The interaction between antibody and a particulate antigen results in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Agglutination reactions are similar in principle to precipitation reactions; they depend on the crosslinking of polyvalent antigens. Just as an excess of antibody inhibits precipitation reactions, such excess can also inhibit agglutination reactions; this inhibition is called the prozone effect. Because prozone effects can be encountered in many types of immunoassays, understanding the basis of this phenomenon is of general importance.
Several mechanisms can cause the prozone effect. First, at high antibody concentrations, the number of antibody binding sites may greatly exceed the number of epitopes. As a result, most antibodies bind antigen only univalently instead of multivalently. Antibodies that bind univalently cannot crosslink one antigen to another. Prozone effects are readily diagnosed by performing the assay at a variety of antibody (or antigen) concentrations. As one dilutes to an optimum antibody concentration, one sees higher levels of agglutination or whatever parameter is measured in the assay being used. When one is using polyclonal antibodies, the prozone effect can also occur for another reason. The antiserum may contain high concentrations of antibodies that bind to the antigen but do not induce agglutination; these antibodies, called incomplete antibodies, are often of the IgG class. At high concentrations of IgG, incomplete antibodies may occupy most of the antigenic sites, thus blocking access by IgM, which is a good agglutinin. This effect is not seen with agglutinating monoclonal antibodies. The lack of agglutinating activity of an incomplete antibody may be due to restricted flexibility in the hinge region, making it difficult for the antibody to assume the required angle for optimal cross-linking of epitopes on two or more particulate antigens. Alternatively, the density of epitope distribution or the location of some epitopes in deep pockets of a particulate antigen may make it difficult for the antibodies specific for these epitopes to agglutinate certain particulate antigens. When feasible, the solution to both of these problems is to try different antibodies that may react with other epitopes of the antigen that do not present these limitations.
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