epitopes.
Activation of complement
Antibodies that bind to surface antigens on, for example, a bacterium attract the first component
of the complement cascade with their Fc region and initiate activation of the "classical"
complement system.[21] This results in the killing of bacteria in two ways.[6] First, the binding of
the antibody and complement molecules marks the microbe for ingestion by phagocytes in a
process called opsonization; these phagocytes are attracted by certain complement molecules
generated in the complement cascade. Secondly, some complement system components form a
membrane attack complex to assist antibodies to kill the bacterium directly.[22]
Activation of effector cells
To combat pathogens that replicate outside cells, antibodies bind to pathogens to link them
together, causing them to agglutinate. Since an antibody has at least two paratopes it can bind
more than one antigen by binding identical epitopes carried on the surfaces of these antigens. By
coating the pathogen, antibodies stimulate effector functions against the pathogen in cells that
recognize their Fc region.[6]
Those cells which recognize coated pathogens have Fc receptors which, as the name suggests,
interacts with the Fc region of IgA, IgG, and IgE antibodies. The engagement of a particular
antibody with the Fc receptor on a particular cell triggers an effector function of that cell;
phagocytes will phagocytose, mast cells and neutrophils will degranulate, natural killer cells will
release cytokines and cytotoxic molecules; that will ultimately result in destruction of the
invading microbe. The Fc receptors are isotype-specific, which gives greater flexibility to the
immune system, invoking only the appropriate immune mechanisms for distinct pathogens.[3]
Natural antibodies
Humans and higher primates also produce “natural antibodies” which are present in serum before
viral infection. Natural antibodies have been defined as antibodies that are produced without any
previous infection, vaccination, other foreign antigen exposure or passive immunization. These
antibodies can activate the classical complement pathway leading to lysis of enveloped virus
particles long before the adaptive immune response is activated. Many natural antibodies are
directed against the disaccharide galactose α(1,3)-galactose (α-Gal), which is found as a terminal
sugar on glycosylated cell surface proteins, and generated in response to production of this sugar
by bacteria contained in the human gut.[23] Rejection of xenotransplantated organs is thought to
be, in part, the result of natural antibodies circulating in the serum of the recipient binding to αGal antigens expressed on the donor tissue.[24]
Immunoglobulin diversity
Virtually all microbes can trigger an antibody response. Successful recognition and eradication
of many different types of microbes requires diversity among antibodies; their amino acid
composition varies allowing them to interact with many different antigens.[25] It has been
estimated that humans generate about 10 billion different antibodies, each capable of binding a
distinct epitope of an antigen.[26] Although a huge repertoire of different antibodies is generated
in a single individual, the number of genes available to make these proteins is limited by the size
of the human genome. Several complex genetic mechanisms have evolved that allow vertebrate
B cells to generate a diverse pool of antibodies from a relatively small number of antibody
genes.[27]
Domain variability
The hypervariable regions of the heavy chain are shown in red, PDB 1IGT
The region (locus) of a chromosome that encodes an antibody is large and contains several
distinct genes for each domain of the antibody—the locus containing heavy chain genes (IGH@)
is found on chromosome 14, and the loci containing lambda and kappa light chain genes (IGL@
and IGK@) are found on chromosomes 22 and 2 in humans. One of these domains is called the
variable domain, which is present in each heavy and light chain of every antibody, but can differ
in different antibodies generated from distinct B cells. Differences, between the variable
domains, are located on three loops known as hypervariable regions (HV-1, HV-2 and HV-3) or
complementarity determining regions (CDR1, CDR2 and CDR3). CDRs are supported within the
variable domains by conserved framework regions. The heavy chain locus contains about 65
different variable domain genes that all differ in their CDRs. Combining these genes with an
array of genes for other domains of the antibody generates a large cavalry of antibodies with a
high degree of variability. This combination is called V(D)J recombination discussed below.[28]
V(D)J recombination
Simplistic overview of V(D)J recombination of immunoglobulin heavy chains
Somatic recombination of immunoglobulins, also known as V(D)J recombination, involves the
generation of a unique immunoglobulin variable region. The variable region of each
immunoglobulin heavy or light chain is encoded in several pieces—known as gene segments.
These segments are called variable (V), diversity (D) and joining (J) segments.[27] V, D and J
segments are found in Ig heavy chains, but only V and J segments are found in Ig light chains.
Multiple copies of the V, D and J gene segments exist, and are tandemly arranged in the
genomes of mammals. In the bone marrow, each developing B cell will assemble an
immunoglobulin variable region by randomly selecting and combining one V, one D and one J
gene segment (or one V and one J segment in the light chain). As there are multiple copies of
each type of gene segment, and different combinations of gene segments can be used to generate
each immunoglobulin variable region, this process generates a huge number of antibodies, each
with different paratopes, and thus different antigen specificities.[2]
After a B cell produces a functional immunoglobulin gene during V(D)J recombination, it cannot
express any other variable region (a process known as allelic exclusion) thus each B cell can
produce antibodies containing only one kind of variable chain.[3][29]
Somatic hypermutation and affinity maturation
Following activation with antigen, B cells begin to proliferate rapidly. In these rapidly dividing
cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate
of point mutation, by a process called somatic hypermutation (SHM). SHM results in
approximately one nucleotide change per variable gene, per cell division.[5] As a consequence,
any daughter B cells will acquire slight amino acid differences in the variable domains of their
antibody chains.
This serves to increase the diversity of the antibody pool and impacts the antibody’s antigenbinding affinity.[30] Some point mutations will result in the production of antibodies that have a
weaker interaction (low affinity) with their antigen than the original antibody, and some
mutations will generate antibodies with a stronger interaction (high affinity).[31] B cells that
express high affinity antibodies on their surface will receive a strong survival signal during
interactions with other cells, whereas those with low affinity antibodies will not, and will die by
apoptosis.[31] Thus, B cells expressing antibodies with a higher affinity for the antigen will
outcompete those with weaker affinities for function and survival. The process of generating
antibodies with increased binding affinities is called affinity maturation. Affinity maturation
occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T
cells.[32]
Mechanism of class switch recombination that allows isotype switching in activated B cells
Class switching
Isotype or class switching is a biological process occurring after activation of the B cell, which
allows the cell to produce different classes of antibody (IgA, IgE, or IgG). The different classes
of antibody, and thus effector functions, are defined by the constant (C) regions of the
immunoglobulin heavy chain. Initially, naïve B cells express only cell-surface IgM and IgD with
identical antigen binding regions. Each isotype is adapted for a distinct function, therefore, after
activation, an antibody with a IgG, IgA, or IgE effector function might be required to effectively
eliminate an antigen. Class switching allows different daughter cells from the same activated B
cell to produce antibodies of different isotypes. Only the constant region of the antibody heavy
chain changes during class switching; the variable regions, and therefore antigen specificity,
remain unchanged. Thus the progeny of a single B cell can produce antibodies, all specific for
the same antigen, but with the ability to produce the effector function appropriate for each
antigenic challenge. Class switching is triggered by cytokines; the isotype generated depends on
which cytokines are present in the B cell environment.[33]
Class switching occurs in the heavy chain gene locus by a mechanism called class switch
recombination (CSR). This mechanism relies on conserved nucleotide motifs, called switch (S)
regions, found in DNA upstream of each constant region gene (except in the δ-chain). The DNA
strand is broken by the activity of a series of enzymes at two selected S-regions.[34][35] The
variable domain exon is rejoined through a process called non-homologous end joining (NHEJ)
to the desired constant region (γ, α or ε). This process results in an immunoglobulin gene that
encodes an antibody of a different isotype.[36]
INTRODUCTION:
Antigen (Ant Installer Generator) is a tool to take an Ant build script, combine it with a GUI and
wrap it up as an executable jar file. Its primary purpose is to create powerful graphical
installers from Ant scripts.
ANTIGENS PROPERTIES:
Antigens- may possess:

