Immunology
TEACHING OBJECTIVES
To describe the basis of cellular defense
To describe the nature of antigen-antibody reactions
To compare and contrast antibody affinity and avidity
To delineate the basis for antibody specificity and cross reactivity
To discuss the principles of commonly used tests for antigen/antibody
reactions
Types of Immuity
Non-specific (innate) Immunity
 Body’s response is effective against a variety
of “attackers”
Involves antimicrobial cells and proteins
•Specific (acquired) Immunity
Body’s response is tailored for a specific
“attacker”
Involves antibodies
Non-Specific Immunity
 Mediated by host cells
Phagocytosis (by phagocytes)
Non-phagocytic cells
 Mediated by host proteins
Complement system
Interferons
Each of these play a role following a microbial
infection and/or a wound to tissue.
Non-specific Immunity-Phagocytosis
Phagocytosis Def: Ingestion of infecting
microbes by phagocytic white blood cells
(i.e., leukocytes)
Neutrophils – short-lived; 60-70% of
leukocytes
Macrophages – long-lived; develop from
monocytes
Phagocytosis
Non-phagocytic Cells
Killing is by means other than phagocytosis
 Eosinophils or Eosinophil granulocytes, usually called
eosinophils (or, less commonly, acidophils), are white blood
cells that are one of the immune system components
responsible for combating infection and parasites in
vertebrates.
•Attach to parasite and
discharge destructive
enzymes
•Along with mast cells, they also
control mechanisms associated
with allergy and asthma. They
are granulocytes that develop
during Haematopoiesis in the
bone marrow before migrating
into blood.
Non-phagocytic Cells
Natural Killer (NK) Cells – destroy infected
cells or precancerous cells by destroying the
cell membrane
 a type of cytotoxic lymphocyte
that constitute a major
component of the innate immune
system.
 NK cells play a major role in the
rejection of tumors and cells
infected by viruses.
 The cells kill by releasing small
cytoplasmic granules of proteins
called perforin and granzyme that
cause the target cell to die by
apoptosis or necrosis.
Interferons
 Interferons (IFNs) are natural proteins produced by the
cells of the immune system of most vertebrates in
response to challenges by foreign agents such as
viruses, parasites and tumor cells.
 Interferons belong to the large class of glycoproteins
known as cytokines.
 Interferons are produced by a wide variety of cells in
response to the presence of double-stranded RNA, a key
indicator of viral infection.
 Interferons assist the immune response by inhibiting viral
replication within host cells, activating natural killer cells
and macrophages, increasing antigen presentation to
lymphocytes, and inducing the resistance of host cells to
viral infection.
Specific Immunity - Antibody-Antigen Interaction
Antigen - any agent
capable of eliciting an
immune response
Isolated molecules
Molecules on surface of
cell or virus
A specific antibody
molecule will be able to
recognize a specific
epitope of an antigen
Antibody binds to antigen
Antibody Structure
 There are 5 classes of human antibodies: IgG,
IgM, IgA, IgD, and IgE. The simplest antibodies,
such as IgG, IgD, and IgE, are "Y"-shaped
macromolecules called monomers .
 A monomer is composed of four glycoprotein
chains: two identical heavy chains and two
identical light chains . The two heavy chains
have a high molecular weight that varies with the
class of antibody.
 The four glycoprotein chains are connected to
one another by disulfide (S-S) bonds and
noncovalent bonds.
Antibody Structure
 The Fab portion of the
antibody has specificity for
binding an epitope of an
antigen. The Fc portion
directs the biological activity
of the antibody.
 Epitope: that part of an
antigenic molecule to which
the T-cell receptor responds,
a site on a large molecule
against which an antibody will
be produced and to which it
will bind.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
 A. Lock and Key Concept - The combining site
of an antibody is located in the Fab portion of the
molecule and is constructed from the
hypervariable regions of the heavy and light
chains.
 X-Ray crystallography studies of antigens (Ag)
and antibodies (Ab) interacting shows that the
antigenic determinant nestles in a cleft formed
by the combining site. Thus, our concept of AgAb reactions is one of a key (i.e. the Ag) which
fits into a lock (i.e. the Ab).
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
 B. Non-covalent Bonds - The bonds that hold
the Ag in the antibody combining site are all noncovalent in nature. These include hydrogen
bonds, electrostatic bonds, Van der Waals forces
and hydrophobic bonds. Multiple bonding
between the Ag and the Ab ensures that the Ag
will be bound tightly to the Ab.
 C. Reversible - Since Ag-Ab reactions occur via
non-covalent bonds, they are by their nature
reversible.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
Antibody Structure
 Additional S-S bonds fold the individual glycoprotein
chains into a number of distinct globular domains. The
area where the top of the "Y" joins the bottom is called the
hinge. This area is flexible to enable the antibody to bind
to pairs of epitopes various distances apart on an antigen.
 The two tips of the "Y" monomer are referred to as the
Fab portions of the antibody. The amino acid
sequence in the tips of the "Y" varies greatly among
different antibodies. This variable region, composed of
110-130 amino acids, give the antibody its specificity
for binding antigen. The variable region includes the
ends of the light and heavy chains.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
Antibody Structure
Treating the antibody with a protease can
cleave this region, producing Fab or
fragment antigen binding that include
the variable ends of an antibody.
