Last Update: 2 November 2017 P-I

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ANTIGEN-ANTIBODY INTERACTIONS

Antigen-antibody interactions are similar to enzymatic reactions in that they are reversible.

Nature of Ag-Ab 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 and antibodies
interacting shows that the antigenic determinant
nestles in a cleft formed by the combining site of
the antibody as illustrated in Figure 1. Thus, our
concept of Ag-Ab reactions is one of a key (i.e. the
Ag) which fits into a lock (i.e. the Ab).

B. Non-covalent Bonds - The bonds that hold the

Ag in the antibody combining site are all non-
covalent 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.
Fig.1
C. Reversible - Since Ag-Ab reactions occur via non-covalent bonds they are by their nature reversible.

Affinity and avidity

Every antibody displays an affinity for its antigen.

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
thecombining site of the antibody as illustrated in Figure 2.

Interactions between antigen and antibody involve non-covalent binding of an antigenic determinant (epitope) to the variable
region (complementarity determining region, CDR) of both the heavy and light immunoglobulin chains. These interactions
are analogous to those observed in enzyme-substrate interactions and they can be defined similarly. To describe the strength
of the antigen-antibody interaction, one can define the affinity constant (K) as shown:

[Ab - Ag]
Affinity K = = 104 to 1012 L/mol
[Ab] [Ag]

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 If the interaction between antigen and antibody were totally random, one would expect the concentrations of free
antigen, free antibody and bound Ag-Ab complex to all be equivalent. In other words,
1
Affinity K = = 100 L/mol
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Affinity is the strength of the total bonds between the antigen binding sites and the antigen.
Antibodies with low affinity bind their antigens weakly and disassociate easily. Antibodies with high affinity bind
their antigens stronger and do not disassociate easily.
Another term used to describe antigen-antibody interaction is AVIDITY.

Avidity is the strength of multiple interactions between antigen and antibody with multiple binding sites.

All antibodies have at least two antigen binding sites represented as their (Fab) 2. But we know that some antibodies
(IgM and IgA) exist in secreted form as a multi-antibody complex. Thus, pentameric IgM has 10 potential binding sites and
dimeric IgA has 4 potential binding sites, whereas IgG only has 2 binding sites. The sum of all the binding sites equals
avidity.
Avidity can make up for affinity. For example, IgM may have low affinity
but extremely high avidity.

SPECIFICITY AND CROSS REACTIVITY

A. 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 the primary structure of an antigen, isomeric forms
of an antigen, and secondary and tertiary structure of an antigen.

B. Cross reactivity :
Cross reactivity can occur when two (or more) antigens share similar structural features. Consider three different
antigens, as shown on the right. Antibody produced in response to Ag1 is very specific and would, therefore, have a large
affinity constant (K) when combining with Ag1. However, Ag2 is similar in shape to Ag1 and is capable of interacting with
anti-Ag1 antibody via two of three sites. The interaction between Ab and Ag2 is not as strong as the interaction between Ab
and Ag 1 (i.e. K is much smaller) but is still strong enough to allow binding. Hence, Ag1 and Ag2 are said to cross-react.
Ag3, in contrast, cannot interact very well with anti-Ag1 antibody and would have a K value so low that significant binding
would not occur. Ag3, therefore, would not cross-react with Ag1.
Crossreactivity also forms the basis for several diagnostic tests. For example, infection with Treponema pallidum
(syphilis) causes the production of antibodies that cross-react with a subsatnce found in cardiac muscle, cardiolipin. Since it
is much easier to obtain pure cardiolipin than pure Treponemal antigens, this cross-reaction is used to test for syphilis
(Wassermann test). Likewise, antibodies produced against certain Rickettsia cross-react with antigens from Proteus. Since
the latter are much easier to obtain, they can be used to test for the former.
Antibodies are antigen specific, but sometimes they are also cross-reactive. This means that they recognize
other antigens that look similar to the one for which they are specific.

