Antibody Structure and
Function
W. Robert Fleischmann, Ph.D.
Department of Urologic Surgery
University of Minnesota Medical School
rfleisch@umn.edu
(612) 626-5034
Objectives
• Understand the basic structure and function
of antibodies
• Understand how antigens and antibodies
interact
• Understand the role of antibodies in
protection from toxins, bacteria, viruses and
parasites
• Understand the mechanisms by which
antibodies provide this protection
Charlie Reiser, age 72, visits his internist with
complaints of back and chest pain. The
internist conducts a thorough physical
examination that is entirely normal. The pains
do not become worse with movement or
pressure. His heart sounds are normal.
The internist is aware that Charlie has had a
series of bacterial infections over the past
several months.
What would you do next?
The bone density X-ray shows areas of diffuse osteoporosis.
The results of the troponin test are normal. The EKG and
sonogram of Charlie’s heart are normal.
The blood test results have some unusual findings.
RBCs (with some stacking) 3.0 million/µl
(4.3-6.2 million/µl)
Hct
27%
(40-50%)
Hb
8.4 gm/dL
(13.2-16.2 gm/dL)
Mean cell HB
33 gm/dL
(31-35 gm/dL)
RBC Mean Cell Vol
90 fL
(82-102 fL)
Creatinine
BUN
Uric Acid
2.2 mg/dL
30 mg/dL
10 mg/dL
What do the test results suggest?
What would you do next?
(0.5-1.4 mg/dL)
(7-21 mg/dL)
(3.5-8.5 mg/dL)
First Consult:
•Hypercalcemia
•Bladder cancer
•Acute lymphoblastic leukemia
•Multiple myeloma
•Neuroblastoma
•Myelodysplastic syndrome
•Evaluation of hepatomegaly
•Wilm’s tumor
•Renal cell carcinoma
•Prostate cancer
•Chronic myelogenous leukemia
•Breast cancer
•Sickle cell disease
•Non-Hodgkin lymphoma
•Chronic fatigue syndrome
•Zollinger-Ellison syndrome (peptic ulcer disease)
The Purpose of Adaptive Immunity
• Innate immunity deals with infectious
agents when we first come into contact
with them.
• Adaptive immunity deals with infectious
agents, toxins, and foreign cells
– that persist in our body for an extended
period of time or
– that enter our body a second time.
Components of Adaptive Immunity
• Adaptive immunity has two components.
– Humoral immunity is mediated by B cells and plasma
cells.
– Cell-mediated immunity is mediated by CD8+
cytotoxic T cells.
• Adaptive immunity must be carefully regulated.
– Under-stimulation of adaptive immunity can fail to
generate sufficient memory to last a lifetime.
– Over-stimulation of adaptive immunity can lead to the
development of autoimmunity or blood cell cancers.
Development of Humoral Immunity
• Monocytes, dendritic cells, and other professional antigenpresenting cells present a specific antigen to a subset of
CD4+ helper T cells (Th2 cells) that carry a T cell receptor
that recognizes (binds to) that specific antigen.
• Antigen-presenting cells provide additional signals to
activate Th2 cells.
• Antigen-presenting cells and activated Th2 cells interact
with B cells that recognize the specific antigen.
– Antigen-presenting cells present specific antigen to B cells that
carry an antibody on their surface that recognizes that specific
antigen.
– Th2 cells secrete cytokines that activate B cells.
• B cells divide and differentiate to become antibodyproducing plasma cells.
Summary of Acquired Immunity
M
IL-1
IL-12
IL-1
CD4 Th1
CD4 Th2
IL-2
IL-3
IFN-
IL-4
IL-5
IL-6
IL-9
IL10
IL-13
CD8 T cell
Cytolysis:
FAS/FAS ligand
TNF
Granzyme B
Perforin
B cell/Plasma cell
Antibody
Antigens
Antigens: Definition
• Antigens are infectious agents, toxins,
and foreign cells that contain unique
molecular structures that can be
recognized by humoral immunity.
• Antigens are recognized by T cell
receptors and by antibodies.
• Antigens may be proteins,
carbohydrates or lipids, though the best
antigens are proteins.
Antigens: Characteristics
• Antigens have one or more immune
recognition sites called epitopes.
• The more complex the antigen, the
more epitopes it has.
Epitopes: Characteristics
• Epitopes are generally hydrophilic regions of an antigen.
– Epitopes recognized by B cells are linear or conformational regions
located on the surface of the antigen that are available for
recognition and binding by antibody.
– Epitopes recognized by T cells are linear regions of an antigen that
have been cut from the antigen and presented to the T cell receptor
in association with an MHC molecule.