The first 110 amino acids or first domain of
both the heavy and light chain of the Fab
region of the antibody provide specificity
for binding an epitope on an antigen.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
Antibody Structure
 The amino acid sequence of the first domain of
both the light chain and the heavy chain shows
tremendous variation from antibody to antibody and
constitutes the variable domains of the antibody.
 This is because each B-lymphocyte, early in its
development, becomes genetically programmed
through a series of gene-splicing reactions to
produce a Fab with a unique 3-dimensional shape
capable of fitting some epitope with a
corresponding shape.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
 Figure 2
 The Fab portion of the
antibody has the
complementarity-determining
regions (red) providing
specificity for binding an
epitope of an antigen. The Fc
portion (purple) directs the
biological activity of the
antibody. (S-S = disulfide
bond; N = amino terminal of
glycoprotein; C = carboxy
terminal of glycoprotein;
CHO = carbohydrate.)
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
 The various genes the cell splices together determine the
order of amino acids of the Fab portion of both the light and
heavy chain; the amino acid sequence determines the final
3-dimensional shape.
 Therefore, different antibody molecules produced by
different B-lymphocytes will have different orders of amino
acids at the tips of the Fab to give them unique shapes for
binding epitope.
 The antigen-binding site is large enough to hold an epitope
of about 5-7 amino acids or 3-4 sugar residues.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
Figure 3
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
 The bottom part of the "Y", the C terminal region of
each glycoprotein chain, is called the Fc portion.
The Fc portion, as well as one domain of both the
heavy and light chain of the Fab region has a
constant amino acid sequence that defines the
class and subclass of each antibody. The Fc portion
is responsible for the biological activity of the
antibody.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
AFFINITY AND AVIDITY
 A. Affinity - Antibody affinity is the strength of
the reaction between a single antigenic
determinant and a single combining site on the
antibody. It is the sum of the attractive and
repulsive forces operating between the antigenic
determinant and the combining site of the
antibody as illustrated in Figure 4.
Affinity is the equilibrium constant that describes the
Ag-Ab reaction as illustrated in Figure 5. Most
antibodies have a high affinity for their antigens.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
 B. Avidity - Avidity is a measure of the overall
strength of binding of an antigen with many
antigenic determinants and multivalent
antibodies.
Affinity refers to the strength of binding between a
single antigenic determinant and an individual
antibody combining site whereas avidity refers to
the overall strength of binding between multivalent
antigens and antibodies.
Avidity is influenced by both the valence of the antibody
and the valence of the antigen. Avidity is more than the
sum of the individual affinities. This is illustrated in the
on the next page.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
SPECIFICITY AND CROSS REACTIVITY
 Specificity - Specificity refers to the ability of an
individual antibody combining site to react with only
one antigenic determinant or the ability of a
population of antibody molecules to react with only
one antigen.
 In general, there is a high degree of specificity in
Ag-Ab reactions. Antibodies can distinguish
differences in
 1) the primary structure of an antigen
 2) isomeric forms of an antigen
 3) secondary and tertiary structure of an antigen.
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
 Cross reactivity - Cross reactivity refers to the ability of an individual
antibody combining site to react with more than one antigenic
determinant or the ability of a population of antibody molecules to react
with more than one antigen. The figure below illustrates how cross
reactions can arise. Cross reactions arise because the cross reacting
antigen shares an epitope in common with the immunizing antigen or
because it has an epitope which is structurally similar to one on the
immunizing antigen (multispecificity).
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Factors affecting measurement of Ag/Ab
reactions
The only way that one knows that an antigenantibody reaction has occurred is to have
some means of directly or indirectly
detecting the complexes formed between
the antigen and antibody. The ease with
which one can detect antigen-antibody
reactions will depend on a number of
factors.
1. Affinity - The higher the affinity of the
antibody for the antigen, the more stable
will be the interaction. Thus, the ease with
which one can detect the interaction is
enhanced.
2. Avidity - Reactions between multivalent
antigens and multivalent antibodies are
more stable and thus easier to
detect. Figure 6
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
3. Ag:Ab ratio - The ratio between the antigen and antibody influences the
detection of Ag/Ab complexes because the sizes of the complexes
formed is related to the concentration of the antigen and antibody. This
is depicted in Figure 6.
4. Physical form of the antigen - The physical form of the antigen
influences how one detects its reaction with an antibody. If the antigen
is a particulate, one generally looks for agglutination of the antigen by
the antibody. If the antigen is soluble one generally looks for the
precipitation of the antigen after the production of large insoluble Ag/Ab
complexes. Figure 7
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Agglutination Tests
 Agglutination/Hemagglutination - When the antigen is
particulate the reaction of an antibody with the antigen
can be detected by agglutination (clumping) of the
antigen. When the antigen is an erythrocyte the term
hemagglutination is used.