Immunogenicity,

Antigenicity,

Allerogenicity, or

Tolerogenicity
Immunogenicity –
Property that allows a substance to induce a detectable immune response (humoral or
cellular) when introduced into an animal. Such substances are termed Immunogens.
Antigenicity –
Property that allows a substance to combine specifically with antibodies or TcR,whether
or not they are immunogenic. Therefore, all immunogens are antigens but not all antigens are
immunogens.
Allerogenicity-
Property that allows a substnace to induce an allergic response. Such substances are
termed allergens.
Tolerogenicity-
Property that allows a substance to induce specific immunologic non-responsiveness in
either the humoral or cell-mediated branch. Such substances are termed tolerogens.
Haptens –
Low-molecular weight compounds including many drugs and antibiotics, are nonimmunogenic but when coupled to immunogenic proteins, the resulting conjugates stimulate the
production of antibodies which can bind to the low-molecular weight component. Such
molecules are termed haptens.
Epitope –
The part of an antigen that combines with a specific antibody or T cell receptor. Previous
term used was antigenic determinant.
Immunogens
For the induction of humoral immunity (antibody response), the most potent immunogens
are macromolecular proteins or glycoproteins, but polysaccharides, synthetic peptides, and
other synthetic polymers such as polyvinylpyrrolidone are immunogenic under appropriate
conditions. Pure nucleic acids or lipids are not immunogenic but antibodies which react with
them can be induced by immunization with nucleoproteins or lipoproteins.
In general, proteins serve as immunogens for T cell-mediated immunity. (Recall that these
proteins must be processed into peptides and the peptides must be presented by an APC in
association with MHC proteins.
Requirements for Immunogenicity
The requirements are somewhat dependent upon the experimental conditions (mode of
immunization, organism being immunized, sensitivity of detection methods, etc.). However,
certain conditions must be met in order for a molecule to be immunogenic:
A. Foreignness: [Rabbit albumin not immunogenic in another rabbit but would be immunogenic
in a mouse.]
B. Molecular Size: [Certain minimum size is required for immunogenicity. The most potent
immunogens are macromolecular proteins with
molecular weights greater than 100,000. Substances <1000 are not usually immunogenic]
C. Chemical Complexity: [Homopolymers consisting of repeating units of a single amino acid
are poor immunogens regardless of their size]
Co-polymers of 2 or 3 amino acids may be good immunogens. A co-polymer of glutamic acid
and lysine must be 30-40,000 to be immunogenic
Add tyrosine - reduce size limitation to 10-20,000
Add tyrosine and phenylalanine and reduce to, 4000
-- In general, immunogenicity increases with structural complexity.
Aromatic amino acids contribute more to immunogenicity than non-aromatic (ex: tyrosine or
phenylalanine).
D. Degradability: Macromolecules that cannot be degraded and processed by APCs are poor
immunogens. For example, polymers of D-amino acids are not immunogenic. Proteolytic
enzymes can only degrade proteins containing L-amino acids.
E. Genetic constitution of the Animal: Immune response is under genetic control. Individuals
differ in their ability to respond to immunogens.
Animal may not have an appropriate Ig, may not have an appropriate TcR, or may not have an
appropriate MHC.
F. Method of Antigen Administration: Whether an antigen will induce an immune response
depends on the dose and the mode of administration.
Antigen can be delivered in a variety of ways: ip, sc, iv, im, id, etc. The repsonse will vary
depending upon the route chosen. In addition, too little antigen may not stimulate a strong
response, whereas too much antigen may stimulate tolerance (specific non-responsiveness). The
use of an adjuvant can also enhance the immunogenicity of an antigen. Adjuvants (such as
Freunds) prolong the persistence of the antigen in the body, induce a strong inflammatory
response which can lead to granuloma formation, and can stimulate lymphocyte proliferation.
T cells and B cells exhibit fundamental differences in epitope recognition.
B cell epitopes [epitopes recognized by membrane-bound Ig, or secreted Ig]
The binding of antigen to Ig involves weak non-covalent interactions so there must be
complementarity.
Properties of B cell epitopes:
1]Epitopes may be associated with soluble immunogens or particulate immunogens.
2]Epitopes tend to be accessible and on the exposed surface of the immunogen.
3]Epitopes generally contain hydrophilic amino acids
4]Often the epitopes are found where the molecule bends or where there is a high degree of
segmental mobility [atomic mobility]
5]The epitope may consist of either sequential or non-sequential amino acids. If non-sequential,
the 3D conformation of the epitope if vital.
6] Complex proteins contain multiple overlapping epitopes
7] The size of a B cell epitope is determined by the size of the antigen binding site on the
antibody molecule. Conformationally determined epitopes tend to require a larger epitope.
Smaller ligands (carbohydrates, peptides, haptens, etc.) tend to fit within a deep concave pocket
or crevice. Larger globuluar antigens tend to interact with Ig across a large planar face.
Protrusions on the antigen binding site would be complementary with depressions on the epitope
and vice versa. This type of interaction is obviously highly dependent upon the 3D conformation
of the globular antigen.
T cell epitopes [epitopes recognized by membrane-bound TcR only]
T cells recognize processed peptides associated with MHC on the surface of APCs (Class II
MHC) or altered self cells (Class I MHC). In other words, T cells exhibit MHC RESTRICTED
ANTIGEN RECOGNITION
CD4+ T cells are restricted to Class II MHC
CD8+ T cells are restricted to Class I MHC
More recent evidence suggests that a small population of T cells may possess TcRs capable of
recognizing lipids or glycolipids. Little is known about this type of recognition and we will
restrict our discussion to peptide epitopes.
1]The binding of the TcR to MHC + peptide represents a tri-molecular complex.
2]Only oligomeric peptides serve as epitopes. Epitopes which bind to Class I MHC have an
optimal size of 9 amino acids with a range from 8-11. Epitopes which bind to Class II MHC
have an optimal size range from 12-25 amino acids.
3) Epitopes are often internal and only exposed by processing within APCs or target cells.
4] Antigen processing is required to generate the peptides that interact specifically with MHC
molecules.
5] T cell epitopes must have two binding regions. The region of the peptide which binds to MHC
is termed the agretope while the region which binds to the TcR is termed the epitope.
6) Complex proteins may contain multiple, overlapping epitopes.
7 ) Immunodominant T cell epitopes are determined by the set of MHC molecules which are
expressed by an individual.
Experimentally it has been demonstrated that there is a correlation between the ability of a
peptide to bind to a particular MHC molecule and the T
cell response to that peptide.
MITOGENS
Mitogens are agents that are able to induce cell division (mitosis) in a high percentage
of T or B cells. This proliferation is described as polyclonal activation (as opposed to clonal
expansion following the specific encounter with a conventional immunogen). There are T cell
mitogens and B cell mitogens.
A number of common mitogens are lectins. Lectins are proteins which bind to specific
carbohydrate groups (moieties). They bind to glycoproteins on the surface of the lymphocytes
and cause activation. Important...they do not act via conventional TcR-epitope or Ig-epitope
interactions.
Lectin Examples:
Con A - T cell mitogen
PHA - T cell mitogen
PWM - T and B cell mitogen
Another important mitogen which is NOT a lectin is LPS (lipopolysaccharide).
This
polysaccharide component of the outer membrane of gram - bacteria is also known as endotoxin.
LPS is a very potent mitogen for B cells.
SUPERANTIGENS
Among the most potent T cell mitogens known!!
Bind differently to the TcR and MHC so a large # of T cells are activated. In some cases as many
as 1/5 T cells may be activated to proliferate and to secrete cytokines. See figure in text for
nature of interaction of superantigen + TCR and MHC. Many microbial pathogens (including
Streptococcus and Staphylococcus) produce toxins which function as superantigens.
The
symptoms of staphylococcal food poisoning are due to the massive T cell proliferation and
cytokine secretion which occurs in the GALT due to the presence of staphylococcal enterotoxin
in food. Toxic shock syndrome and Toxic shock-like syndrome are both due to the action of
bacterial superantigens.
GENETIC BASIS OF ANTIBODY DIVERSITY
OBJECTIVES: When you finish this section, you should be able to: 1. Define the following
terms: allelic exclusion, isotype switching, affinity maturation, antibody repertoire, alternative
RNA splicing, recombination signal sequence.2. Describe the genes that encode Ig Heavy and
Light chains. 3. Describe the sequence of Ig gene rearrangement that occurs during B cell
differentiation. 4. Discuss how diversity in antibody specificity is achieved. 5. Discuss the
mechanisms of heavy chain class switching. 5. Calculate the number of possible Igs which can
be produced from a
given number of V, J, D, and C genes.
GENETIC BASIS OF ANTIBODY DIVERSITY
•Estimates of antibody specificities in an individual range between 106 - 108 .
•If 1 gene encoded 1 immunoglobulin of a given specificity, approximately 106 - 108 genes
would be required to JUST encode antibodies.
•This is impossible because the entire human genome is made up of 3.2 x 109
base pairs.
•Average protein = 107 amino acids = 107 x 3 = 3.2 x 102 base pairs
•If 1 gene encodes 1 protein, the total # of genes will be equivalent to
3.2 x 109
3.2 x 102 = 1 x 107 genes
• However, estimates are that only 31,000 to 35,000 genes in the human genome
actually encode proteins!
� This number of genes is unable to encode ALL antibody specificities
Organization of Immunoglobulin Genes
�Numerous V region genes are preceded by
Leader or signal sequences (60-90 bp)
exons interspersed with introns.
�Heavy chain contains V (Variable), D
(Diversity), J (Joining) and C (Constant)
region gene segments.
•V-D-J-C
�Light chain contains V, J, and C region
gene segments
•V-J-C
•Constant region genes are sub-divided into
exons encoding domains
(CH1,CH2, CH3, CH4)
Genomic organization of the heavy and light chain gene segments in humans
CHARACTERISTICS OF IMMUNOGLOBULIN GENE RE-ARRANGEMENT
1. Involves Allelic Exclusion.
– Only one of two parental alleles of Ig is expressed in a B cell.
– Either kappa or lambda light chain is expressed by a B cell (light chain isotype
exclusion).
2. Ig rearrangement occurs prior to antigen exposure.
• A. Heavy chain re-arrangement
– Re-arrangement occurs in a precise order:
– Heavy chain re-arranges before Light chain.
– D-J joining occurs first to form DJ and is followed by V-DJ joining to form VDJ.
– Production of μ heavy chain by re-arrangement of one allele inhibits re-arrangement
on other allele.
� If re-arrangement on first allele is non-productive (due to mutations, deletions or
frame shifts that generate stop codons), then re-arrangement on the second
allele is stimulated.
� Therefore, in any antibody-producing B cell, one allele is productively rearranged
and the other is either not re-arranged (in germ line configuration) or is
aberrantly re-arranged.
5
B. Light chain re-arrangement i.Kappa chain (κ) rearranges before lambda (λ) chain Vjoining occurs.
ii. Productive arrangement on one allele blocks re-arrangement on other allele.
iii. If kappa protein is produced, re-arrangement of lambda chain is blocked. Otherwise
lambda chain undergoes re-arrangement.
GENETIC BASIS OF ANTIBODY DIVERSITY
OBJECTIVES: When you finish this section, you should be able to:
1. Define the following terms: allelic exclusion, isotype switching, affinity
maturation, antibody repertoire, alternative RNA splicing, recombination
signal sequence.
2. Describe the genes that encode Ig Heavy and Light chains.
3. Describe the sequence of Ig gene rearrangement that occurs during B
cell differentiation.
4. Discuss how diversity in antibody specificity is achieved.
5. Discuss the mechanisms of heavy chain class switching.
5. Calculate the number of possible Igs which can be produced from a
given number of V, J, D, and C genes.
GENETIC BASIS OF ANTIBODY DIVERSITY
•Estimates of antibody specificities in an individual range between 106 - 108 .
•If 1 gene encoded 1 immunoglobulin of a given specificity, approximately 106 108 genes would be required to JUST encode antibodies.
•This is impossible because the entire human genome is made up of 3.2 x 109
base pairs.
•Average protein = 107 amino acids = 107 x 3 = 3.2 x 102 base pairs
•If 1 gene encodes 1 protein, the total # of genes will be equivalent to
3.2 x 109
3.2 x 102 = 1 x 107 genes
• However, estimates are that only 31,000 to 35,000 genes in the human genome
actually encode proteins!
� This number of genes is unable to encode ALL antibody specificities!
DILEMMA
Since only 31-35 thousand genes in the human genome actually
encode proteins,
How then is antibody diversity (between 1 million to 100 million
Organization of Immunoglobulin Genes
Numerous V region genes are preceded by nLeader or signal sequences (60-90 bp) exons
interspersed with introns. Heavy chain contains V (Variable), D (Diversity), J (Joining) and C
(Constant) region gene segments. • V - D - J - C
�Light chain contains V, J, and C region gene segments • V - J - C
•Constant region genes are sub-divided into exons encoding domains
(CH1,CH2, CH3, CH4) Genomic organization of the heavy and light chain gene segments in
humans
CHARACTERISTICS OF IMMUNOGLOBULIN GENE RE-ARRANGEMENT
1. Involves Allelic Exclusion.
– Only one of two parental alleles of Ig is expressed in a B cell.
– Either kappa or lambda light chain is expressed by a B cell (light chain isotype
exclusion).
2. Ig rearrangement occurs prior to antigen exposure.
• A. Heavy chain re-arrangement
– Re-arrangement occurs in a precise order:
– Heavy chain re-arranges before Light chain.
– D-J joining occurs first to form DJ and is followed by V-DJ joining to form VDJ.
– Production of μ heavy chain by re-arrangement of one allele inhibits re-arrangement
on other allele.
� If re-arrangement on first allele is non-productive (due to mutations, deletions or
frame shifts that generate stop codons), then re-arrangement on the second
allele is stimulated.
� Therefore, in any antibody-producing B cell, one allele is productively rearranged
and the other is either not re-arranged (in germ line configuration) or is
aberrantly re-arranged.
B. Light chain re-arrangement
i.Kappa chain (κ) rearranges before lambda (λ) chain V-joining occurs.
ii. Productive arrangement on one allele blocks re-arrangement
on other allele. iii. If kappa protein is produced, re-arrangement of lambda chain
is blocked. Otherwise lambda chain undergoes re-arrangement.
MECHANISM OF IMMUNOGLOBULIN RE-ARRANGEMENT
� Occurs principally via looping out (excision) of intervening gene
sequences followed by ligation of Ig gene segments.
� Controlled by recombination signal sequences (RSS) located at joining
sites.
� Consist of heptamer/nonamer (7/9) sequences interspersed by 12/23
base pair spacers.
� Recognized by Recombinases (enzymes with endonuclease and ligase
activities). Consists of RAG1,2 proteins (lymphocyte-specific, and nonlymphocytespecific DNA repair proteins (DNA ligase IV, DNAdependent
protein kinase (DNA-PK) and Ku, a protein that associates
with DNA-PK
� Genes encoding recombinases are present in all cell types but are
expressed only in lymphoid (B &T) cells.
� Recombination activating genes 1 and 2 (RAG-1, RAG-2) have been
identified which stimulate Ig gene rearrangement. Have endonuclease
activity.
MECHANISMS FOR GENERATING ANTIBODY DIVERSITY
• Presence of multiple V genes in the germ line.
• Combinatorial Diversity - due to potentially different associations of different V, D and J
gene segments.
• Junctional Diversity
• i. Imprecise joining
• ii. N/P region (insertional) diversity occurs in VDJ joining (heavy chain) as well as
VJ join of light chain. Arises from addition of up to 20 nucleotides by terminal
deoxynucleotidyl transferase (TdT).
• Somatic Hypermutation
• i. Occurs randomly after antigenic stimulation and principally in CDR1,
CDR2, CDR3 regions (more frequent in CDR3). Introduces point mutations at a
higher rate than for normal mammalian genes. Mutation rate of V genes is 1 base
pair change per 103 base pairs/cell division; it is 10-7 in other mammalian genes.
• ii. Can give rise to Ig with different (new) antigen specificities leading to
high or low affinity Abs. High affinity B cell clones are selectively
expanded (Affinity Maturation).
• iii. Affinity maturation is associated with isotype switching.
• Random Assortment of H and L chains.
Mechanisms Contributing to the Generation of Antibody Diversity in Humans
Germ line genes H κ λ V segments 65 40 30 J segments 6 5 4 D segments 27 0 0
Combinatorial Joining V x J (x D) 11,000 200 120 H-L chain associations H x κ 2.2 x 106
from 177 H x λ 1.3 x 106 segments EXPRESSION OF DIFFERENT CLASSES AND TYPES
OF IMMUNOGLOBULINS
•Co-expression of IgM and IgD
• Both IgM and IgD are co-expressed on the surface of a mature B lymphocyte.
• Occurs by alternative RNA splicing.
ISOTYPE SWITCHING
Is the conversion of an immunoglobulin from one isotype to another
(e.g. IgG to IgE) while retaining the same antigen specificity.
�Switching is dependent on antigenic stimulation and is induced by
cytokines released by helper T cells and requires engagement of CD40L
[e.g. IL-4 triggers switching from IgM to IgE or IgG4 (humans); IFN-γ
triggers switching from IgM to IgG2a (mice)]. Cyokines are thought to
alter chromatin structure making switch sites more accessible to
recombinases for gene transcription.
�Involves switch sites located in introns upstream of each CH segment
(except Cδ).
�Switch sites consist of multiple copies of conserved repeat sequences
[(GAGCT)n GGGGGT)] where n can vary from 3-7.
�Class switching occurs usually in activated B cells (including memory
cells) and not in naïve B cells and involves heavy chain genes. These
cells (you will recall) already have rearranged VDJ genes at the DNA
level and were producing IgM and IgD.
ISOTYPE SWITCHING (Contd.)
• If this B cell chooses to class switch to say IgG at the DNA level (by DNA
deletion, which is irreversible), then the switch regions of IgM and IgD (recall that
they are both controlled by one switch region) join with that of IgG to excise the
IgM and IgD constant region gene segments.
• When this occurs what you have left is the recombined VDJ gene cluster still
separated from the IgG constant region gene segments by intronic DNA.
• The switched gene will now be transcribed into primary RNA that undergoes
splicing to now bring the VDJ and C regions together to form VDJC.
• The take home lesson is that VDJ (or VJ) segments do not join to C region genes
at the DNA level whether it is during classical gene re-arrangement or class
switching. The joining of VDJ to C to form VDJC occurs only at the RNA level!
� Mechanism of switching is unclear. However, class specific recombinases
are thought to recognize and bind to switch sites to facilitate
recombination.
Isotype switching can occur by:
A. Switch recombination (Deletion of
DNA)
-primary mechanism of isotype switching -is irreversible
B. Alternative splicing of primary RNA transcript (rare)
-Explains co-expression of multiple isotypes by a single B cell.
Anti antibodies
Anti-nuclear antibodies (ANAs, also known as anti-nuclear factor or ANF) are autoantibodies
directed against contents of the cell nucleus.
They are present in higher than normal numbers in autoimmune disease. The ANA test measures
the pattern and amount of autoantibody which can attack the body's tissues as if they were
foreign material. Autoantibodies are present in low titers in the general population, but in about
5% of the population, their concentration is increased, and about half of this 5% have an
autoimmune disease.
One can check for the presence of ANAs in blood serum by means of a laboratory test. There are
also additional tests that allow one to test for individual ANAs. The general ANA test is usually
one of two types: indirect immunofluorescence or ELISA. The indirect immunofluoresence is
considered to be the more accurate of the two,[citation needed] but the ELISA version is gaining
popularity because of its lower cost.[citation needed]
[edit] Associated diseases
The normal titer of ANA is 1:40 or less. Higher titers are indicative of an autoimmune disease.
The presence of ANA is indicative of lupus erythematosus (present in 80-90% of cases), though
they also appear in some other auto-immune diseases such as[citation needed] Sjögren's syndrome
(60%), rheumatoid arthritis, autoimmune hepatitis, scleroderma and polymyositis &
dermatomyositis (30%), and various non-rheumatological conditions associated with tissue
damage. ANA are also directed to the nuclear pore complex in primary biliary cirrhosis.[citation
needed]
Other conditions with high ANA titre include[citation needed] Addison disease, Idiopathic
thrombocytopenic purpura (ITP), Hashimoto's, Autoimmune hemolytic anemia, Type I diabetes
mellitus, Mixed connective tissue disorder.
[edit] Sensitivity
The following table list the sensitivity of different types of ANAs for different diseases, in this
case what percentage of those with the disease have the ANA. Some ANAs appear in several
types of disease, resulting in lower specificity of the test.
ANA type Tar Sen
get siti
anti vity
gen
SLE Dru Diff Lim Sjö Infl
g- use ited gre am
ind syst Scl n ma
uce emi ero syn tor
d c der dro y
LE scle ma me my
rosi
opa
s
thy
All Vari>95 >95 70- 70- 50- 40AN ous
90 90 80 60
As
(by
indir
ect
IF)
Ant DN 40- i- A 60
dsD
NA
-
-
-
-
Ant Cor 20- i- e 30
Sm pro
tein
s of
snR
NPs
-
-
-
-
Ant Hist 50- >95 i- one 70
hist s
one
-
-
-
Ant An 30- i- snR 40
U1 NP
RN
P
15 10 -
-
Ant Typ i eI
Scl- top
70 ois
om
era
se
-
28- 10- 70 18
-
Ant Cen i- tro
cen me
tro ric
me pro
re tein
s
-
22- 90 26
-
SS- RN 30- A Ps 50
(Ro
)
-
-
70- 10
95
SS- RN 10- B Ps 15
(La)
-
-
60- 90
Jo- Hist 1 idin
etRN
A
liga
se
-
-
-
-
25
- = less than 5% sensitivity
Unless else specified in boxes, then ref is: [2]
About 30% to 40% of people with rheumatoid arthritis have a high ANA titer.
UNIT-III
INTRODUCTION
A substace that can produce a specific immune response when it is introduced in to the tissues of an animal
and that can react specifically with specific immune cell is known as an antigen.Antibody is the
immunological substance produced against the antigen.The interaction between antigen and antibody is
called antigen-antibody reaction.The antibody binds with the antigen form a complex molecule called
immune complex or antigen-antibody complex.
Strength of antigen-antibody binding
The reaction between antigen and antibody is highly specific.Specificity refers to the discriminate abi
of a particular antibody to combine with only one type of antigen.The strength of antigen –antibody can be def
by the following factors,
1. Binding forces of antigen and antibody
2. Avidity
3. Bonus Effect
4. Cross Effects
1.BINDING FORCES OF ANTIGEN-ANTIBODY
The binding between antigen and antibody reaction is due to three factors namely,
a , Closeness between antigen and antibody
b , Intermolecular forces
c , Affinity of antibody
A. when the antigen and antibody are closely fit,the strength of binding
is great.When they are apart,the binding strength is low.
B. The force which operate between two macromolecules,as for example between protein and protein ,also e
between antigen and antibody ag-ab reaction.The intermolecular forces which operate between antigen
antibody are non-covalent forces and are of 4 types
1.Electrostatic forces
2.Hydrogen bonding
3.Hydrophobic bonding
4.Vander Waals bonding
C. Affinity refers to the strength of binding between a particular molecule of antibody and a single antige
determinant.Antibodies which bond strongly to the antigenic determinantare called high affinity antibodies.Affin
is used to denote the binding capacity of an antibody with a univalent antigen.
2.AVIDITY
Avidity refers to the capacity of an antiserum containing various antibodies to combine with the wh
antigen that stimulated the production of antibodies.Avidity is used to denote the overall capacity of antibodies
combine with multivalent antigen.
Avidity is the strength of the bond after the formation of antigen-antibody complex.
A multivalent antigen has many types of antigenic determinants.When it is injected into the blood ,e
antigenic determinants stimulates the production of a particular antibody.The various antibodies produced by sin
antigen combine with the different antigenic determinants of the antigen.
3. BONUS EFFECT
The antigen-antibody reaction,the antibody not only binds with the antigen but also the antigens are brid
by a single antibody.In some cases two antigens may be bridged by a single antibody.Such binding is weak and
Ag-Ab reaction may be reversible in such cases.
But when two antigens are bridged by two antibodies,the binding will be strong and the Ag-Ab complex w
not dissociate.
This phenomenon of giving extra-strength to the antigen-antibody complex by the binding of two antibod
to the two antigen molecule is called bonus effect.
The bonus effect is highly possible because the antigens are multivalent and there are many types
antibodies.
Bonus effect increases the binding strength of antigen and antibody molecule.
4. CROSS REACTION
An antiserum raised against a given antigen may sometimes react with another closely related antigen.T
reaction is called cross reaction and the antigen which produces the cross reaction is called cross reaction antig
The cross reaction is due to the presence of one or more identical antigenic determinants on the related antigen.
Eg:The antiserum raised against the albumin of hen’s egg cross react with the albumin of duck’s egg.
Avidity and affinity
In proteins, avidity is a term used to describe the combined strength of multiple bond
interactions. Avidity is distinct from affinity, which is a term used to describe the strength of a
single bond. As such, avidity is the combined synergistic strength of bond affinities rather than
the sum of bonds.
It is commonly applied to antibody interactions in which multiple antigen binding sites
simultaneously interact with a target. Individually, each binding interaction may be readily
broken, however, when many binding interactions are present at the same time, transient
unbinding of a single site does not allow the molecule to diffuse away, and binding of that site is
likely to be reinstated. The overall effect is synergistic, strong binding of antigen to antibody
(e.g. IgM is said to have low affinity but high avidity because it has 10 weak binding sites as
opposed to the 2 strong binding sites of IgG, IgE and IgD).
If the clustered proteins form a matrix, such as a clathrin-coat, the interaction is described by
the term matricity.
Antigen
The basic principle of any immunochemical technique is that a specific antibody will combine
with its specific antigen to give an exclusive antibody-antigen complex
An ANTIGEN is defined as “any foreign substance that elicits an immune response (e.g., the
production of specific antibody molecules) when introduced into the tissues of a susceptible
animal and is capable of combining with the specific antibodies formed”. Antigens are generally
of high molecular weight and commonly are proteins or polysaccharides. Polypeptides, lipids,
nucleic acids and many other materials can also function as antigens. Immune responses may
also be generated against smaller substances, called haptens, if these are chemically coupled to a
larger carrier protein, such as bovine serum albumin, keyhole limpet hemocyanin (KLH) or other
synthetic matrices. A variety of molecules such as drugs, simple sugars, amino acids, small
peptides, phospholipids, or triglycerides may function as haptens. Thus, given enough time, just
about any foreign substance will be identified by the immune system and evoke specific antibody
production. However, this specific immune response is highly variable and depends much in part
on the size, structure and composition of antigens. Antigens that elicit strong immune responses
are said to be strongly immunogenic.
Antigen-Antibody Interaction
The specific association of antigens and antibodies is dependent on hydrogen bonds,
hydrophobic interactions, electrostatic forces, and van der Waals forces. These are all bonds of a
weak, non-covalent nature, yet some of the associations between antigen and antibody can be
quite strong. Like antibodies, antigens can be multivalent, either through multiple copies of the
same epitope, or through the presence of multiple epitopes that are recognized by multiple
antibodies. Interactions involving multivalency can produce more stabilized complexes, however
multivalency can also result in steric difficulties, thus reducing the possibility for binding. All
antigen-antibody binding is reversible, however, and follows the basic thermodynamic principles
of any reversible bimolecular interaction:
where KA is the affinity constant, Ab and Ag are the molar concentrations of unoccupied binding
sites on the antibody or antigen respectively, and Ab–Ag is the molar concentration of the
antibody-antigen complex.
The time taken to reach equilibrium is dependent on the rate of diffusion and the affinity of the
antibody for the antigen, and can vary widely. The affinity constant for antibody-antigen binding
can span a wide range, extending from below 105 mol–1 to above 1012 mol–1. Affinity constants
can be affected by temperature, pH and solvent. Affinity constants can be determined for
monoclonal antibodies, but not for polyclonal antibodies, as multiple bondings take place
between polyclonal antibodies and their antigens. Quantitative measurements of antibody affinity
for antigen can be made by equilibrium dialysis. Repeated equilibrium dialyses with a constant
antibody concentration but varying ligand concentration are used to generate Scatchard plots,
which give information about affinity valence and possible cross-reactivity.
Affinity describes the strength of interaction between antibody and antigen at single antigenic
sites. Within each antigenic site, the variable region of the antibody “arm” interacts through
weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the
affinity. Avidity is perhaps a more informative measure of the overall stability or strength of the
antibody-antigen complex. It is controlled by three major factors: antibody-epitope affinity; the
valence of both the antigen and antibody; and the structural arrangement of the interacting parts.
Ultimately these factors define the specificity of the antibody, that is, the likelihood that the
particular antibody is binding to a precise antigen epitope. Cross-reactivity refers to an antibody
or population of antibodies binding to epitopes on other antigens. This can be caused either by
low avidity or specificity of the antibody or by multiple distinct antigens having identical or very
similar epitopes. Cross-reactivity is sometimes desirable when one wants general binding to a
related group of antigens or when attempting cross-species labeling when the antigen epitope
sequence is not highly conserved in evolution.
Immunofluorescence
Definition
Immunofluorescence is a laboratory technique to identify specific antibodies or antigens.
Antibody identification is usually performed on blood (serum).
ELISA
Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay
(EIA), is a biochemical technique used mainly in immunology to detect the presence of an
antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine
and plant pathology, as well as a quality control check in various industries. In simple terms, in
ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is
applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme,
and in the final step a substance is added that the enzyme can convert to some detectable signal.
Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon
the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the
sample can be inferred through the magnitude of the fluorescence.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The
sample with an unknown amount of antigen is immobilized on a solid support (usually a
polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically
(via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the
antigen is immobilized the detection antibody is added, forming a complex with the antigen. The
detection antibody can be covalently linked to an enzyme, or can itself be detected by a
secondary antibody which is linked to an enzyme through bioconjugation. Between each step the
plate is typically washed with a mild detergent solution to remove any proteins or antibodies that
are not specifically bound. After the final wash step the plate is developed by adding an
enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the
sample.
Traditional ELISA typically involves chromogenic reporters and substrates which produce some
kind of observable color change to indicate the presence of antigen or analyte. Newer ELISAlike techniques utilize fluorogenic, electrochemiluminescent, and real-time PCR reporters to
create quantifiable signals. These new reporters can have various advantages including higher
sensitivities and multiplexing. Technically, newer assays of this type are not strictly ELISAs as
they are not "enzyme-linked" but are instead linked to some non-enzymatic reporter. However,
given that the general principles in these assays are largely similar, they are often grouped in the
same category as ELISAs.
Applications
ELISA results using S-OIV A neuraminidase antibody at 1 μg/ml to probe the immunogenic and
the corresponding seasonal influenza A neuraminidase peptides at 50, 10, 2 and 0 ng/ml.
Because the ELISA can be performed to evaluate either the presence of antigen or the presence
of antibody in a sample, it is a useful tool for determining serum antibody concentrations (such
as with the HIV test or West Nile Virus). It has also found applications in the food industry in
detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. ELISA can
also be used in toxicology as a rapid presumptive screen for certain classes of drugs.
The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an
ELISA, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens are
attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The
plate is then washed to remove all other components of the serum. A specially prepared
"secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate,
followed by another wash. This secondary antibody is chemically linked in advance to an
enzyme. Thus, the plate will contain enzyme in proportion to the amount of secondary antibody
bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a
change in color or fluorescence. ELISA results are reported as a number; the most controversial
aspect of this test is determining the "cut-off" point between a positive and negative result.
A cut-off point may be determined by comparing it with a known standard. If an ELISA test is
used for drug screening at workplace, a cut-off concentration, 50 ng/mL, for example, is
established, and a sample will be prepared which contains the standard concentration of analyte.
Unknowns that generate a signal that is stronger than the known sample are "positive". Those
that generate weaker signal are "negative."
ELISA can also be used to determine the level of antibodies in faecal content...specifically the
direct method
Types
"Indirect" ELISA
The steps of "indirect" ELISA follows the mechanism below:






A buffered solution of the protein antigen to be tested for is added to each well of a
microtiter plate, where it is given time to adhere to the plastic through charge
interactions.
A solution of non-reacting protein, such as bovine serum albumin, or casein is added to
block any plastic surface in the well that remains uncoated by the protein antigen.
Then the serum is added, which contains a mixture of the serum donor's antibodies, of
unknown concentration, some of which may bind specifically to the test antigen that is
coating the well.
Afterwards, a secondary antibody is added, which will bind any antibody produced by a
member of the donor's species (for example, an antibody produced in a mouse that will
bind any rabbit antibody). This secondary antibody often has an enzyme attached to it,
which has a negligible effect on the binding properties of the antibody.
A substrate for this enzyme is then added. Often, this substrate changes color upon
reaction with the enzyme. The color change shows that secondary antibody has bound to
primary antibody, which strongly implies that the donor has had an immune reaction to
the test antigen. This can be helpful in a clinical setting, and in R&D.
The higher the concentration of the primary antibody that was present in the serum, the
stronger the color change. Often a spectrometer is used to give quantitative values for
color strength.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the
enzyme molecules will produce many signal molecules. Within common-sense limitations the
enzyme can go on producing color indefinitely, but the more primary antibody is present in the
donor serum, the more secondary antibody + enzyme will bind, and the faster color will develop.
A major disadvantage of the indirect ELISA is that the method of antigen immobilization is nonspecific; when serum is used as the source of test antigen, all proteins in the sample may stick to
the microtiter plate well, so small concentrations of analyte in serum must compete with other
serum proteins when binding to the well surface. The sandwich or direct ELISA provides a
solution to this problem, by using a "capture" antibody specific for the test antigen to pull it out
of the serum's molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple
positive or negative result (yes or no) for a sample. The cutoff between positive and negative is
determined by the analyst and may be statistical. Two or three times the standard deviation (error
inherent in a test) is often used to distinguish positive from negative samples. In quantitative
ELISA, the optical density (OD) of the sample is compared to a standard curve, which is
typically a serial dilution of a known-concentration solution of the target molecule. For example
if your test sample returns an OD of 1.0, the point on your standard curve that gave OD = 1.0
must be of the same analyte concentration as your sample.
Sandwich ELISA
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any
antigen present binds to capture antibody; (3) enzyme linked capture antibody used as detecting
antibody is added, and binds to antigen; (4) substrate is added, and is converted by enzyme to
detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample
antigen. The steps are as follows:
1.
2.
3.
4.
5.
Prepare a surface to which a known quantity of capture antibody is bound.
Block any non specific binding sites on the surface.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.
Apply enzyme linked primary antibodies as detection antibodies which also bind
specifically to the antigen.
6. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
7. Apply a chemical which is converted by the enzyme into a color or fluorescent or
electrochemical signal.
8. Measure the absorbency or fluorescence or electrochemical signal (e.g., current) of the
plate wells to determine the presence and quantity of antigen.
The image to the right includes the use of a secondary antibody conjugated to an enzyme, though
technically this is not necessary if the primary antibody is conjugated to an enzyme. However,
use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked
antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that
binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a
variety of situations. Without the first layer of "capture" antibody, any proteins in the sample
(including serum proteins) may competitively adsorb to the plate surface, lowering the quantity
of antigen immobilized.Use of the purified specific antibody to attach the antigen to the plastic
eliminates a need to purify the antigen from complicated mixtures before the measurement,
simplifying the assay, and increasing the specificity and the sensitivity of the assay.
Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat
different than the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen. (Sample)
2. These bound antibody/antigen complexes are then added to an antigen coated well.
3. The plate is washed, so that unbound antibody is removed. (The more antigen in the
sample, the less antibody will be able to bind to the antigen in the well, hence
"competition.")
4. The secondary antibody, specific to the primary antibody is added. This second antibody
is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual
signal. The major advantage of a competitive ELISA is the ability to use crude or impure
samples and still selectively bind any antigen that may be present.
(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzymelinked antibody. The labeled antigen competes for primary antibody binding sites with your
sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained
in the well and the weaker the signal).
Commonly the antigen is not first positioned in the well.
Reverse ELISA
A new technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12
protruding ogives. The entire device is immersed in a test tube containing the collected sample
and the following steps (washing, incubation in conjugate and incubation in chromogenous) are
carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.
The advantages of this technique are as follows:
1. The ogives can each be sensitized to a different reagent, allowing the simultaneous
detection of different antibodies and different antigens for multi-target assays;
2. The sample volume can be increased to improve the test sensitivity in clinical (saliva,
urine), food (bulk milk, pooled eggs) and environmental (water) samples;
3. One ogive is left unsensitized to measure the non-specific reactions of the sample;
4. The use of laboratory supplies for dispensing sample aliquots, washing solution and
reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.
5. Enzyme-linked immunosorbent assay (ELISA): ELISA stands for "enzyme-linked
immunosorbent assay." This is a rapid immunochemical test that involves an enzyme (a
protein that catalyzes a biochemical reaction). It also involves an antibody or antigen
(immunologic molecules).
6. ELISA tests are utilized to detect substances that have antigenic properties, primarily
proteins (as opposed to small molecules and ions such as glucose and potassium). Some
of these include hormones, bacterial antigens and antibodies.
7. There are variations of this test, but the most basic consists of an antibody attached to a
solid surface. This antibody has affinity for (will latch on to) the substance of interest, for
example, human chorionic gonadotropin (HGC), the commonly measured protein which
indicates pregnancy. A mixture of purified HCG linked (coupled) to an enzyme and the
test sample (blood, urine, etc) are added to the test system. If no HCG is present in the
test sample, then only HCG with linked enzyme will bind. The more HCG which is
present in the test sample, the less enzyme linked HCG will bind. The substance the
enzyme acts on is then added, and the amount of product measured in some way, such as
a change in color of the solution.
8. ELISA tests are generally highly sensitive and specific and compare favorably with
radioimmune assay (RIA) tests. They have the added advantages of not needing
radioisotopes or a radiation-counting apparatus
ELISA Procedure
Polyoxyethylene sorbitol monolaurate (Tween 20) was purchased from BioRad Laboratories
(Hercules, CA). Skim milk powder was obtained from Difco/Becton Dickinson (Atlanta, GA).
Horseradish peroxidase (HRPO)-conjugated mouse anti-human IgG (affinity purified, -chain
specific monoclonal clone HP6043) was obtained from Hybridoma Reagent Laboratories
(Baldwin, MD). Peroxidase substrate 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS),
hydrogen peroxide (H2O2), and peroxidase stop solution were obtained from Kirkegaard & Perry
Laboratories (KPL, Gaithersburg, MD). All other laboratory reagents were obtained from Sigma
Chemical Co. (St. Louis, MO) unless otherwise specified. Sterile, Type I endotoxin-free water
was used for all ELISA procedures.
Immulon II-HB flat-bottom 96-well microtiter plates (Thermo Labsystems, Franklin, MA), were
coated for 16 hrs at +4°C with 100 µL/well of rPA at a concentration of 2.0 µg/mL in 0.01 M
phosphate-buffered saline (PBS), pH 7.4 (Life Technologies, Gaithersburg, MD). Plates were
stored at +4°C without blocking and used within 7 days of preparation. Antigen-coated plates
were then washed three times (ELX405 microplate washer, BioTek Instruments Inc., Winooski,
VT) with PBS containing 0.1% Tween 20 and blotted dry by inversion on clean paper towels.
Control and serum antibodies were tested without a separate blocking step. Serum standards and
sera for testing were prepared at the appropriate dilutions in PBS containing 5% skim milk and
0.5% Tween 20, pH 7.4. The human standard reference serum and test sera were serially diluted
twofold in the plate in the same buffer solution. The minimum dilution of test serum was 1/50.
Three positive control sera from three separate donors and one negative control serum were each
used at single dilution factors selected to give a range of optical density (OD) values across the
standard reference curve. The final volume in all wells was 100 µL.
Test and standard sera were incubated in a humidified chamber (covered tray) for 60 min at
37°C, and the plates were then washed three times with PBS containing 0.1% Tween 20. Bound
anti-PA IgG was then detected by using HRPO-conjugated mouse anti-human IgG Fc PAN
monoclonal HP6043 diluted in PBS containing 5% skim milk and 0.5% Tween 20 (100
µL/well), and plates were incubated in a humidified chamber (covered tray) for 60 min at 37°C.
Plates were again washed three times with PBS containing 0.1% Tween 20, and bound conjugate
was detected colorimetrically by using ABTS/H2O2 substrate (100 µL/well). Color development
was over 30 min (±5 min) and was stopped by addition of 100 µL of Peroxidase Stop Solution
(KPL) to all wells of the test plates. OD values were read within 30 min of addition of the stop
solution with a MRX Revelation microtiter plate reader (Thermo Labsystems, Franklin, MA) at a
wavelength of 410 nm with a 610-nm reference filter. Data were analyzed by using a fourparameter (4-PL) logistic-log curve fitting model with ELISA for Windows software. A
calibration factor for the standard reference serum was used to determine the concentration of
anti-PA IgG in micrograms per milliliter of serum (µg/mL).
Radioimmunoassay
Radioimmunoassay (RIA) is a very sensitive technique used to measure concentrations of
antigens (for example, hormone levels in the blood) without the need to use a bioassay.
Although the RIA technique is extremely sensitive and extremely specific, it requires specialized
equipment and is costly. It also requires special precautions, since radioactive substances are
used. Therefore, today it has been largely supplanted by the ELISA method, where the antigenantibody reaction is measured using colorimetric signals instead of a radioactive signal.
The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect
the causative allergen for an allergy.