– Linear epitopes are 5-7 aa in size.
– Confirmational epitopes may include discontinuous aa groups
spread over a 15-22 aa sequence.
• Epitopes vary in their ability to stimulate an immune
response.
– Immunodominant epitopes induce a greater immune response
than other epitopes on an antigen.
Visualization of Epitopes
• Epitopes are regions on an
antigen that can be
recognized by the paratope
(antigen-binding site) of an
antibody or by a T cell
receptor.
• Epitopes are also called
antigenic determinants.
• In the picture, epitopes on a
whole poliovirus virion and
on the isolated poliovirus
VP1 protein are shown in
white.
• As can be seen for the
individual VP1 protein in the
bottom picture, a given
protein can have multiple
epitopes.
Biological Molecules Have Differing
Immunogenicity
• Thymus-Dependent Antigens
– Proteins are the most immunogenic.
• Thymus-Independent Antigens
– Polysaccharides are potentially immunogenic.
• ABO blood groups.
– Nucleic Acids are poorly antigenic unless bound to
protein carriers.
• Anti-DNA antibodies are seen in Lupus and other autoimmune diseases.
– Lipids are not usually antigenic unless bound to
proteins.
– Thymus-independent antigens involve recognition
of conformational epitopes.
Haptens: a Special Class of Small
Antigens
• Some molecules are too small to
stimulate an immune response but can
still bind to antibody.
– Thus, these small molecules are antigens
but not immunogens.
– Such molecules are called haptens.
– Haptens can stimulate immunity when
combined (covalently or noncovalently)
with a suitable carrier molecule.
– Example: penicillin allergy, where penicillin
is the hapten
Antibodies
Antibodies Are Members of the
Immunoglobulin Supergene Family
• Other members of the immunoglobulin
supergene family (IgSF) include a variety of
cell adhesion proteins
– ICAM, VCAM, T cell receptor, MHC molecules,
CD4, CD8, and CD28 (binds to B7), IL-1 receptor
• Members of the IgSF have one or more
immunoglobulin domains
– Two or more -pleated sheets arranged in opposite
directions that are stabilized by one or more
disulfide bonds (aka -barrel)
Antibodies: Definition
• Antibodies are members
of a family of proteins that
are collectively known as
immunoglobulins (Igs).
– Antibodies are present in
the third peak of
electrophoretically
separated serum globulin
proteins (that are heavier
than albumin), hence they
are also called “gamma”
globulins.
– All antibodies have similar
structural features.
Albumin

Globulins


Migration distance
Multiple Myeloma
• Antibodies are produced by B
cells and plasma cells.
• When a single plasma cell
becomes transformed into a
cancerous cell, it causes
myeloma.
– Myeloma patients over-produce a
homogeneous Ig produced by a
single plasma cell. This can be
observed as a heightened peak of
Ig by electrophoresis of blood
proteins.
– Myeloma patients also have some
immunoglobulin proteins that spill
over into their urine. These
Bence-Jones proteins are
dimers of kappa or lambda light
chains.
Albumin
Globulins



IgX from
Myeloma
patients
Migration distance
Antibodies: General Features
• Antibodies recognize unique regions on
antigens called epitopes.
• Each antibody recognizes a single epitope by
its unique paratope or antigen-binding site.
• The sum of all of the epitopes recognized by
all of the different antibodies that a person
can produce is called the person’s repertoire.
– Every individual has their own repertoire.
– Some individuals respond better to certain
epitopes and therefore to certain antigens than to
others.
Antibody Structure
• Antibodies are hetero-tetramers of four
peptide chains.
• Antibodies contain two identical light
chains and two identical heavy chains.
– Each light chain has a molecular weight of
about 22,000 daltons.
– Each heavy chain has a molecular weight
of about 55,000 daltons.
– The total molecular weight of an antibody
is about 150-160 daltons.
General Structure of an Antibody
Light chains:
Kappa chains are found twice as
frequently in Abs as are lambda chains.
Heavy chains:
These undergo class switching
as the B cell undergoes differentiation.
Light chain
(kappa or lambda)
Heavy chain
(isotypic region:
,  , , or )
Light Chains
• MWs of light chains are about 22,000 daltons
(about 200 amino acids).
• From study of Bence-Jones proteins of
different multiple myeloma patients:
– The amino terminal half of the proteins varied from
patient to patient; e.g. variable region.
– The carboxy terminal half of the proteins varied
among two groups; constant region.
• 60% are  chains, with no subtypes
• 40% are  chains, with 1, 2, 3, and 4 subtypes
differing in only a few amino acids
• Antibody contains two copies of the same
light chain
Heavy Chains
• MWs of heavy chains are about 55,000 daltons
(about 500 amino acids).