Cross-reactivity occurs when 2 different antigens share the same or similar epitopes.
One good example is the Jenner small pox vaccine. Jenner used antigens from the cow pox virus to vaccinate against
small pox. This worked because the two viruses had similar or shared epitopes. Antibody responses to the cow pox virus
cross reacted with the small pox virus.

TYPES OF ANTIGEN-ANTIBODY INTERACTIONS

In vitro interactions between antigen and antibody (serological assays) are widely used for diagnostic purposes.
Immunoassays can detect serum antibodies to specific infectious agents and identify microorganisms in clinical specimens.
Antigen-antibody interactions can result in a variety of consequences, including precipitation of soluble antigens,
agglutination of particulate antigens, neutralization of toxins and viruses, and activation of complement.
Cross-linking of multiple antigen molecules by antibody is required for precipitation, agglutination or complement
fixation, and it is possible only if the antigen is multivalent and the antibody is at least bivalent [either intact or F(ab')2].
Described below are Ag-Ab reactions widely used in diagnosis. Many others, not included here, are largely variations of
these.

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I. Agglutination reactions
The reactions of antibody with a multivalent antigen that is particulate (i.e. an insoluble particle) results in the cross-
linking of the various antigen particles by antibodies. This cross-linking eventually results in the clumping (agglutination)
of the antigen particles by the antibodies.
When the antigen is an erythrocyte the term hemagglutinin is often used,and this event is called hemagglutination.

An agglutination reaction between a multideterminant, multivalent antigen and an antiserum containing antibodies
to determinants A, B and C.
Antigen and antibody must be present in the correct proportion for agglutination to occur. Agglutination will not
occur in reaction mixtures containing too much antibody (prozone effect) or too little antibody (postzone). The reciprocal of
the highest dilution to cause agglutination of the antigen particles is known as the antiserum's titer.
To agglutinate bacteria, erythrocytes or other cells in suspension, serial dilutions of antiserum are added to tubes or
wells of an assay plate containing known, constant amounts of particulate antigen. Positive reactions are visualized by the
formation of a mat-like latticework in the bottom of the tube or well. A negative reaction appears as a tight, round "button"
of particles.

DILUTION FACTOR

Diagrammatic representation of an hemagglutination

reaction performed in a microtiter plate. The objective of the
study is to determine the existence and titer of
hemagglutinating antibodies in three different samples. In the
first step, each sample is sequentially diluted from 1:10 to
1:20,480 in a separate row of wells. In the second step, a
fixed amount of red cells is added to each serum dilution. In the row containing saline, no agglutination can be seen, and
the sample is considered negative. In the row containing Patient A serum, the first three dilutions do not show agglutination
(prozone), but the next dilutions through 1:5120 are positive; the titer of this sample is 5120. In the row containing Patient
B serum, agglutination occurs through the 1:320 dilution; the titer of this sample is 320.

Zeta potential
Some particles, e.g. red blood cells, possess charges on their surfaces that prevent them from getting close enough together
for agglutination by IgG. This electrical repulsion is termed zeta potential. The IgG molecule is too short to overcome the
zeta potential that separates two negatively-charged red blood cells, but in the 1950's, Coombs devised a method for
circumventing this problem. He found that RBCs coated with IgG could be agglutinated by the addition of anti-IgG
antibodies. The anti-IgG antibodies cross-link
the IgG molecules on relatively distant
erythrocytes, across the separation induced by
the zeta potential, and cause agglutination.

Coombs test (Antiglobulin Test)

There are two versions of the Coombs
test (also called the anti-immunoglobulin test or
antiglobulin test).
The direct Coombs test is used test the
erythrocytes of babies suspected of having
erythroblastosis fetalis (hemolytic disease of
the newborn) for the presence of maternal anti-
RhIgG.

The indirect Coombs test is used to test the

serum of an Rh- woman for anti-Rh antibodies.