Method
To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently
by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled
antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two
chemically bind to one another. Then, a sample of serum from a patient containing an unknown
quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the
serum to compete with the radiolabeled antigen ("hot") for antibody binding sites.
As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing
the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free
radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the
radioactivity of the free antigen remaining in the supernatant is measured. Using known
standards, a binding curve can then be generated which allows the amount of antigen in the
patient's serum to be derived.
History
It was developed by Rosalyn Yalow and Solomon Aaron Berson in the 1950s. In 1977, Rosalyn
Sussman Yalow received the Nobel Prize in Medicine for the development of the RIA for
insulin: the precise measurement of minute amounts of such a hormone was considered a
breakthrough in endocrinology.
With this technique, separating bound from unbound antigen is crucial. Initially, the method of
separation employed was the use of a second "anti-antibody" directed against the first for
precipitation and centrifugation. The use of charcoal suspension for precipitation was extended
but replaced later by Drs. Werner and Acebedo at Columbia University for RIA of T3 and T4.
An ultramicro RIA for human TSH was published in BBRC (1975) by
Radioimmunoassay
The technique of
radioimmunoassay has
revolutionized research and
clinical practice in many areas,
e.g.,



blood banking
diagnosis of allergies
endocrinology
The technique was introduced in
1960 by Berson and Yalow as
an assay for the concentration of
insulin in plasma. It represented
the first time that hormone
levels in the blood could be
detected by an in vitro assay.
The Technique






A mixture is prepared of
o radioactive
antigen
 Because
of the
ease with
which
iodine
atoms can
be
introduced into tyrosine residues in a protein, the radioactive isotopes 125I
or 131I are often used.
o antibodies against that antigen.
Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These
compete for the binding sites of the antibodies.
At increasing concentrations of unlabeled antigen, an increasing amount of radioactive
antigen is displaced from the antibody molecules.
The antibody-bound antigen is separated from the free antigen in the supernatant fluid,
and
the radioactivity of each is measured.
From these data, a standard binding curve, like this one shown in red, can be drawn.
Radioimmunoassay (RIA)
RIA involves the separation of the drug using the specificity of antibody - antigen binding and
quantitation using radioactivity.
Components of RIA Assay Kit



Drug
Antibody
Labelled Drug
General Procedure for Performing a RIA Analysis





Mix sample containing drug with fixed quantity of labelled drug and antibody
Allow to equilibrate - incubate
Separate drug bound to antibody from unbound drug
o Charcoal adsorption of antibody (and bound drug)
o Antibody - antibody binding precipitates bound drug
o Antibody bonded to container
Measure radioactivity associated with bound labelled drug
o low drug concentration means more bound radioactivity and higher measurement
o high drug concentration means less bound radioactivity and lower measurement
Determine standard curve
o Non-linear plot of radioactivity versus concentration
o Logit-log concentration plot is linear
A Blank and Three Standard Samples
RIA before and after Incubation - Blank and Three Standard Samples
Table 3.7.1 Bound and Free Drug Concentrations
Tot Bo Fre
al un e
[Dr d [Dr
ug] [Dr ug]
ug]
0
6 0
3
4 2
6
3 3
12 2 4
FLOW CYTOMETRY
Flow cytometry (abbreviated: FCM) is a technique for counting and
examining microscopic particles, such as cells and chromosomes, by
suspending them in a stream of fluid and passing them by an electronic
detection apparatus. The technology has applications in a number of fields,
including molecular biolog y, pathology, immunology, plant biology and
marine biology. It is used in diagnose blood cancer and application in
research & clinical practice. Light of single wave length is directed onto
hydro dynamically focused stream of fluid. These fluorescent light rays
scattered because of the presence of suspended particle present in the fluid.
These fluorescent particles are picked by a no. of detector, which is
connected to the instrument. The instrument used for flow cytometry is
flow cytometre.
Both ELISA & flow cytometry have application in immunology, clinical,
diagnostic fields.
ELISA ( Enzyme-Linked Immunosorbent Assay)
A 96-well microtiter plate being used for ELISA.
Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme
immunoassay (EIA), is a biochemical technique used mainly in
immunology to detect the presence of an antibody or an antigen in a
sample. The ELISA has been used as a diagnostic tool in medicine and
plant pathology, as well as a quality control check in various industries. In
simple terms, in ELISA, an unknown amount of antigen is affixed to a
surface, and then a specific antibody is applied over the surface so that it
can bind to the antigen. This antibody is linked to an enzyme, and in the
final step a substance is added that the enzyme can convert to some
detectable signal. Thus in the case of fluorescence ELISA, when light of
the appropriate wavelength is shone upon the sample, any antigen/antibody
complexes will fluoresce so that the amount of antigen in the sample can
be inferred through the magnitude of the fluorescence.
Performing an ELISA involves at least one antibody with specificity for a
particular antigen. The sample with an un known amount of antigen is
immobilized on a solid support (usually a polystyrene microtiter plate)
either non-specifically (via adsorption to the surface) or specifically (via
capture by another antibody specific to the same antigen, in a "sandwich"
ELISA). After the antigen is immobilized the detection antibody is added,
forming a complex with the antigen. The de tection antibody can be
covalently linked to an enzyme, or can itself be detected by a secondary
antibody which is linked to an enzyme through bioconjugation. Between
each step the plate is typically washed with a mild detergent solution to
remove any proteins or antibodies that are not specifically bound. After the
final wash step the plate is developed by adding an enzymatic substrate to
produce a visible signal, which indicates the quantity of antigen in the
sample.
Traditional ELISA typically involves chromogenic reporters and substrates
which produce some kind of observable color change to indicate the
presence of antigen or analyte. Newer ELISA -like techniques utilize
fluorogenic, electrochemiluminescent, and real-time PCR reporters to
create quantifiable signals. These new reporters can have various
advantages including higher sensitivities and multiplexing [ 1 ] [ 2 ] .
Technically, newer assays of this type are not strictly ELISAs as they are
not "enzyme-linked" but are instead linked to some non -enzymatic
reporter. However, given that the general principles in these assays are
largely similar, they are often grouped in the same category as ELISAs .
Applications
ELISA results using S-OIV A neuraminidase antibody at 1 μg/ml to probe
the immunogenic and the corresponding seasonal influenza A
neuraminidase peptides at 50, 10, 2 and 0 ng/ml.
Because the ELISA can be performed to evaluate either the presence of
antigen or the presence of antibody in a sample, it is a useful tool for
determining serum antibody concentrations (such as with the HIV test [ 3 ] or
West Nile Virus). It has also found applications in the food industry in
detecting potential food allergens such as milk, peanuts, walnuts, almonds,
and eggs. [ 4 ] ELISA can also be used in toxicology as a rapid presumptive
screen for certain classes of drugs.
The ELISA was the first screening test widely used for HIV because of its
high sensitivity. In an ELISA, a person' s serum is diluted 400-fold and
applied to a plate to which HIV antigens are attached. If antibodies to HIV
are present in the serum, they may bind to these HIV antigens. The plate is
then washed to remove all other components of the serum. A specially
prepared "secondary antibody" — an antibody that binds to other
antibodies — is then applied to the plate, followed by another wash. This
secondary antibody is chemically linked in advance to an enzyme. Thus,
the plate will contain enzyme in proportion to the amount of secondary
antibody bound to the plate. A substrate for the enzyme is applied, and
catalysis by the enzyme leads to a change in color or fluorescence. ELISA
results are reported as a number; the most controversial aspect of this test
is determining the "cut-off" point between a positive and negative result.
A cut-off point may be determined by comparing it with a known standard.
If an ELISA test is used for drug screening at workplace, a cut -off
concentration, 50 ng/mL, for example, is established , and a sample will be
prepared which contains the standard concentration of analyte. Unknowns
that generate a signal that is stronger than the known sample are
"positive". Those that generate weaker signal are "negative."
ELISA can also be used to determi ne the level of antibodies in faecal
content...specifically the direct method
History
Before the development of the EIA/ELISA, the only option for conducting
an immunoassay was radioimmunoassay, a technique using radioactivelylabeled antigens or antibodies. In radioimmunoassa y, the radioactivity
provides the signal which indicates whether a specific antigen or antibody
is present in the sample. Radioimmunoassay was first described in a paper
by Rosalyn Sussman Yalow and Solomon Berson published in 1960 [ 5 ] .
Because radioactivity poses a potential health threat, a safer alternative
was sought. A suitable alternative to radioimmunoassay would substitute a
non-radioactive signal in place of the radioactive signal. When enzymes
(such as peroxidase) react with appropriate substrates (such as ABTS or
3,3’,5,5’-Tetramethylbenzidine ), this causes a change in color, which is
used as a signal. However, the signal has to be associated with the
presence of antibody or antigen, which is why the enzyme has to be linked
to an appropriate anti body. This linking process was independently
developed by Stratis Avrameas and G.B. Pierce [ 6 ] . Since it is necessary to
remove any unbound antibody or antigen by washing, the a ntibody or
antigen has to be fixed to the surface of the container, i.e. the
immunosorbent has to be prepared. A technique to accomplish this was
published by Wide and Jerker Porath in 1966. [ 7 ]
In 1971, Peter Perlmann and Eva Engvall at Stockholm University in
Sweden, and Anton Schuurs and Bauke van Weemen in The Netherlands,
independently published papers which synthesized this knowledge into
methods to perform EIA/ELISA.
Types
 "Indirect" ELISA
The steps of "indirect" ELISA foll ows the mechanism below:






A buffered solution of the protein antigen to be tested for is added
to each well of a microtiter plate, where it is given time to adhere to
the plastic through charge interactions.
A solution of non-reacting protein, such as bovine serum albumin, or
casein is added to block any plastic surface in the well that remains
uncoated by the protein antigen.
Then the serum is added, which contains a mixture of the serum
donor's antibodies, of unknown concentration, some of which may
bind specifically to the test antigen that is coating the well.
Afterwards, a secondary antibody is added, which will bind any
antibody produced by a member of the donor's species (for example,
an antibody produced in a mouse that will bind any rabbit antibod y).
This secondary antibody often has an enzyme attached to it, which
has a negligible effect on the binding properties of the antibody.
A substrate for this enzyme is then added. Often, this substrate
changes color upon reaction with the enzyme. The colo r change
shows that secondary antibody has bound to primary antibody,
which strongly implies that the donor has had an immune reaction
to the test antigen. This can be helpful in a clinical setting, and in
R&D.
The higher the concentration of the primary antibody that was
present in the serum, the stronger the color change. Often a
spectrometer is used to give quantitative values for color strength.
The enzyme acts as an amplifier; even if only few enzyme -linked
antibodies remain bound, the enzyme molecules will produce many signal
molecules. Within common -sense limitations the enzyme can go on
producing color indefinitely, but the more primary antibody is present in
the donor serum, the more secondary antibody + enzyme will bind, and the
faster color will develop. A major disadvantage of the indirect ELISA is
that the method of antigen immobilization is non -specific; when serum is
used as the source of test antigen, all proteins in the sample may stick to
the microtiter plate well, so small concentrations of analyte in serum must
compete with other serum proteins when binding to the well surface. The
sandwich or direct ELISA provides a solution to this problem, by using a
"capture" antibody specific for the test a ntigen to pull it out of the serum's
molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative
results provide a simple positive or negative result (yes or no) for a
sample. The cutoff between positive and negative is determi ned by the
analyst and may be statistical. Two or three times the standard deviation
(error inherent in a test) is often used to distinguish positive from negative
samples. In quantitative ELISA, the optical density (OD) of the sample is
compared to a standard curve, which is typically a serial dilution of a
known-concentration solution of the target molecule. For example if your
test sample returns an OD of 1.0, the point on your standard curve that
gave OD = 1.0 must be of the same analyte concentration a s your sample.
 Sandwich ELISA
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2)
sample is added, and any antigen present binds to capture antibody; (3)
enzyme linked capture antibody used as detecting antibody is added, and
binds to antigen; (4) substrate is added, and is converted by enzyme to
detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is used
to detect sample antigen. The steps are as follows:
1. Prepare a surface to which a known quantity of cap ture antibody is
bound.
2. Block any non specific binding sites on the surface.
3. Apply the antigen -containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply enzyme linked primary antibodies as detection antibodies
which also bind specifically to the antigen.
6. Wash the plate, so that the unbound antibody -enzyme conjugates
are removed.
7. Apply a chemical which is converted by the enzyme into a color or
fluorescent or electrochemical signal.
8. Measure the absorbency or fluorescenc e or electrochemical signal
(e.g., current) of the plate wells to determine the presence and
quantity of antigen.
The image to the right includes the use of a secondary antibody conjugated
to an enzyme, though technically this is not necessary if the prim ary
antibody is conjugated to an enzyme. However, use of a secondary antibody conjugate avoids the expensive process of creating enzyme -linked
antibodies for every antigen one might want to detect. By using an enzyme linked antibody that binds the Fc regio n of other antibodies, this same
enzyme-linked antibody can be used in a variety of situations. Without the
first layer of "capture" antibody, any proteins in the sample (including
serum proteins) may competitively adsorb to the plate surface, lowering
the quantity of antigen immobilized.Use of the purified specific antibody
to attach the antigen to the plastic eliminates a need to purif y the antigen
from complicated mixtures before the measurement, simplif ying the assay,
and increasing the specificity and the sensitivity of the assay.
 Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this
ELISA are somewhat different than the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen.
(Sample)
2. These bound antibody/antigen complexes are then added to an
antigen coated well.
3. The plate is washed, so that unbound antibody is removed. (The
more antigen in the sample, the less antibody will be able to bind to
the antigen in the well, hence "competiti on.")
4. The secondary antibody , specific to the primary antibody is added.
This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.
For competitive ELISA, the higher the sample antigen concentration, the
weaker the eventual signal. The major advantage of a competitive ELISA is
the ability to use crude or impure samples and still selectively bind any
antigen that may be present.
(Note that some competitive ELISA kits include enzym e-linked antigen
rather than enzyme -linked antibody. The labeled antigen competes for
primary antibody binding sites with your sample antigen (unlabeled). The
more antigen in the sample, the less labeled antigen is retained in the well
and the weaker the signal).
Commonly the antigen is not first positioned in the well.
 Reverse ELISA
A new technique uses a solid phase made up of an immunosorbent
polystyrene rod with 4 -12 protruding ogives. The entire device is immersed
in a test tube containing the collecte d sample and the following steps
(washing, incubation in conjugate and incubation in chromogenous) are
carried out by dipping the ogives in microwells of standard microplates
pre-filled with reagents.
The advantages of this technique are as follows:
1. The ogives can each be sensitized to a different reagent, allowing
the simultaneous detection of different antibodies and different
antigens for multi-target assays;
2. The sample volume can be increased to improve the test sensitivity
in clinical (saliva, urine), food (bulk milk, pooled eggs) and
environmental (water) samples;
3. One ogive is left unsensitized to measure the non -specific reactions
of the sample;
4. The use of laboratory supplies for dispensing sample aliquots,
washing solution and reagents in microwel ls is not required,
facilitating ready-to-use lab-kits and on-site kits.
INTRODUCTION
The complement system is a biochemical cascade that helps, or “complements”, the ability of
antibodies to clear pathogens from an organism. It is part of the immune system called the innate
immune system that is not adaptable and does not change over the course of an individual's
lifetime. However, it can be recruited and brought into action by the adaptive immune system.
The complement system consists of a number of small proteins found in the blood, generally
synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When
stimulated by one of several triggers, proteases in the system cleave specific proteins to release
cytokines and initiate an amplifying cascade of further cleavages. The end-result of this
activation cascade is massive amplification of the response and activation of the cell-killing
membrane attack complex. Over 25 proteins and protein fragments make up the complement
system, including serum proteins, serosal proteins, and cell membrane receptors. These proteins
are synthesized mainly in the liver, and they account for about 5% of the globulin fraction of
blood serum.
Three biochemical pathways activate the complement system: the classical complement
pathway, the alternative complement pathway, and the mannose-binding lectin pathway.
History
In the late 19th century, Hans Ernst August Buchner found that blood serum contained a "factor"
or "principle" capable of killing bacteria. In 1896, Jules Bordet, a young Belgian scientist in
Paris at the Pasteur Institute, demonstrated that this principle had two components: one that
maintained this effect after being heated, and one that lost this effect after being heated. The
heat-stable component was responsible for the immunity against specific microorganisms,
whereas the heat-sensitive (heat-labile) component was responsible for the non-specific
antimicrobial activity conferred by all normal serum. This heat-labile component is what we now
call "complement."
The term "complement" was introduced by Paul Ehrlich in the late 1890s, as part of his larger
theory of the immune system. According to this theory, the immune system consists of cells that
have specific receptors on their surface to recognize antigens. Upon immunization with an
antigen, more of these receptors are formed, and they are then shed from the cells to circulate in
the blood. These receptors, which we now call "antibodies," were called by Ehrlich
"amboceptors" to emphasize their bifunctional binding capacity: They recognize and bind to a
specific antigen, but they also recognize and bind to the heat-labile antimicrobial component of
fresh serum. Ehrlich, therefore, named this heat-labile component "complement," because it is
something in the blood that "complements" the cells of the immune system. In the early half of
the 1930s, a team led by the renowned Irish researcher, Jackie Stanley, stumbled upon the allimportant opsonization-mediated effect of C3b. Building off Ehrlich's work, Stanley's team
proved the role of complement in both the innate as well as the cell-mediated immune response.
Ehrlich believed that each antigen-specific amboceptor has its own specific complement,
whereas Bordet believed that there is only one type of complement. In the early 20th century,
this controversy was resolved when it became understood that complement can act in
combination with specific antibodies, or on its own in a non-specific way.
Functions of the Complement
The following are the basic functions of the complement
1. Opsonization- enhancing phagocytosis of antigens
2. Chemotaxis- attracting macrophages and neutrophils
3. Lysis- rupturing membranes of foreign cells
4. Clumping of antigen bearing agents
5. Altering the molecular structure of viruses
Lectin pathway (MBL - MASP)
The lectin pathway is homologous to the classical pathway, but with the opsonin, mannosebinding lectin (MBL), and ficolins, instead of C1q. This pathway is activated by binding
mannose-binding lectin to mannose residues on the pathogen surface, which activates the MBLassociated serine proteases, MASP-1, and MASP-2 (very similar to C1r and C1s,
respectively),which can then split C4 into C4a and C4b and C2 into C2a and C2b. C4b and C2a
then bind together to form the C3-convertase, as in the classical pathway. Ficolins are
homologous to MBL and function via MASP in a similar way. In invertebrates without an
adaptive immune system, ficolins are expanded and their binding specificities diversified to
compensate for the lack of pathogen-specific recognition molecules.
Activation of complements by antigen-associated antibody
In the classical pathway, C1 binds with its C1q subunits to Fc fragments (made of CH2 region)
of IgG or IgM which has formed a complex with antigens. C4b and C3b are also able to bind to
antigen-associated IgG or IgM, to its Fc portion .
Such immunoglobulin-mediated binding of the complement may be interpreted as that the
complement uses the ability of the immunoglobulin to detect and bind to non-self antigens as its
guiding stick. The complement itself is able to bind non-self pathogens after detecting their
pathogen-associated molecular patterns (PAMPs). however, utilizing specificity of antibody,
complements are able to detect non-self enemies much more specifically. There must be
mechanisms that complements bind to Ig but would not focus its function to Ig but to the antigen.
Figure 2 shows the classical and the alternative pathways with the late steps of complement
activation schematically. Some components have a variety of binding sites. In the classical
pathway C4 binds to Ig-associated C1q and C1r2s2 enzyme cleave C4 to C4b and 4a. C4b binds
to C1q, antigen-associated Ig (specifically to its Fc portion), and even to the microbe surface.
C3b binds to antigen-associated Ig and to the microbe surface. Ability of C3b to bind to antigenassociated Ig would work effectively against antigen-antibody immune complexes to make them
soluble. In the figure, C2b refers to the larger of the C2 fragments.
Regulation of the complement system
The complement system has the potential to be extremely damaging to host tissues, meaning its
activation must be tightly regulated. The complement system is regulated by complement control
proteins, which are present at a higher concentration in the blood plasma than the complement
proteins themselves. Some complement control proteins are present on the membranes of selfcells preventing them from being targeted by complement. One example is CD59, also known as
protectin which inhibits C9 polymerisation during the formation of the membrane attack
complex.
Role in disease
It is thought that the complement system might play a role in many diseases with an immune
component,
such
as
Barraquer-Simons
Syndrome,
asthma,
lupus
erythematosus,
glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis,
inflammatory bowel disease, and ischemia-reperfusion injuries. The complement system is also
becoming increasingly implicated in diseases of the central nervous system such as Alzheimer's
disease and other neurodegenerative conditions.
Deficiencies of the terminal pathway predispose to both autoimmune disease and infections
(particularly Neisseria meningitis, due to the role that the C56789 complex plays in attacking
Gram-negative bacteria).
Mutations in the complement regulators factor H and membrane cofactor protein have been
associated with atypical haemolytic uraemic syndrome. Moreover, a common single nucleotide
polymorphism in factor H (Y402H) has been associated with the common eye disease agerelated macular degeneration. Both of these disorders are currently thought to be due to aberrant
complement activation on the surface of host cells.
Mutations in the C1 inhibitor gene can cause hereditary angioedema, an autoimmune condition
resulting from reduced regulation of the complement pathway.
Mutations in the MAC components of complement--esp. C8--are often implicated in recurrent
Nessaria infection.
.Th
e
Cla
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Co
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hw
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nat
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h
a
s
c
o
m
p
l
e
m
e
n
t
p
r
o
t
e
i
n
s
,
a
c
u
t
e
p
h
a
s
e
p
r
o
t
e
i
n
s
,
a
n
d
c
y
t
o
k
i
n
e
s
.
Ex
am
ple
s of
inn
ate
im
mu
nit
y
incl
ude
ana
to
mic
al
bar
rier
s,
me
cha
nic
al
re
mo
val,
bac
teri
al
ant
ago
nis
m,
patt
ern
rec
ogn
itio
n
rec
ept
ors,
anti
gen
non
spe
cifi
c
def
ens
e
che
mic
als,
the
co
mp
lem
ent
pat
hw
ays
,
pha
goc
yto
sis,
infl
am
mat
ion
,
and
fev
er.
In
the
nex
t
sev
eral
sec
tio
ns
we
wil
l
loo
k at
eac
h
of
the
se
in
gre
ater
det
ail.
We
wil
l
no
w
tak
ea
clo
ser
loo
k at
the
3
pat
hw
ays
of
the
co
mp
lem
ent
sys
tem
.
Th
e
co
mp
lem
ent
sys
tem
ref
ers
to a
seri
es
of
pro
tein
s
circ
ulat
ing
in
the
blo
od
and
bat
hin
g
the
flui
ds
sur
rou
ndi
ng
tiss
ues
.
Th
e
pro
tein
s
circ
ulat
e in
an
ina
ctiv
e
for
m,
but
in
res
pon
se
to
the
rec
ogn
itio
n
of
mo
lec
ula
r
co
mp
one
nts
of
mic
roo
rga
nis
m,
the
y
bec
om
e
seq
uen
tial
ly
acti
ved
,
wo
rki
ng
in a
cas
cad
e
wh
ere
in
the
bin
din
g
of
one
pro
tein
pro
mo
tes
the
bin
din
g
of
the
nex
t
pro
tein
in
the
cas
cad
e.
Th
ere
are
3
co
mp
lem
ent
pat
hw
ays
that
ma
ke
up
the
co
mp
lem
ent
sys
tem
:
the
cla
ssic
al
co
mp
le
me
nt
pat
hw
ay,
the
lect
in
pat
hw
ay,
an
d
the
alt
ern
ati
ve
co
mp
le
me
nt
pat
hw
ay.
Th
e
pat
hw
ays
diff
er
in
the
ma
nn
er
in
wh
ich
the
y
are
act
iva
ted
an
d
ulti
ma
tel
y
pro
du
ce
a
key
enz
ym
e
call
ed
C3
con
ver
tas
e:
We
wil
l
no
w
tak
ea
clo
ser
loo
k at
the
cla
ssic
al
co
mp
lem
ent
pat
hw
ay.
Th
e
Cla
ssic
al
Co
mp
le
me
nt
Pat
hw
ay
Alt
hou
gh
at
lea
st
21
diff
ere
nt
ser
um
pro
tein
s
hav
e
thu
s
far
bee
n
ide
ntif
ied
as
par
t of
the
cla
ssic
al
co
mp
lem
ent
pat
hw
ay,
one
can
loo
k at
it
as
a
pat
hw
ay
that
is
pri
ma
rily
act
iva
ted
by
eit
her
Ig
G
or
Ig
M
bin
din
g
to
an
ant
ige
n
and
inv
olv
es
11
ma
jor
ser
um
pro
tei
n
co
mp
one
nts
.
Ig
G
and
Ig
M
are
cla
sse
s of
anti
bod
y
mo
lec
ule
s
that
wil
l be
dis
cus
sed
in
gre
ater
det
ail
in
Uni
t 3,
but
as
me
nti
one
d
pre
vio
usl
y,
one
of
the
maj
or
def
ens
es
aga
inst
mic
rob
es
is
the
im
mu
ne
def
ens
es'
pro
du
cti
on
of
ant
ibo
dy
m
ole
cul
es
aga
ins
t
tha
t
mi
cro
be.
Th
e
"tip
s"
of
the
anti
bod
y
(th
e
Fa
b
por
tio
n
hav
e
sha
pes
that
are
co
mp
lem
ent
ary
to
epit
ope
s por
tio
ns
of
mic
rob
ial
pro
tein
s
and
gly
cop
rot
ein
s
fou
nd
on
the
sur
fac
e
of
the
mic
rob
e.
Th
e
Fc
por
tio
n
of
Ig
G
and
Ig
M
can
acti
vat
e
the
cla
ssic
al
co
mp
lem
ent
pat
hw
ay
by
ena
bli
ng
the
firs
t
enz
ym
e in
the
pat
hw
ay,
C1,
to
ass
em
ble.
Th
e
rea
ctio
ns
are
as
foll
ow
s:
a.
Ty
pic
ally
to
act
iva
te
the
cla
ssic
al
co
mp
lem
ent
pat
hw
ay,
Ig
G
or
Ig
M
is
ma
de
in
res
pon
se
to
an
anti
gen
.
Th
e
Fa
b
por
tio
n
of
Ig
G
(2
mo
lec
ule
s)
or
Ig
M
(1
mo
lec
ule
)
rea
cts
wit
h
epi
top
es
of
that
anti
gen
.A
pro
tein
call
ed
C1
q
firs
t
bin
ds
to
the
Fc
po
rtio
n
of
anti
gen
bou
nd
Ig
G
or
Ig
M
afte
r
whi
ch
C1r
and
C1
s
atta
ch
to
for
m
C1,
the
firs
t
enz
ym
e
of
the
pat
hw
ay
b.
Th
e
acti
vat
ed
C1
no
w
enz
ym
atic
ally
cle
ave
s
C4
int
o
C4
a
and
C4
b.
Th
e
C4
b
the
n
bin
ds
to
adj
ace
nt
pro
tein
s
and
car
boh
ydr
ate
s
on
the
sur
fac
e
of
the
anti
gen
and
the
n
bin
ds
C2.
Th
e
acti
vat
ed
C1
cle
ave
s
C2
int
o
C2
a
and
C2
b
for
mi
ng
C4
b2
a,
the
C3
con
ver
tas
e.
No
w
the
cla
ssic
al
co
mp
lem
ent
pat
hw
ay
is
acti
vat
ed.
C3
con
ver
tas
e
can
no
w
cle
ave
hu
nd
red
s of
mo
lec
ule
s of
C3
int
o
C3
a
an
d
C3
b.
c.
So
me
mo
lec
ule
s of
C3
b
bin
d
to
C4
b2a
,
the
C3
con
ver
tas
e,
to
for
m
C4
b2
a3
b,
a
C5
con
ver
tas
e
that
cle
ave
s
C5
int
o
C5
a
and
C5
b.
d.
C5
b
bin
ds
to
the
sur
fac
e
of
the
tar
get
cell
and
sub
seq
uen
tly
bin
ds
C6,
C7,
C8,
and
a
nu
mb
er
of
mo
no
me
rs
of
C9
to
for
m
C5
b6
789
n,
the
Me
mb
ran
e
Att
ack
Co
mp
lex
(M
AC
).
As
me
nti
one
d
abo
ve,
co
mp
one
nts
of
the
co
mp
lem
ent
pat
hw
ays
car
ry
out
6
be
nef
icia
l
inn
ate
def
ens
e
fun
cti
ons
.
Th
ese
incl
ude
:
a.
tri
gge
rin
g
infl
am
ma
tio
n