• From heavy chains of antibodies of different myeloma
patients:
– The amino terminal 20% of the proteins varied greatly from
patient to patient; e.g. variable region.
– The carboxy terminal part of the proteins varied primarily
among two groups:  and  chains.
– We now know that there are five types of heavy chain
•
•
•
•
•
IgD, containing the  heavy chain
IgM, containing the  heavy chain
IgG, containing the  heavy chain, with 1, 2, 3, 4 subtypes
IgA, containing the  heavy chain, with 1 and 2 subtypes
IgE, containing the  heavy chain
• Antibody contains two copies of the same heavy
chain which may pair with either type of light chain
Molecular Model of IgG
Antigen Binding
Carbohydrate
Light Chain
Heavy
Chain
Fab and Fc Regions of an Antibody
Light chain
(kappa or lambda)
Heavy chain
(isotypic region:
,  , , or )
•
•
Fab = variable region
aka VDJ region
aka idiotypic region
aka paratope region
Fc = constant region
Papain cleaves just above the double disulfide bond, giving 2 Fab
fragments and 1 Fc fragment.
Pepsin cleaves just below the double disulfide bond and within the
constant region, giving 1 Fab2 fragment and degraded Fc.
Fc Versus Fab and Fab2
• Fab and Fab2 fragments get their name
because they contain one or two
antigen binding sites of the antibody.
• The Fc fragment gets its name because
it was noted to crystallize from solution
when chilled.
Fab Versus Fab2
• Fab cannot precipitate antigen, because
it has only one antigen combining site
and can combine with only one antigen
epitope.
• Fab2 can precipitate antigen, because it
has two antigen combining sites and
can combine with two antigen epitopes
(cross-linking antigens).
Functions of Antibody Defined by Studies
of Fab and Fc Regions
• Fab regions: Antigen binding to antigenic
epitopes occurs at the variable regions of the
Fab, located at the amino terminal regions of
the heavy and light chains.
• Fc region: Various effector activities occur at
the constant regions, located at the carboxy
terminal regions of the heavy chains.
– Binding to Fc receptors on macrophages
– Binding to and activation of complement factor C1
(C1qr2s2) (requires conformational change that
occurs with binding of Ag to Fab region of Ab)
The Immunoglobulin Domain
• Immunoglobulins possess several homologous
Ig domains of about 110 aa, stabilized by a
disulfide bond.
– Light chain has 1 variable domain and 1 constant
domain.
– Heavy chain has 1 variable domain and 3 or 4
constant domains.
• Either 3 constant domains and a hinge structure as is seen
for , , and  heavy chains. (Hinge structure gives more
flexibility in the two arms.)
• Or 4 constant domains without a hinge structure for  and 
heavy chains.
Stylized Domain Structure
• The Ig Domain has an intra-domain loop of
about 60 aa held together by a disulfide bond.
Kuby Immunology, 6th Edition
Structure of an Ig Domain
• The Ig domains are folded into a
characteristic structure called the
immunoglobulin fold.
• The immunoglobulin fold is a
“sandwich” composed of two Constant domain
pleated sheets containing
alternating antiparallel  strands
connected by loops of varying
length. This gives a relatively
rigid structure to the
immunoglobulin folds.
• Note that the blue loops of the
CDR
variable region form three
complementarity-determining
regions (CDRs) that together
form the antibody’s paratope or
antigen-binding site (epitope).
Variable domain
CDR
Kuby Immunology
Complementarity Determining
Regions (CDRs)
• There are 3 CDRs on the light chain
and 3 CDRs on the heavy chain.
• The CDRs are also called hypervariable
regions because they show the greatest
amount of variability from antibody to
antibody.
• The fact that they are on the loops of
the Ig domain indicate that, while they
may have slight flexibility, they are held
in place by a relatively rigid structure.
Antigen-Antibody Binding
Antigen Binding to Antibody
Kuby Immunology
Characteristics of Ag-Ab Binding
• The binding of antigen to antibody is a
non-covalent interaction that involves
– Electrostatic forces (salt bridges; ionic
bonds)
– Hydrogen bonds
– Van der Waals forces
– Hydrophobic forces
• Although each individual interaction is
weak, the binding strength of an
antigen to its antibody is due to the
sum of a large number of low energy
interactions.
• Thus, the binding between the
paratope of the antibody and its
antigen epitope can be very strong
(dissociation constant 10-7-10-10).
Kuby, Immunology
Antibody Avidity
• This is the sum of all of the affinity interactions
between a given antigen and its antibody.
– Depends upon the binding affinity of each paratope
for its cognate epitope.
