A DIRECT NONCOMPETITIVE IDIOMETRIC ENZYME-IMMUNOASSAY FOR SERUM ESTRADIOL

We report a novel non-competitive enzyme immunoassay for oestradiol ba sed on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary anti -oestradiol idiotypic antibody (Ab(1)). The first anti-idiotype, the b etatype, competes with the analyte for an epitope of the primary antib ody at the binding site. On the other hand, the second anti-idiotype, the alphatype, binds to the Ab(1) in the presence of analyte but does not bind to the betatype/Ab(1) complex because of steric hindrance. In the present format the biotinylated alphatype was captured onto anti- biotin IgG which was adsorbed on the surface of microtitre wells. Reac tion mixtures containing the Ab(1) complexed sequentially with an enzy me labelled second antibody reagent, with oestradiol standards or seru m samples and with the betatype anti-idiotypic antibody were then allo wed to react with the immobilized alphatype anti-idiotypic antibody. T he enzyme activity of the bound fraction measured at 405 nm increased with increasing oestradiol concentrations over the range 0.06-2.5 ng/m l. The detection limit of the assay was 28 pg/ml. The intra-assay vari ation ranged from 3.5 to 12.4%, and inter-assay variation from 6 to 13 .4%. The results obtained by the colorimetric idiometric immunoassay c orrelated well with those obtained by a direct radioimmunoassay (n = 8 5, r = 0.97). This non-competitive immunoassay, termed idiometric assa y, for haptens permits the development of sensitive immunoassays with a wide working range, and a variety of end-point determinations depend ing on the label used (e.g., enzyme, chemiluminescent or fluorogenic c ompound).