C
5
a
i
s
t
h
e
m
o
s
t
p
o
t
e
n
t
c
o
m
p
l
e
m
e
n
t
p
r
o
t
e
i
n
t
r
i
g
g
e
r
i
n
g
i
n
f
l
a
m
m
a
t
i
o
n
.
I
t
c
a
u
s
e
s
m
a
s
t
c
e
l
l
s
t
o
r
e
l
e
a
s
e
v
a
s
o
d
i
l
a
t
o
r
s
s
u
c
h
a
s
h
i
s
t
a
m
i
n
e
s
o
t
h
a
t
b
l
o
o
d
v
e
s
s
e
l
s
b
e
c
o
m
e
m
o
r
e
p
e
r
m
e
a
b
l
e
;
i
t
i
n
c
r
e
a
s
e
s
t
h
e
e
x
p
r
e
s
s
i
o
n
o
f
a
d
h
e
s
i
o
n
m
o
l
e
c
u
l
e
s
o
n
l
e
u
k
o
c
y
t
e
s
a
n
d
t
h
e
v
a
s
c
u
l
a
r
e
n
d
o
t
h
e
l
i
u
m
s
o
t
h
a
t
l
e
u
k
o
c
y
t
e
s
c
a
n
s
q
u
e
e
z
e
o
u
t
o
f
t
h
e
b
l
o
o
d
v
e
s
s
e
l
s
a
n
d
e
n
t
e
r
t
h
e
t
i
s
s
u
e
(
d
i
a
p
e
d
e
s
i
s
)
;
i
t
c
a
u
s
e
s
n
e
u
t
r
o
p
h
i
l
s
t
o
r
e
l
e
a
s
e
t
o
x
i
c
o
x
y
g
e
n
r
a
d
i
c
a
l
s
f
o
r
e
x
t
r
a
c
e
l
l
u
l
a
r
k
i
l
l
i
n
g
;
a
n
d
i
t
i
n
d
u
c
e
s
f
e
v
e
r
.
T
o
a
l
e
s
s
e
r
e
x
t
e
n
t
C
3
a
a
n
d
C
4
a
a
l
s
o
p
r
o
m
o
t
e
i
n
f
l
a
m
m
a
t
i
o
n
.
A
s
w
e
w
i
l
l
s
e
e
l
a
t
e
r
i
n
t
h
i
s
u
n
i
t
,
i
n
f
l
a
m
m
a
t
i
o
n
i
s
a
p
r
o
c
e
s
s
i
n
w
h
i
c
h
b
l
o
o
d
v
e
s
s
e
l
s
d
i
l
a
t
e
a
n
d
b
e
c
o
m
e
m
o
r
e
p
e
r
m
e
a
b
l
e
,
t
h
u
s
e
n
a
b
l
i
n
g
b
o
d
y
d
e
f
e
n
s
e
c
e
l
l
s
a
n
d
d
e
f
e
n
s
e
c
h
e
m
i
c
a
l
s
t
o
l
e
a
v
e
t
h
e
b
l
o
o
d
a
n
d
e
n
t
e
r
t
h
e
t
i
s
s
u
e
s
.
b.
che
mo
tac
tic
all
y
att
rac
tin
g
ph
ago
cyt
es
to
the
inf
ecti
on
site

C
5
a
a
l
s
o
f
u
n
c
t
i
o
n
s
a
s
a
c
h
e
m
o
a
t
t
r
a
c
t
a
n
t
f
o
r
p
h
a
g
o
c
y
t
e
s
.
P
h
a
g
o
c
y
t
e
s
w
i
l
l
m
o
v
e
t
o
w
a
r
d
s
i
n
c
r
e
a
s
i
n
g
c
o
n
c
e
n
t
r
a
t
i
o
n
s
o
f
C
5
a
a
n
d
s
u
b
s
e
q
u
e
n
t
l
y
a
t
t
a
c
h
,
v
i
a
t
h
e
i
r
C
R
1
r
e
c
e
p
t
o
r
s
t
o
t
h
e
C
3
b
m
o
l
e
c
u
l
e
s
a
t
t
a
c
h
e
d
t
o
t
h
e
a
n
t
i
g
e
n
.
T
h
i
s
w
i
l
l
b
e
d
i
s
c
u
s
s
e
d
i
n
g
r
e
a
t
e
r
d
e
t
a
i
l
l
a
t
e
r
i
n
t
h
i
s
u
n
i
t
u
n
d
e
r
p
h
a
g
o
c
y
t
o
s
i
s
.
c.
pro
mo
tin
g
the
att
ach
me
nt
of
ant
ige
ns
to
ph
ago
cyt
es
(en
ha
nce
d
att
ach
me
nt
or
ops
oni
zat
ion
)

C
3
b
a
n
d
t
o
a
l
e
s
s
e
r
e
x
t
e
n
t
,
C
4
b
c
a
n
f
u
n
c
t
i
o
n
a
s
o
p
s
o
n
i
n
s
,
t
h
a
t
i
s
,
t
h
e
y
c
a
n
a
t
t
a
c
h
a
n
t
i
g
e
n
s
t
o
p
h
a
g
o
c
y
t
e
s
.
O
n
e
p
o
r
t
i
o
n
o
f
t
h
e
C
3
b
b
i
n
d
s
t
o
p
r
o
t
e
i
n
s
a
n
d
p
o
l
y
s
a
c
c
h
a
r
i
d
e
s
o
n
m
i
c
r
o
b
i
a
l
s
u
r
f
a
c
e
s
;
a
n
o
t
h
e
r
p
o
r
t
i
o
n
a
t
t
a
c
h
e
s
t
o
C
R
1
r
e
c
e
p
t
o
r
s
o
n
p
h
a
g
o
c
y
t
e
s
,
B
l
y
m
p
h
o
c
y
t
e
s
,
a
n
d
d
e
n
d
r
i
t
i
c
c
e
l
l
s
f
o
r
e
n
h
a
n
c
e
d
p
h
a
g
o
c
y
t
o
s
i
s
.
.
A
c
t
u
a
l
l
y
,
C
3
b
m
o
l
e
c
u
l
e
c
a
n
b
i
n
d
t
o
p
r
e
t
t
y
m
u
c
h
a
n
y
p
r
o
t
e
i
n
o
r
p
o
l
y
s
a
c
c
h
a
r
i
d
e
.
H
u
m
a
n
c
e
l
l
s
,
h
o
w
e
v
e
r
,
p
r
o
d
u
c
e
F
a
c
t
o
r
H
t
h
a
t
b
i
n
d
s
t
o
C
3
b
a
n
d
a
l
l
o
w
s
F
a
c
t
o
r
I
t
o
i
n
a
c
t
i
v
a
t
e
t
h
e
C
3
b
.
O
n
t
h
e
o
t
h
e
r
h
a
n
d
,
s
u
b
s
t
a
n
c
e
s
s
u
c
h
a
s
L
P
S
o
n
b
a
c
t
e
r
i
a
l
c
e
l
l
s
f
a
c
i
l
i
t
a
t
e
t
h
e
b
i
n
d
i
n
g
o
f
F
a
c
t
o
r
B
t
o
C
3
b
a
n
d
t
h
i
s
p
r
o
t
e
c
t
s
t
h
e
C
3
b
f
r
o
m
i
n
a
c
t
i
v
a
t
i
o
n
b
y
F
a
c
t
o
r
I
.
I
n
t
h
i
s
w
a
y
,
C
3
b
d
o
e
s
n
o
t
i
n
t
e
r
a
c
t
w
i
t
h
o
u
r
o
w
n
c
e
l
l
s
b
u
t
i
s
a
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THE CLASSICAL PATHWAY:
The classical pathway of the complement system is a major effector of the humoral branch of the
human immune response. The trigger for the classical pathway is either IgG or IgM antibody
bound to antigen. Binding of antibody to antigen exposes a site on the antibody which is a
binding site for the first complement component, C1.
C1 is a macromolecular complex made up of the molecules C1q, C1r, and C1s. By itself,
C1q is a hexameric molecule comprised of "stalks" and "knobs". The protruding knobs of the
C1q molecule bind to exposed sites on antigen-bound antibody molecules. Pairs of C1r and C1s
molecules associate with one another to make a figure-8 shape. This figure-8 fits over the knobs
of the C1q molecule to make a complete, intact C1 molecule
When the intact macromolecular C1 binds to the exposed regions of at least two antigen-bound
antibodies, the C1r and C1s subunits are activated. Activated C1s is responsible for the cleavage
of the next two involved complement components, C4 and C2. (Remember, the numbers indicate
the order in which the components were discovered, not the order in which they activate in the
cascade.) C4 is cleaved into two fragments. The larger C4b molecule attaches to the target
membrane nearby while the small C4a molecule floats away. An exposed site on deposited C4b
is available to interact with the next complement component, C2. Again, activated C1s cleaves
the C2 molecule into two pieces. In this case, the fragment that remains is C2a. The smaller C2b
fragment floats away. What remains bound to the membrane is C4b2a, also known as the C3
convertase because its role is to convert the next complement component, C3, into its active
form.
The C3 convertase of the classical pathway splits C3 into two fragments, C3a and C3b. The
convertase has the ability to cleave multiple C3 molecules, forming hundreds of C3a and C3b
fragments. The C3a fragments float away and have a role in inducing an inflammatory response
(more on this later). Of critical importance, some of the C3b binds to the C4b2a to form
C4b2a3b - a.k.a. the C5 convertase. The C5 convertase, much like the C3 convertase before it,
catalyzes the cleavage of hundreds to thousands of C5 complement component into C5a and
C5b before it reverts to inactivity. C5a floats away and contributes to inflammation while the
C5b fragment binds to the antigen surface. This binding of C5b is the initial step in the formation
of the membrane attack complex (MAC).
THE MEMBRANE ATTACK COMPLEX:
The membrane-bound complement component
C5b is bound by the next complement molecule, C6. The resulting bimolecular complex next
binds C7 and then C8. The C5b-8 complex acts as a receptor for a variable number of
membrane-disrupting C9 molecules. The resultant C5b-8 complex and poly-C9 is given the
name "membrane attack complex." The MAC creates a transmembrane pore leading to the lysis
of the target cell.
CONCLUSION
The alternative complement pathway does not require antibody for its activation. Rather, a
variety of antigens such as bacterial lipopolysaccharide and components of viruses and other
pathogens have the ability to activate this pathway. It is thought to have evolved earlier than the
classical pathway, which depends on the relatively recently evolved antibody molecule. Like the
classical pathway, the alternative pathway produces both a C3 and a C5 convertase which leads
to the production of C5b and then to the formation of the MAC. The specific molecular players
and the path followed along the way are, however, different.
The complement component C3 is spontaneously cleaved at low levels. This means that there are
C3a and C3b fragments freely floating in serum. The C3b component can attach to a number of
different surfaces, both foreign and host cells alike. C3b is quickly inactivated by the sialic acid
found on most mammalian cell surfaces. Microbes, most of which lack sialic acid, are stable sites
for C3b deposition. (Sialic acid inactivation of C3b is one type of complement evasion strategy
utilized by certain pathogens, discussed below.) Membrane-bound C3b fragments are bound by
Factor B which is, in turn, cleaved by Factor D. The fragment Ba floats away, while Bb stays
associated with C3b. The resulting C3bBb molecule is the alternative pathway C3 convertase.
The C3 convertase of the alternative pathway is, however, not particularly stable. In order to
effectively split a relevant number of C3 molecules, the C3 convertase requires the stabilization
of another molecule, properdin (P), which binds to the C3bBb complex and extends the half-life
of its activity. Hybridoma technology
Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by
fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected
for its ability to grow in tissue culture and for an absence of antibody chain synthesis. The
antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal
antibodies (in contrast to polyclonal antibodies). The production of monoclonal antibodies was
invented by Cesar Milstein, Georges J. F. Köhler and Niels Kaj Jerne in 1975.
C
o
nt
en
ts
[hid
e]