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a) Direct Coombs Test :
When antibodies bind to erythrocytes, they do not always result in agglutination. This can result from the Ag/Ab
ratio being in antigen excess or antibody excess or in some cases electrical charges on the red blood cells preventing the
effective cross linking of the cells. These antibodies that bind to but do not cause agglutination of red blood cells are
sometimes referred to as incomplete antibodies. In know way is this meant to indicate that the antibodies are different in their
structure, although this was once thought to be true. Rather it is functional definition only. In order to detect the presence of
non-agglutinating antibodies on red blood cells, one simply adds a second antibody directed against the immunoglobulin
(Ab) coating the red cells. This anti-immunoglobulin can now cross link the red blood cells and result in agglutination. This
test is illustrated in Figure 10 and is known as the Direct Coombs test.

c) Indirect Coombs Test :

If it is necessary to know whether a serum sample has antibodies directed against a particular red blood cell and you
want to be sure that you also detect potential non agglutinating antibodies in the sample, an Indirect Coombs test is
performed (Figure 11). This test is done by incubating the red blood cells with the serum sample, washing out any unbound
antibodies and then adding a second anti-immunoglobulin reagent to cross link the cells.

Natural isohemagglutinins (IgM) agglutinate red blood cells by binding to ABO blood group antigens on the
erythrocyte surface. IgM-Fab regions are far enough apart to overcome the zeta potential. This property, together with its
pentavalent structure, makes IgM a most effective agglutinating antibody.
ABO typing by natural isohemagglutinins is an example of direct agglutination
because the antigens are natural constituents of the particle being agglutinated. Agglutination of particles to which soluble
antigens have been artificially attached is referred to as passive agglutination. When red blood cells are used as the indicator
particles, the test is referred to as passive hemagglutination.

II. Precipitation reactions

Precipitin reaction in liquid

The precipitation reaction takes
place when antibodies and soluble antigens
are mixed in the correct proportions. The
lattice theory states that precipitation is a
result of the increase in size of antigen-
antibody aggregates such that each
molecule of antigen is linked to more than
one antibody molecule and vice versa.
When the aggregates exceed some critical
volume, they settle out of solution
(precipitate) spontaneously.

Fig. A precipitation curve for a system of

one antigen and its antibodies. The plot of
the amount of antibody precipitated versus
increasing antigen concentration (at
constant total antibody) reveals three
zones: a zone of antibody excess in which
precipitation is inhibited and excess antibody can be detected in the supernatant; an equivalence zone of maximal
precipitation in which antibody and antigen form large insoluble complexes (shaded in blue) and neither antibody nor
antigen can be detected in the supernatant; and a zone of antigen excess in which precipitation is inhibited and excess
antigen can be detected in the supernatant.
Precipitation reactions can be performed in either liquids or gels. If performed in liquid, the assay is called the
precipitin test. The precipitin test is performed by adding increasing amounts of antigen to a series of tubes containing a
constant volume of antiserum. The amount of protein precipitated increases with the amount of antigen added, to a
maximum beyond which larger amounts of antigen lead to progressively less precipitation. In the equivalence zone, a
maximal amount of antibody is precipitated by an optimal amount of antigen. Like agglutination reactions, precipitation
reactions are subject to the prozone and postzone effects.
B. Precipitation reactions in gels
When soluble antigen and antibodies are placed in wells cut in an agar
gel, the reactants diffuse in the gel and form gradients of concentration, with
the highest concentrations closest to the well. Somewhere between the two
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wells, the reacting antigen and antibodies will be present at proportions that are optimal for formation of a precipitate
(equivalence). This assay is called the Ouchterlony or immuno-double diffusion technique.

I. Ouchterlony or immuno-double diffusion technique:

POSSIBLE TYPES OF INTERACTIONS

(A). Test of identity
Here in Figure (a) the two antigen wells have the same antigen and antibody cannot diffuse through the line of
precipitation.
(B) Test of non-identity
Here in figure (b) there are two different antigens and the antibodies in the center well react with both, but the
independence of the two lines indicates that the two antigens share not common epitopes.
(C). Test of partial identity
Here in figure(c) there is a line of identity in the same curved shape as seen in (a) and in addition a "spur" linked to
the left well which shows there is an antibody that can react with the left and NOT the right.