– Depends upon the number of identical combining
sites (epitopes) on an antigen. Some antigens may
have a single epitope for a given paratope.
– Depends upon the number of combining sites
(paratopes) on an antibody. For example, IgG has
two paratopes while IgM has ten. Thus, even if an
individual IgM paratope had five-fold less affinity
than an IgG paratope, they could have equal avidity
(if there were 10 identical epitopes on the antigen).
Antigen Binding Induces a
Conformational Change in the Antibody
• Binding of the antigen epitope to the
antibody’s paratope causes changes
the conformation of the antibody.
– Antibody with no bound antigen does not
bind complement, while antibody with
bound antigen does bind complement.
– Much like the change in enzyme
conformation by the binding of its
substrate.
Advantages of the Hinge Region
• In the , , and  heavy chains of IgG, IgD, and IgA,
there is a hinge region between the first and second
constant domains.
– This allows wide or narrow separation of the two arms of the
antibodies.
– Can form interactions between paratopes on different arms of
an antibody and epitopes on different antigens.
– If the antigen has multiple epitopes, the antigen:antibody
complexes can form large cross-linked structures (latice-like
structures).
– The large cross-linked structure can then precipitate because
they become too large to stay in solution.
Note: IgM antibody can precipitate antigens because it has
a multimeric structure. Soluble IgE does not precipitate
antigens, as it is bound to mast cells and basophils.
Also, cross-linking of IgE antibodies on mast cells and
basphils is key to degranulation.
Cross-Reactivity of Antigens
• Some antigens have within them the same
sequence of amino acids and thus share a
common epitope.
• Cross-reactivity can also be seen as a slight
mismatch of epitope and paratope, that differ by a
similar amino acid (for example, ala vs. leu).
• Medical application: Antibodies to Group A
streptococci (Streptococcus pyogenes) crossreact with certain proteins in the heart values and
can cause post-streptococcal rheumatic fever.
Site of Antibody Expression
• Most types of antibodies are expressed as
soluble antibodies that have been secreted
into blood, lymph, or bodily secretions such
as saliva, tears, milk, intestinal secretions,
and urogenital secretions.
• Some types of antibodies are expressed on
the surface of immune cells.
– IgD is bound to B cells
– Some forms of IgM are bound to B cells; others
are soluble
– IgE is bound to mast cells and basophils
Antibody Functions
Antibody Functions
•
•
•
•
•
•
•
Mediation of uptake and processing of antigens
Neutralization of antigens
Opsonization of antigens
Agglutination of antigens
Precipitation of antigens
Activation of complement
Evocation of antibody-dependent cell-mediated
cytotoxicity
• Establishment of passive immunity via transfer in
placenta and colostrum
Uptake And Processing of Ag
• B cells expressing IgD or IgM bind antigen.
• The Ag:Ab complex is internalized.
• The antibody is degraded or recycled and the
antigen is digested (processed) to peptides in
phagolysosomes.
• Specific peptide sequences (epitopes) of the
antigen are bound to MHC class II and
presented to helper T cells to trigger either
more antibody synthesis or cytotoxic T cell
activation.
Neutralization of Antigens
• Certain epitopes on antigens are involved in
antigen binding to target cells or molecules.
– If the antigen is a toxin, the antibody is called an
antitoxin.
– Serum containing antitoxins is called antiserum.
• Antibodies that recognize and bind to these
epitopes block the ability of the antigen to
bind to the target cell or molecule.
– This can keep bacteria from being able to adhere
to tissue cells.
– The antigen is said to be neutralized.
Opsonization of Antigens
• Binding of antibodies to antigens can facilitate the uptake of
the antigens by phagocytic cells (opsonization).
• The antibodies bind to antigens by the Fab portion of the
antibody, leaving the Fc portion of the antibody free to bind
to Fc receptors on the surface of phagocytic cells.
• The antibodies bind via Fc: Fc receptor binding and the
antigen is phagocytosed.
Agglutination of Antigens
• Individual antigens can be bound
by multiple antibodies, either
recognizing the repeated or
different epitopes.
• If multiple antibodies bind to a
given antigen and if an antibody
binds to two or more antigens,
the antigens can be clumped
together or agglutinated.
• Agglutinated antigens can be
more readily phagocytosed than
individual antigens.
Precipitation of Antigens
• When the concentration of soluble antigens
and antibodies are about equal, large Ag:Ab
complexes can form (agglutination).
• If the Ag:Ab complex is sufficiently large, it
can precipitate from solution.
• Precipitated Ag:Ab complexes can be more
readily removed by phagocytes.