1
M
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
2
A
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[edit] Method
(1) Immunisation of a mouse
(2) Isolation of B cells from the spleen
(3) Cultivation of myeloma cells
(4) Fusion of myeloma and B cells
(5) Separation of cell lines
(6) Screening of suitable cell lines
(7) in vitro (a) or in vivo (b) multiplication
(8) Harvesting
Laboratory animals (mammals, e.g. mice) are first exposed to an antigen to which we are
interested in isolating an antibody against. Usually this is done by a series of injections of the
antigen in question, over the course of several weeks. Once splenocytes are isolated from the
mammal's spleen, the B cells are fused with immortalized myeloma cells. The myeloma cells are
selected beforehand to ensure they are not secreting antibody themselves and that they lack the
hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene, making them sensitive to the
HAT medium (see below). The fusion is accomplished using polyethylene glycol or the Sendai
virus. It is performed by making the cell membranes more permeable.[clarification needed]
Fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine medium) for
roughly 10 to 14 days. Aminopterin blocks the pathway that allows for nucleotide synthesis.
Hence, unfused myeloma cells die, as they cannot produce nucleotides by the de novo or salvage
pathways because they lack HGPRT. Removal of the unfused myeloma cells is necessary
because they have the potential to outgrow other cells, especially weakly established
hybridomas. Unfused B cells die as they have a short life span. In this way, only the B cellmyeloma hybrids survive, since the HGPRT gene coming from the B cells is functional. These
cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells).
The incubated medium is then diluted into multi-well plates to such an extent that each well
contains only one cell. Since the antibodies in a well are produced by the same B cell, they will
be directed towards the same epitope, and are thus monoclonal antibodies.
The next stage is a rapid primary screening process, which identifies and selects only those
hybridomas that produce antibodies of appropriate specificity. The hybridoma culture
supernatant, secondary enzyme labeled conjugate, and chromogenic substrate, are then
incubated, and the formation of a colored product indicates a positive hybridoma. Alternatively,
immunocytochemical screening can also be used.[1]
The B cell that produces the desired antibodies can be cloned to produce many identical daughter
clones. Supplemental media containing interleukin-6 (such as briclone) are essential for this step.
Once a hybridoma colony is established, it will continually grow in culture medium like RPMI1640 (with antibiotics and fetal bovine serum) and produce antibodies.[1]
Multiwell plates are used initially to grow the hybridomas, and after selection, are changed to
larger tissue culture flasks. This maintains the well-being of the hybridomas and provides enough
cells for cryopreservation and supernatant for subsequent investigations. The culture supernatant
can yield 1 to 60 µg/ml of monoclonal antibody, which is maintained at 20 °C or lower until
required.[1]
By using culture supernatant or a purified immunoglobulin preparation, further analysis of a
potential monoclonal antibody producing hybridoma can be made in terms of reactivity,
specificity, and cross-reactivity.[1]
Applications
This section may need to be wikified to meet Wikipedia's quality standards. Please help by adding
relevant internal links, or by improving the section's layout. (January 2010)
It
has
bee
n
sug
ges
ted
tha
t
this
arti
cle
or
sec
tion
be
me
rge
d
into
Mo
noc
lon
al
anti
bod
ies.
(Dis
cus
s)
The use of monoclonal antibodies is numerous and includes the prevention, diagnosis, and
treatment of disease. For example, monoclonal antibodies can distinguish subsets of B cells and
T cells, which is helpful in identifying different types of leukaemias.
In diagnostic histopathology
With the help of monoclonal antibodies, tissues and organs can be classified based on their
expression of certain defined markers, which reflect tissue or cellular genesis. Prostate specific
antigen, placental alkaline phosphatase, human chorionic gonadotrophin, α-fetoprotein and
others are organ-associated antigens and the production of monoclonal antibodies against these
antigens helps in determining the nature of a primary tumor.[1]
Monoclonal antibodies are especially useful in distinguishing morphologically similar lesions,
like mesothelioma and adenocarcinoma, and in the determination of the organ or tissue origin of
undifferentiated metastases. Selected monoclonal antibodies help in the detection of occult
metastases by immuno-cytological analysis of bone marrow, other tissue aspirates, as well as
lymph nodes and other tissues.[1]
One study[2] performed a sensitive immuno-histochemical assay on bone marrow aspirates of 20
patients with localized prostate cancer. Three monoclonal antibodies (T16, C26, and AE-1),
capable of recognizing membrane and cytoskeletal antigens expressed by epithelial cells to
detect tumour cells, were used in the assay. Bone marrow aspirates of 22% of patients with
localized prostate cancer (stage B, 0/5; Stage C, 2/4), and 36% patients with metastatic prostate
cancer (Stage D1, 0/7 patients; Stage D2, 4/4 patients) had antigen-positive cells in their bone
marrow. It was concluded that immuno-histochemical staining of bone marrow aspirates are very
useful to detect occult bone marrow metastases in patients with apparently localized prostate
cancer.
Although immuno-cytochemistry using tumor-associated monoclonal antibodies has led to an
improved ability to detect occult breast cancer cells in bone marrow aspirates and peripheral
blood, further development of this method is necessary before it can be used routinely.[3] One
major drawback of immuno-cytochemistry is that only tumor-associated and not tumor-specific
monoclonal antibodies are used, and as a result, some cross-reaction with normal cells can
occur.[4]
The detection of small quantities of invasive or metastatic cells by normal histopathological
staining with haematoxylin and eosin is not always sensitive. The use of monoclonal antibodies
increases the sensitivity to a large extent. For example, the use of monoclonal antibodies to
cytokeratin in the investigation of the sentinel axillary lymph node for metastatic breast cancer
increases nodal positivity by up to 10%.[1]
In order to effectively stage breast cancer and assess the efficacy of purging regimens prior to
autologous stem cell infusion, it is important to detect even small quantities of breast cancer
cells. Immuno-histochemical methods are ideal for this purpose because they are simple,
sensitive, and quite specific. Franklin et al.[5] performed a sensitive immuno-cytochemical assay
by using a combination of four monoclonal antibodies (260F9, 520C9, 317G5 and BrE-3)
against tumor cell surface glycoproteins to identify breast tumour cells in bone marrow and
peripheral blood. They concluded from the results that immuno-cytochemical staining of bone
marrow and peripheral blood is a sensitive and simple way to detect and quantify breast cancer
cells.
One of the main reasons for metastatic relapse in patients with solid tumours is the early
dissemination of malignant cells. The use of monoclonal antibodies (mAbs) specific for
cytokeratins can identify disseminated individual epithelial tumor cells in the bone marrow.
One study[6] reports on having developed an immuno-cytochemical procedure for simultaneous
labeling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). This
would help in the further characterization of disseminated individual epithelial tumor cells in
patients with prostate cancer. The twelve control aspirates from patients with benign prostatic
hypertrophy showed negative staining, which further supports the specificity of CK18 in
detecting epithelial tumour cells in bone marrow.
In most cases of malignant disease complicated by effusion, neoplastic cells can be easily
recognized. However, in some cases, malignant cells are not so easily seen or their presence is
doubtful to call it a positive report. The use of immuno-cytochemical techniques increases
diagnostic accuracy in these cases.
Ghosh, Mason and Spriggs[7] analysed 53 samples of pleural or peritoneal fluid from 41 patients
with malignant disease. Conventional cytological examination had not revealed any neoplastic
cells. Three monoclonal antibodies (anti-CEA, Ca 1 and HMFG-2) were used to search for
malignant cells. Immunocytochemical labelling was performed on unstained smears, which had
been stored at -20°C up to 18 months.
Twelve of the forty-one cases in which immuno-cytochemical staining was performed, revealed
malignant cells. The result represented an increase in diagnostic accuracy of approximately 20%.
The study concluded that in patients with suspected malignant disease, immuno-cytochemical
labeling should be used routinely in the examination of cytologically negative samples and has
important implications with respect to patient management.
The use of immuno-cytochemical techniques can help to avoid performing procedures, which are
painful, uncomfortable and expensive to the patient. It can also help to speed up the start of
appropriate treatment.
Another application of immuno-cytochemical staining is for the detection of two antigens in the
same smear. Double staining with light chain antibodies and with T and B cell markers can
indicate the neoplastic origin of a lymphoma.[8]
One study has reported the isolation of a hybridoma cell line (clone 1E10), which produces a
monoclonal antibody (IgM, k isotype). This monoclonal antibody shows specific immunocytochemical staining of nucleoli.[9]
Tissues and tumours can be classified based on their expression of certain markers, with the help
of monoclonal antibodies. They help in distinguishing morphologically similar lesions and in
determining the organ or tissue origin of undifferentiated metastases. Immuno-cytological
analysis of bone marrow, tissue aspirates, lymph nodes etc. with selected monoclonal antibodies
help in the detection of occult metastases. Monoclonal antibodies increase the sensitivity in
detecting even small quantities of invasive or metastatic cells. Monoclonal antibodies (mAbs)
specific for cytokeratins can detect disseminated individual epithelial tumour cells in the bone
marrow. Immuno-cytochemical staining can also detect the presence of two antigens in the same
smear.
UNIT - IV
MHC antigens - types and functions
AUTOIMMUNITY
.
Autoimmunity is the failure of an organism to recognize its own
constituent parts as self, which allows an immune response against its own
cells and tissues. Any disease that results from such an aberrant immune
response is termed an autoimmune disease . Prominent examples include
Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus
erythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome ,
Hashimoto's thyroiditis , Graves' disease, idiopathic thrombocytopenic
purpura, and rheumatoid arthritis (RA). See List of autoimmune di seases.
The misconception that an individual's immune system is totally incapable
of recognizing self antigens is not new. Paul Ehrlich, at the beginning of
the twentieth century, proposed the concept of horror autotoxicus ,
wherein a 'normal' body does not mount an immune response against its
own tissues. Thus, any autoimmune response was perceived to be abnormal
and postulated to be connected with human disease. Now, it is accepted
that autoimmune responses are an integral part of vertebrate immune
systems (sometimes termed 'natural autoimmunity'), normally prevented
from causing disease by the phenomenon of immunological tolerance to
self-antigens. Autoimmunity should not be confused with alloimmunity.
Low-level autoimmunity
While a high level of autoimmunity is unhealthy, a low level of
autoimmunity may actually be beneficial. First, low -level autoimmunity
might aid in the recognition of neoplastic cells by CD8+ T cells, and thus
reduce the incidence of cancer.
Second, autoimmunity may have a role in allowing a rapid immune
response in the early stages of an infection when the availability of foreign
antigens limits the response (i.e., when there are few pathogens present).
In their study, Stefanova et al. (2002) injected an anti -MHC Class II
antibody into mice expressing a single type of MHC Class II molecule (H 2 b ) to temporarily prevent CD4+ T cell -MHC interaction. Naive CD4+ T
cells (those that have not encountered any antigens before) recovered from
these mice 36 hours post -anti-MHC administration showed decreased
responsiveness to the antigen pigeon cytochrome C peptide, as determined
by Zap-70 phosphorylation, proliferation, and Interleukin-2 production.
Thus Stefanova et al. (2002) d emonstrated that self -MHC recognition
(which, if too strong may contribute to autoimmune dise ase) maintains the
responsiveness of CD4+ T cells when foreign antigens are absent. [ 1 ] This
idea of autoimmunity is conceptually similar to play -fighting. The playfighting of young cubs (TCR and self -MHC) may result in a few scratches
or scars (low-level-autoimmunity), but is beneficial in the long -term as it
primes the young cub for proper fights in the future.
Immunological tolerance
Pioneering work by Noel Rose and Witebsky in New York, and Roitt and
Doniach at University College London provided clear evidence that, at
least in terms of antibody-producing B lymphocytes, diseases such as
rheumatoid arthritis and thyrotoxicosis are associated with of loss of
immunological tolerance , which is the ability of an individual to ignore
'self', while reacting to 'non -self'. This breakage leads to the immune
system's mounting an effective and specific immune response against self
determinants. The exact genesis of immunological tolerance is still elusive,
but several theories have been proposed since the mid -twentieth century to
explain its origin.
Three hypotheses have gained widespread attention among immuno logists:


Clonal Deletion theory, proposed by Burnet, according to which
self-reactive lymphoid cells are destroyed during the development
of the immune system in an individual. For their work Frank M.
Burnet and Peter B. Medawar were awarded the 1960 Nobel Prize in
Physiology or Medicine "for discovery of acquired immunological
tolerance".
Clonal Anergy theory , proposed by Nossal, in which self -reactive Tor B-cells become inactivated in the normal individual and cannot
amplify the immune response. [ 2 ]

Idiotype Network theory , proposed by Jerne, wherein a network of
antibodies capable of neutralizing self -reactive antibodies exists
naturally within the body. [ 3 ]
In addition, two other theories are under intense investigation:


The so-called "Clonal Ignorance" theory, according to which host
immune responses are directed to ignore self -antigens [ 4 ]
The "Suppressor population" or " Regulatory T cell " theories, wherein
regulatory T-lymphocytes (commonly CD4 + FoxP3 + cells, among
others) function to prevent, downregulate, or limit autoaggressive
immune responses cdimmune System.
Tolerance can also be differentiated into 'Central' and 'Peripheral'
tolerance, on whether or not the above -stated checking mechanisms operate
in the central lymphoid organs (Thymus and Bone Marrow) or the
peripheral lymphoid organs (lymph node, spleen, etc., where self -reactive
B-cells may be destroyed). It must be emphasised that these theories are
not mutually exclusive, and evidence has been mounting suggesting that all
of these mechanisms may actively contribute to vertebrate immunological
tolerance.
A puzzling feature of the documented loss of tolerance seen in spontaneous
human autoimmunity is that it is almost entirely restricted to the
autoantibody responses produced by B lymphocytes. Loss of tolerance by T
cells has been extremely hard to demonstrate, and where there is evidence
for an abnormal T cell response it is usually not t o the antigen recognised
by autoantibodies. Thus, in rheumatoid arthritis there are autoantibodies to
IgG Fc but apparently no corresponding T cell response. In systemic lupus
there are autoantibodies to DNA, which cannot evoke a T cell response,
and limited evidence for T cell responses implicates nucleoprotein
antigens. In Celiac disease there are autoantibodies to tissue
transglutaminase but the T cell response is to the foreign protein gliadin.
This disparity has led to the idea that human autoimmune di sease is in
most cases (with probable exceptions including type I diabetes) based on a
loss of B cell tolerance which makes use of normal T cell responses to
foreign antigens in a variety of aberrant ways [ 5 ] .
Genetic Factors
Certain individuals are genetically susceptible to developing autoimmune
diseases. This susceptibility is associated with multiple genes plus other
risk factors. Genetically-predisposed individuals d o not always develop
autoimmune diseases.
Three main sets of genes are suspected in many autoimmune diseases.
These genes are related to:



Immunoglobulins
T-cell receptors
The major histocompatibility complexes (MHC).
The first two, which are involved in the recognition of antigens, are
inherently variable and susceptible to recombination. These variations
enable the immune system to respond to a very wide variety of invaders,
but may also give rise to lymphocytes capable of self-reactivity.
Scientists such as H. McDevitt, G.
also provided strong evidence to
allotypes are strongly correlated



Nepom, J. Bell and J. Todd have
suggest that certain MHC class II
with
HLA DR2 is strongly positively correlated with Systemic Lupus
Erythematosus, narcolepsy [ 6 ] and multiple sclerosis, and negatively
correlated with DM Type 1.
HLA DR3 is correlated strongly with Sjögren's syndrome , myasthenia
gravis, SLE, and DM Type 1.
HLA DR4 is correlated with the genesis of rheumatoid arthritis, Type
1 diabetes mellitus, and pemphigus vulgaris.
Fewer correlations exist with MHC class I molecules. The most notable and
consistent is the association between HLA B27 and ankylosing spondylitis.
Correlations may exist between polymorphisms within class II MHC
promoters and autoimmune disease.
The contributions of genes outside the MHC compl ex remain the subject of
research, in animal models of disease (Linda Wicker's extensive genetic
studies of diabetes in the NOD mouse), and in patients (Brian Kotzin's
linkage analysis of susceptibility to SLE).
 Sex
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A person's sex also seems to have some role in the development of
autoimmunity, classif ying most autoimmune diseases as sex-related
diseases. Nearly 75% [ 7 ] of the more than 23.5 million Americans who
suffer from autoimmune disease are women, although it is less -frequently
acknowledged that millions of men also suffer from these disea ses.
According to the American Autoimmune Related Diseases Association
(AARDA), autoimmune diseases that develop in men tend to be more
severe. A few autoimmune diseases that men are just as or more likely to
develop as women, include: ankylosing spondylitis, type 1 diabetes
mellitus, Wegener's granulomatosis , Crohn's disease and psoriasis.
The reasons for the sex role in autoimmunity are unclear. Women appear to
generally mount larger inflammatory responses than men when their
immune systems are triggered, increasing the risk of autoimmu nity. [ 7 ]
Involvement of sex steroids is indicated by that many autoimmune disease s
tend to fluctuate in accordance with hormonal changes, for example, during
pregnancy, in the menstrual cycle, or when using oral contraception. [ 7 ] A
history of pregnancy also appears to leave a persistent increased risk for
autoimmune disease. [ 7 ] It has been suggested that the slight exchange of
cells between mothers and their children during pregnancy may induce
autoimmunity. [ 8 ] This would tip the gender balance in the direction of the
female.
Another theory suggests the fema le high tendency to get autoimmunity is
due to an imbalanced X chromosome inactivation . [ 9 ] The X-inactivation
skew theory, proposed by Princeton University's Jeff Stewart, has recently
been confirmed experimentally in scleroderma and autoimmune
thyroiditis. [ 1 0 ] Other complex X -linked genetic susceptibility mechanisms
are proposed and under investigation. [ 7 ]
Environmental Factors
An interesting inverse relationship exists between infectious diseases and
autoimmune diseases. In areas where multiple infectious diseases are
endemic, autoimmune diseases are quite rarely seen. The reverse, to some
extent, seems to hold true. The hygiene hypothesis attributes these
correlations to the immune manipulating strategies of pathogens. Whilst
such an observation has been variously termed as spurious and ineff ective,
according to some studies, parasite infection is associated with reduced
activity of autoimmune disease. [ 1 1 ] [ 1 2 ] [ 1 3 ]
The putative mechanism is that the parasite attenuates the host immune
response in order to protect itself. This may pro vide a serendipitous benefit
to a host that also suffers from autoimmune disease. The details of parasite
immune modulation are not yet known, but may include secretion of anti inflammatory agents or interference with the host immune signaling.
A paradoxical observation has been the strong association of certain
microbial organisms with autoimmune diseases. For example, Klebsiella
pneumoniae and coxsackievirus B have been strongly correlated with
ankylosing spondylitis and diabetes mellitus type 1 , respectively. This has
been explained by the tendency of the infecting organism to produce superantigens that are capable of polyclonal activation of B-lymphoc ytes, and
production of large amounts of antibodies of varying specificities, some of
which may be self-reactive (see below).
Certain chemical agents and drugs can also be associated with the genesis
of autoimmune conditions, or conditions that simulate autoimmune
diseases. The most striking of these is the drug-induced lupus
erythematosus. Usually, withdrawal of the offending drug cures the
symptoms in a patient.
Cigarette smoking is now established as a major risk factor for both
incidence and severity of rheumatoid arthritis. [ c i t a t i o n n e e d e d ] This may relate
to abnormal citrullination of proteins, since the effects of smoking
correlate with the presence of antibodies to citrullinated peptides. [ c i t a t i o n
needed]
Pathogenesis of autoimmunity
Several mechanisms are thought to be operative in the pathogenesis of
autoimmune diseases, against a backdrop of genetic predisposition and
environmental modulation. It is beyond the scope of this article to discuss
each of these mechanisms exhaustively, but a summary of some of the
important mechanisms have been described:

T-Cell Bypass - A normal immune system requires the activation of
B-cells by T-cells before the former can produce antibodi es in large
quantities. This requirement of a T -cell can be bypassed in rare
instances, such as infection by organisms producing super-antigens,
which are capable of initiating polyclonal activation of B -cells, or
even of T-cells, by directly binding to the β -subunit of T-cell
receptors in a non -specific fashion.



T-Cell-B-Cell discordance - A normal immune response is assumed to
involve B and T cell responses to the same antige n, even if we know
that B cells and T cells recognise very different things:
conformations on the surface of a molecule for B cells and pre processed peptide fragments of proteins for T cells. However, there
is nothing as far as we know that requires this. All that is required is
that a B cell recognising antigen X endocytoses and processes a
protein Y (normally =X) and presents it to a T cell. Roosnek and
Lanzavecchia showed that B cells recognising IgGFc could get help
from any T cell responding to an ant igen co-endocytosed with IgG by
the B cell as part of an immune complex. In coeliac disease it seems
likely that B cells recognising tissue transglutamine are helped by T
cells recognising gliadin.
Aberrant B cell receptor-mediated feedback - A feature of human
autoimmune disease is that it is largely restricted to a small group
of antigens, several of which have known signaling roles in the
immune response (DNA, C1q, IgGFc, Ro, Con. A receptor, Peanut
agglutinin receptor(PNAR)). This fact gave rise to the idea that
spontaneous autoimmunity may result when the binding of antibody
to certain antigens leads to aberrant signals being fed back to
parent B cells through membrane bound ligands. These ligands
include B cell receptor (for antigen), IgG Fc receptors , CD21, which
binds complement C3d, Toll -like receptors 9 and 7 (which can bind
DNA and nucleoproteins) and PNAR. More indirect aberrant
activation of B cells can also be envisaged with autoantibodies to
acetyl choline receptor (on thymic myoid cells) and hormone and
hormone binding proteins. Together with the concept of T -cell-B-cell
discordance this idea forms the basis of the hypothesis of self perpetuating autoreactive B cells [ 1 4 ] . Autoreactive B cells in
spontaneous autoimmunity are seen as surviving because of
subversion both of the T cell help pathway and of the feedback
signal through B cell receptor, thereby overcoming the negative
signals responsible for B cell self -tolerance without necessarily
requiring loss of T cell self -tolerance.
Molecular Mimicry - An exogenous antigen may share structural
similarities with certain host antigens; thus, any antibody produced
against this antigen (which mimics the self -antigens) can also, in
theory, bind to the host antigens, and amplify the immune response.
The idea of molecular mimicry a rose in the context of Rheumatic
Fever, which follows infection with Group A beta -haemolytic




streptococci. Although rheumatic fever has been attributed to
molecular mimicry for half a century no antigen has been formally
identified (if anything too many have been proposed). Moreover, the
complex tissue distribution of the disease (heart, joint, skin, b asal
ganglia) argues against a cardiac specific antigen. It remains entirely
possible that the disease is due to e.g. an unusual interaction
between immune complexes, complement components and
endothelium.
Idiotype Cross-Reaction - Idiotypes are antigenic epitopes found in
the antigen-binding portion (Fab) of the immunoglobulin molecule.
Plotz and Oldstone presented evid ence that autoimmunity can arise
as a result of a cross-reaction between the idiotype on an antiviral
antibody and a host cell receptor for the virus in question. In this
case, the host-cell receptor is envisioned as an internal image of the
virus, and the anti-idiotype antibodies can react with the host cells.
Cytokine Dysregulation - Cytokines have been recently divided into
two groups according to the population of cells whose functio ns
they promote: Helper T -cells type 1 or type 2. The second category
of cytokines, which include IL -4, IL-10 and TGF-β (to name a few),
seem to have a role in prevention of exaggeration of proinflammatory immune responses.
Dendritic cell apoptosis - immune system cells called dendritic cells
present antigens to active lymphocytes. Dendritic cells that are
defective in apoptosis can lead to inappropriate systemic
lymphocyte activation and consequent decline in self -tolerance. [ 1 5 ]
Epitope spreading or epitope drift - when the immune reaction
changes from targeting the primary epitope to also targeting other
epitopes. [ 1 6 ] In contrast to molecular mimicry, the other epitopes
need not be structurally similar to the primary one.
The roles of specialized immunoregulatory cell types, such as regulatory T
cells, NKT cells, γδ T-cells in the pathogenesis of autoimmune disease are
under investigation.
Classification
Autoimmune diseases can be broadly divided into systemic and organ specific or localised autoimmune disorders, depending on the principal
clinico-pathologic features of each disease.

Systemic autoimmune diseases include SLE, Sjögren's syndrome,
scleroderma, rheumatoid arthritis, and dermatomyositis. These
conditions tend to be associated with autoantibo dies to antigens
which are not tissue specific. Thus although polymyositis is more or
less tissue specific in presentation, it may be included in this group
because the autoantigen s are often ubiquitous t -RNA synthetases.

Local syndromes which affect a specific organ or tissue:
o Endocrinologic: Diabetes mellitus type 1 , Hashimoto's
thyroiditis, Addison's disease
o Gastrointestinal: Coeliac disease, Pernicious anaemia
o Dermatologic: Pemphigus vulgaris, Vitiligo
o Haematologic: Autoimmune haemolytic anaemia , Idiopathic
thrombocytopenic purpura
o Neurological: Myasthenia gravis
Using the traditional “organ specific” and “non -organ specific”
classification scheme, many dis eases have been lumped together under the
autoimmune disease umbrella. However, many chronic inflammatory
human disorders lack the telltale associations of B and T cell driven
immunopathology. In the last decade it has been firmly established that
tissue "inflammation against self" does not necessarily rely on abnormal T
and B cell responses.
This has led to the recent proposal that the spectrum of autoimmunity
should be viewed along an “immunological disease continuum,” with
classical autoimmune diseases a t one extreme and diseases driven by the
innate immune system at the other extreme. Within this scheme, the full
spectrum of autoimmunity can be included. Many common human
autoimmune diseases can be seen to have a substantial innate immune
mediated immunopathology using this new scheme. This new classification
scheme has implications for understanding disease mechanisms and for
therapy development (see PLoS Medicine article.
Diagnosis
Diagnosis of autoimmune disorders largely rests on accurate history and
physical examination of the patient, and high index of suspicion against a
backdrop of certain abnormalities in routine laboratory tests (example,
elevated C-reactive protein). In several systemic disorders, serological
assays which can detect specific autoantibodies can be employed.
Localised disorders are best diagnosed by immunofluorescence of biopsy
specimens. Autoantibodies are used to diagnose many autoimmune
diseases. The levels of autoantibodies are measured to determine the
progress of the disease .
Treatments
Treatments for autoimmune disease have traditionally been
immunosuppressive , anti-inflammatory, or palliative. [ 4 ] Nonimmunological therapies, such as hormone replacement in Hashimoto's
thyroiditis or Type 1 diabetes mellitus treat outcomes of the autoaggressive
response, thus these are palliative treatments. Dietary manipulation limits
the severity of celiac disease . Steroidal or NSAID treatment limits
inflammatory symptoms of many diseases. IVIG is used for CIDP and GBS.
Specific immunomodulatory therapies, such as the TNFα antagonists (e.g.
etanercept), the B cell depleting agent rituximab, the anti-IL-6 receptor
tocilizumab and the costimulation blocker abatacept have been shown to be
useful in treating RA. Some of these immunotherapies may be associated
with increased risk of adverse effects, such as susceptibility to infection.
Helminthic therapy is an experimental approach that involves inoculation
of the patient with specific parasitic intestinal nematodes (helminths).
There are currently two closely-related treatments available, inoculation
with either Necator americanus, commonly known as hookworms, or
Trichuris Suis Ova, commonly known as Pig Whipworm Eggs.
[17][17][18][19][20][21]
T cell vaccination is also being explored as a possible future therapy for
auto-immune disorders.
.