Comparison of Different Immunoenzymatic Methods for the Determination of the Fine Specificity and Affinity Constants of Polyclonal Antibodies Against Pseudopeptide Haptens

Abstract

We evaluated two phosphinopeptides and one phosphonopeptide, which are transition state analogs of a proteolytic reaction, for their ability to generate murine polyclonal antibodies. The specificity of these antisera was determined by indirect and competitive ELISA. Cross-reactivity analysis by these ELISA showed that the antisera recognized selectively haptens containing a phosphate group. The pseudopeptides recognized by the antisera in the indirect ELISA were not the same, however, as those recognized in the competitive ELISA. The differences between these results are probably due to the presentation forms of the hapten, i.e., passively adsorbed in the former ELISA format and soluble in the latter. The affinity of the antibodies was then determined by using two methods based on competitive ELISA, one described by Friguet et al. and the other by Seligman. The dissociation constant (Kd) values calculated by the two methods, for an antiserum and its homologous hapten, are similar. However, only the middle portion of the inhibition scale in Seligman's method gave access to reliable values. Nevertheless, the Seligman representation allowed us to underscore the large range of affinity constants of the polyclonal antibodies.