The specificity of immunoassays can be improved by using a second antiserum to bind substances that cross-react. Both theory and practice show that the effectiveness of the procedure is dependent of the complex interplay of the concentrations of the two antibodies, the concentrations of the three antigens involved (the labelled tracer, the antigen whose concentration is to be measured and the substance that cross reacts) and the affinities of the antibodies for these antigens. Measured cross-reactions can frequently be reduced to zero in the most important concentration ranges thereby enabling one to perform assays upon unpurified materials which other wise would not be possible. Limitations of the method are discussed.
Practical aspects of the measurement of the specificity of immunoassay are reviewed. Antibody heterogeneity in an antiserum makes a pragmatic rather than a theoretical approach necessary. A new method for the measurement of immunoassay specificity is described. This method is based on the errors caused by the cross-reacting antigens and is directly relevant to the validity of results obtained by immunoassay methods. The effect of selectively blocking the least specific antibodies in antisera raised against steroid haptens is tested. The practical consequences of these considerations are tested using steroid radioimmunoassay and enzyme-immunoassay.
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