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M - 10
ANTIGEN-ANTIBODY INTERACTIONS
Antigen-antibody interactions are similar to enzymatic reactions in that they are reversible.
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.
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.
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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
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.
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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.
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.
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
The table below indicates the lower limits of sensitivity of the assays we have discussed.
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