 The term agglutinin is used to describe antibodies that
agglutinate particulate antigens. When the antigen is an
erythrocyte the term hemagglutinin is often used. All
antibodies can theoretically agglutinate particulate
antigens but IgM due to its high valence is particularly
good agglutinin and one sometimes infers that an
antibody may be of the IgM class if it is a good
agglutinating antibody.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Agglutination Tests
 When the antigen is particulate, the reaction of an antibody
with the antigen can be detected by agglutination (clumping)
of the antigen. The general term agglutinin is used to
describe antibodies that agglutinate particulate antigens.
 When the antigen is an erythrocyte the term
hemagglutination is used. All antibodies can theoretically
agglutinate particulate antigens but IgM, due to its high
valence, is particularly good agglutinin and one sometimes
infers that an antibody may be of the IgM class if it is a good
agglutinating antibody.
 Qualitative agglutination test - Agglutination tests can be
used in a qualitative manner to assay for the presence of an
antigen or an antibody. The antibody is mixed with the
particulate antigen and a positive test is indicated by the
agglutination of the particulate antigen.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
For example, a patient's red blood cells can
be mixed with antibody to a blood group
antigen to determine a person's blood type.
In a second example, a patient's serum is
mixed with red blood cells of a known blood
type to assay for the presence of antibodies
to that blood type in the patient's serum.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Quantitative agglutination test
 Agglutination tests can also be used to measure the
level of antibodies to particulate antigens. In this
test, serial dilutions are made of a sample to be
tested for antibody and then a fixed number of red
blood cells or bacteria or other such particulate
antigen is added.
 Then the maximum dilution that gives agglutination
is determined. The maximum dilution that gives
visible agglutination is called the titer. The results
are reported as the reciprocal of the maximal
dilution that gives visible agglutination. Figure 8
illustrates a quantitative hemagglutination test.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
What are Pos and Neg?
What does titer mean?
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Pos and Neg are controls
Titer – measure of concentration
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Radioimmunoassay (RIA)/Enzyme Linked
Immunosorbent Assay (ELISA)
 Radioimmunoassays (RIA) are assays that are based on
the measurement of radioactivity associated with
immune complexes.
 In any particular test, the label may be on either the antigen or
the antibody.
 Enzyme Linked Immunosorbent Assays (ELISA) are
those that are based on the measurement of an
enzymatic reaction associated with immune complexes.
 In any particular assay, the enzyme may be linked to either the
antigen or the antibody.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Competitive RIA/ELISA for Ag Detection
 The method and principle of RIA and ELISA for
the measurement of antigen is shown below.
 By using known amounts of a
standard unlabeled antigen,
one can generate a standard
curve relating radioactivity
(cpm) (Enzyme) bound versus
amount of antigen.
 From this standard curve, one
can determine the amount of
an antigen in an unknown
sample.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Noncompetitive RIA/ELISA for Ag or Ab
 Noncompetitive RIA and ELISAs are also used for the
measurement of antigens and antibodies.
 As seen in the figure below, the bead is coated with the
antigen and is used for the detection of antibody in the
unknown sample. The amount of labeled second
antibody bound is related to the amount of antibody in
the unknown sample.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Noncompetitive RIA/ELISA for Ag or Ab
 This assay is commonly employed for the measurement of
antibodies of the IgE class directed against particular allergens by
using a known allergen as antigen and anti-IgE antibodies as the
labeled reagent.
 It is called the RAST test
(radioallergosorbent test). In the figure at
right, a bead is coated with antibody and
is used to measure an unknown antigen.
 The amount of labeled second antibody
that binds is proportional to the amount of
antigen that bound to the first antibody
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Flow Cytometry
 Flow cytometry is commonly used in the clinical laboratory to identify
and enumerate cells bearing a particular antigen. Cells in
suspension are labeled with a fluorescent tag by either direct or
indirect immunofluorescence. The cells are then analyzed on the
flow cytometer.
 The figure at right illustrates the principle
of flow cytometry. In a flow cytometer,
the cells exit a flow cell and are
illuminated with a laser beam. The
amount of laser light that is scattered off
the cells as they passes through the
laser can be measured, which gives
information concerning the size of the
cells. In addition, the laser can excite the
fluorochrome on the cells and the
fluorescent light emitted by the cells can
be measured by one or more detectors.
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
 The type of data that is obtained from the flow cytometer is shown
below.
 In a one parameter histogram, increasing amount of fluorescence
(e.g. green fluorescence) is plotted on the x axis and the number of
cells exhibiting that amount of fluorescence is plotted on the y axis.
 The fraction of cells that are
fluorescent can be
determined by integrating
the area under the curve.
 In a two parameter
histogram, the x axis is one
parameter (e.g. red
fluorescence) and the y
axis is the second
parameter (e.g. green
fluorescence). The number
of cells is indicated by the
contour and the intensity of
the color.
Immunology
Information taken from the following
websites:
 http://pathmicro.med.sc.edu/mayer/ab-ag-rx.htm
 http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/
antibody/structure.html
 http://www.hhmi.org/biointeractive/vlabs/immunology/ind
ex.html (this last site is a virtual ELISA activity)