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as an opaque band in an agar plate assay. Agglutination only occurs when there is the

right amount of antibody and antigen present. It relies on the fact that as an antibody

has two binding sites then each of them can be bound to different antigen particles.

As this happens bridges are formed created by the antibody molecules spanning two

antigen molecules. The resulting lattice that is created forms a stable structure where

antigen and antibody particles are suspended in solution by their attachment to each

other. For this to take place there must be a precise amount of antigen and antibody

present and this is known asequivalence. If too much antibody is present then each

antigen molecule will bind multiple antibodies and the meshwork will not develop. If

too much antigen is present then each antibody will bind only one antigen particle

and no lattice will form. For this reason a dilution series of antibody is often made and

a measure of antigen concentration can be made from the end point at which

agglutination occurs.

Modifications of the agglutination reaction involve the use of antibody bound to

red blood cells or latex particles which allow the reaction to be observed more easily

in a liquid phase. Agglutination immunoassays are still used as they provide rapid

results with the minimum of equipment. They are commonly used for the detection of

viral antigens in blood serum. In commercial tests the antibody concentration in the

reagent is provided at a working dilution known to produce a positive for the normal

range of antigen concentration.

The Ouchterlony double diffusion agar plate method is the commonest gel-based

assay system used (Fig. 7.11). Wells are cut in an agar plate which is then used to load

samples and an antibody solution. The antigens and the antibody diffuse through the

agar and if the antibody recognises antigen within the gel then a precipitin band is

formed. A diffusion gradient is formed through the agar as the reagents progress and

so there is no requirement for dilutions to be made to ensure that agglutination occurs.

Antibody excess
no agglutination

Agglutination at point

of equivalence

Antigen excess

no agglutination

Fig. 7.10Agglutination reaction.

284 Immunochemical techniques