II. The radial immunodiffusion (RID) or Mancini test :

The radial immunodiffusion (RID) test is a variation of the immuno-double-diffusion test. In radial
immunodiffusion antibody isincorporated into the agar gel as it is poured and different dilutions of the antigenare placed in
holes punched into the agar. The wells contain antigen at different concentrations, while the antibodies are distributed
uniformly in the agar gel. Thus, a precipitin ring around the well replaces the precipitin line. The diameter of the precipitin
ring is directly proportional to the concentration of the antigen in the well. From the results obtained with known
concentrations, a calibration curve is constructed, permitting quantitation of the Ag concentration.
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III. Immunoelectrophoresis
In immunoelectrophoresis a complex mixture of antigens is placed in a well punched out of an agar gel and the
antigens are electrophoresed so that the antigen are separated according to their charge. After lectrophoresis a trough is cut in
the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone
when an Ag/Ab reaction occurs as illustrated in Figure 14.

This tests is used for the qualitative analysis of complex mixtures of antigens, although a crude measure of quantity
(thickness of the line) can be obtained. This test is commonly used for the analysis of components in a patient' serum. Serum
is placed in the well and antibody to whole serum in the trough. By comparisons to normal serum one can determine whether
there are deficiencies on one or more serum components or whether there is an overabundance of some serum component
(thickness of the line). This test can also be used to evaluate purity of isolated serum proteins.
Immunoelectrophoresis is used to separate antigens in complex mixtures on the basis of their charges in an electrical
field. Antigens are separated in an agar gel by applying an electric charge. The pH is chosen so that positively charged
proteins move toward the negative electrode and negatively charged proteins move toward the positive electrode. A
trough is then cut between the wells and filled with antibody, which is allowed to diffuse. The antigens and antibodies form
precipitin arcs.
This method is often used to characterize human serum proteins.

Countercurrent electrophoresis - In this test the antigen and antibody are placedin wells punched out of an agar gel
and the antigen and antibody are electrophoresed into each other where they form a precipitation line as illustrated in fig. 15

This test only works if conditions

can be found where the antigen and antibody
have opposite charges. This test is primarily
qualitative, although from the thickness of
the band you can get some measure of quantity. It's major advantage
is it's speed.

Ii. Solid phase immunoassays

Solid phase immunoassays are the most sensitive and widely-
used serological assays. In these assays, a solid supportsuch as
plastic or nitrocellulose paperis used to immobilize the reactants.
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A good example of a solid phase assay is the home pregnancy test. An antibody to human chorionic gonadotropin (HCG) is
adsorbed onto a nitrocellulose disc (1). Pregnant womans urine thought to contain HCG is passed through the filter, and, if
present, attaches to the antibody on the disc (2). Next, a second antibody to HCGthis one conjugated to an enzymeis
passed through the filter and attaches to the HCG antigen immobilized by the first antibody (3). During the final step, a
colorless substrate solution is applied to the filter (4). A color change catalyzed by the enzyme attached to the second
antibody indicates that HCG is present in the urine and that the patient is pregnant (5). Conversely, if no color appears, it
must be concluded that the woman is not pregnant, since no enzyme-linked second antibody, and hence no HCG, became
attached to the nitrocellulose. A solid phase immunoassay such as this one, which employs a second antibody or other
indicator ligand linked to an enzyme, is termed an enzyme immunoassay (EIA). When the test is performed in a plastic
micro titer plate, it can also be termed an enzyme-linked immunosorbent assay (ELISA). The colored products produced in
an ELISA can be measured spectrophotometrically. In a radioimmunoassay (RIA), the enzyme label is replaced by a
radioisotope, and binding between reactants can be measured in a scintillation counter. There are many variations of the
solid phase immunoassay. A few are listed below:
A. Double-antibody (sandwich, two-site capture) assay: Employs a "capture" antibody to pull the antigen out of a
complex solution, and a second antibody linked to an enzyme or radioisotope to detect the captured antigen. The home
pregnancy test is an example.
B. Direct assay: Antigen directly adsorbed onto the solid phase is detected by a labeled antibody specific for the antigen.
C. Indirect assay: Antigen directly adsorbed onto the solid phase is first incubated with patient serum, and then with a
labeled antibody specific for human immunoglobulin.
D. Competition assay: Patient serum is mixed with known concentrations of antigen before adding the serum to an
antigen-coated microtiter plate. As the patient's antibodies bind to increasing amounts of the soluble antigen, the amount
of antibodies available to bind to the plastic-adsorbed antigen decreases. This results in a corresponding decrease in the
amount of labeled anti-human antibody bound to the plate.
E. In a western blot or immunoblot, antigens are separated according to molecular mass by polyacrylamide gel
electrophoresis and then transferred electrophoretically to a nitrocellulose membrane. The blot is incubated with patient
antiserum, followed by labeled goat antibody specific for human immunoglobulins. If the goat antibody has an enzyme
label, reactive antigen bands are detected with a precipitable, colored substrate. If the goat antibody is conjugated to a
radioisotope, reactive bands are visualized by exposing the blot to X-ray film. This is the only technique that identifies an
antigen based on its molecular weight.

