Production of highly specific polyclonal and monoclonal antibodies using estradiol-3-O-carboxymethyl ether as hapten

The preparation of high affinity and high specificity polyclonal and monoclonal antibodies to estradiol is described. Monoclonal antibodies were derived from BALB/c mice hyperimmunized with estradiol-3-O-carboxymethyl ether conjugated to bovine serum albumin. Spleen cells were hybridized with mouse myeloma cells. Quite a few monoclonal antibodies showed very good affinity for estradiol. Extended immunization and hyperimmunization were essential for producing a greater number of positive clones secreting high affinity antibodies. Binding constants of the antisera and their cross-reactivities with related steroids were calculated. Both polyclonal and monoclonal antibodies showed very high affinity for estradiol exhibiting little or no cross-reactivities with structurally related steroids indicating that this site of linkage is a good choice for discriminating between differences at the 16-17 position in the D-ring. This monoclonal antibody (44.28.6), having negligible cross-reactivity with estriol and estrone, can be used for diagnostic purposes.