• However, if precipitated Ag:Ab complexes are
not phagocytosized quickly, complement can
be activated and damage can occur to the
site of the precipitated Ag:Ab complexes (cell
surface or basement membrane).
– Example: post-streptococcal glomerulonephritis
Activation of Complement
• If cross-linking of Ag occurs with Ab binding,
complement can be activated.
• If complement is activated by antibody binding to a
bacterium, lysis of the bacterium will occur due to
the deposition of the complement C9 membrane
attack complex.
• If complement is activated by Ag:Ab that has
precipitated on a host cell or basement membrane,
deposition of the complement attack complex can
damage the cell or basement membrane.
– A number of host factors block deposition or activation of complement
on normal tissues (eg, protectin [CD59] and homlogous restriction
factor [HRF]).
– These host factors can be overwhelmed by high levels of activated
complement (post-streptococcal glomerulonephritis).
Ab:Ag Activation
of Complement:
The Classical
Pathway of
Complement
Activation
Kuby et al
Three Mechanisms of Complement
Activation
Mannose-binding lectin
Kuby Immunology
Antibody-Dependent Cell-Mediated
Cytotoxicity
• When Ag:Ab bind and when the Fc portion of the
antibody binds to Fc receptors on Natural Killer
cells, eosinophils, neutrophils, or macrophages,
ADCC can be activated.
• ADCC results in the death of bacteria, viruses,
parasites, and tumor cells.
• The specificity for target cell killing in ADCC is
provided by the antibody, not by the effector cell.
• ADCC differs from opsonization as the antigen is
killed without first being phagocytosed.
Mechanisms of ADCC Killing
Lysosomal
Enzymes
Neutrophils
+
Macrophages
+
Eosinophils
+
NK cells
Perforin
Granzyme
TNF
+
+
+
+
+
Lysosomal enzymes digest target cells.
Perforin punches holes in target cells (like complement C9 membrane attack
complex).
Granzyme and TNF activate apoptosis in target cells.
Passive Immunity From Mother
• Certain antibodies can pass through the placenta
or be secreted in body fluid products (tears;
sweat; milk; and, respiratory, enteric, and
urogenital secretions).
– The mother’s IgG passes through the placenta to
provide a measure of systemic protection for the
newborn.
– IgA and, to a lesser extent, IgG can be secreted into
body fluid products where they protect against
bacterial colonization and invasion.
• IgA is present in particular abundance in colostrum but is also
present in subsequently produced milk.
Interventional Passive Immunity
• Passive immunization involves injection of specific
serum.
– Zoster hyper-immune serum (ZIG) for immunosuppressed
individuals who are infected with varicella-zoster virus.
– Lassa fever hyper-immune serum for individuals infected
with Lassa Fever virus.
– Anti-toxin for individuals who have been infected with
Clostridium tetani.
– Anti-venom for individuals who have been bitten by a
poisonous snake.
• Advantage:
– Provides instant immunity
• Disadvantages:
– Short-lived (weeks to months)
– If from another species, can cause serum sickness
Interventional Active Immunity
• Vaccines can be delivered in a number of forms.
• Inoculation with live, attenuated agent
– Viruses: measles, mumps, rubella, rotavirus, varicella, yellow fever
– Bacteria: tuberculosis (BCG)
• Inoculation with inactivated or killed agent
– Influenza virus, Salk polio, Hep A, rabies
– Bacteria: cholera, plague
• Inoculation with toxoid
– Diphtheria, tetanus
• Inoculation with subunit
– Hep B
– Pertussis
– Streptococcal pneumonia
• Inoculation with antigen conjugate
– Haemophilus influenzae type B
– Streptococcal pneumonia
The Antibody Response Is a Protective Response
That Shows Memory
Note: the memory response is primarily an IgG response.
Kuby Immunology
Charlie Reiser
Kidney function tests show impaired kidney
function.
Urinalysis shows high level of protein in urine.
Bone marrow aspiration and biopsy shows
increased numbers of plasma cells at various
stages of maturation.
What do these results suggest?
What would you do next?
Charlie Reiser
Serum protein electrophoresis shows a tall,
narrow, homogenous spike in the gamma
globulin region.
What does this result indicate?
Charlie Reiser
Charlie is diagnosed with multiple myeloma.
The prognosis is grim. It is likely that he will die of his
disease within 2-5 years, depending upon the stage of the
disease and its susceptibility to treatments.
A number of treatment options may extend life. These
include
Bortezomib- proteosome inhibitor
Lenalidomide- a derivative of thalidomide
Chemotherapy with melphalan, cyclophosphamide,
vincristine, doxorubicin,
Corticosteroids
Autologous stem-cell transplantation
Radiation therapy