Radioimmunoassay (RIA)/Enzyme Linked Immunosorbent Assay (ELISA)

Radioimmunoassays (RIA) are assays which 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.

1. Competitive RIA/ELISA for Ag Detection

The method and principle of RIA and ELISA for
the measurement of antigen is shown in Figure 16. By
using known
amounts of a standard unlabeled antigen one can generate
a standard curve relating cpm (Enzyme) bound vs amount
of antigen. From this standard curve one can determine
the amount of an antigen in an unknown sample.

The key to the assay is the separation of the

immune complexes from the remainder of the
components. This has been accomplished in many
different ways and serves as the basis for the names given
to the assay:

1) Precipitation with ammonium sulphate

Ammonium sulphate (33-50% final concentration)
will precipitate immunoglobulins but not many antigen.
Thus, this can be used to separate the immune complexes
from free antigen. This has been called the Farr
Technique
2) Anti-immunoglobulin antibody
The addition of a second antibody directed against the first antibody can result in the precipitation of the immune complexes
and thus the separation of the complexes from free antigen.
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3) Immobilization of the Antibody
The antibody can be immobilized onto the surface of a plastic bead or coated onto the surface of a plastic plate and
thus the immune complexes can easily be separated from the other components by simply washing the beads or plate (Figure
17). This is the most common method used today and is referred to as Solid phase RIA or ELISA. In the clinical laboratory
competitive RIA and ELISA are commonly used to quantitate serum proteins, hormones, drugs metabolites.

2. Noncompetitive RIA/ELISA for Ag or Ab:

Noncompetitive RIA and ELISAs are also used for the measurement of antigens and antibodies. In Figure 18 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. 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 Figure 19 the 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.

The table below indicates the lower limits of sensitivity of the assays we have discussed.

METHOD ANTIBODY CONCENTRATION (G/ML)

Precipitin reactions in liquid media 20
Precipitation reactions in agar gel 0.05-20
Complement fixation 0.5
Bacterial agglutination 0.01
Passive hemagglutination 0.01
ELISA, EIA, RIA, immunoblot 0.001-0.00001

Epitopes , paratopes (idiotopes ) and agretopes:

The portion of an antigen that binds specifically with the binding site of an antibody or TCR is termed an antigenic
determinant or epitope. Most epitopes are equivalent to 5-7 amino acids (B cell epitopes) or 10-15 amino acids (T cell
epitopes). Antigens may be composed of one or several different epitopes. The part of the antibody molecule that binds to
the epitope is called the idiotope or paratope. On T cells, the recognition site which binds to antigens is termed the agretope
which is located on the MHC molecule

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