A direct non-competitive idiometric enzyme immunoassay for serum oestradiol

We report a novel non-competitive enzyme immunoassay for oestradiol based 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 (Ab1). The first anti-idiotype, the betatype, competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the second anti-idiotype, the alphatype, binds to the Ab1 in the presence of analyte but does not bind to the betatype/Ab1 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. Reaction mixtures containing the Ab1 complexed sequentially with an enzyme labelled second antibody reagent, with oestradiol standards or serum samples and with the betatype anti-idiotypic antibody were then allowed to react with the immobilized alphatype anti-idiotypic antibody. The enzyme activity of the bound fraction measured at 405 nm increased with increasing oestradiol concentrations over the range 0.06-2.5 ng/ml. The detection limit of the assay was 28 pg/ml. The intra-assay variation ranged from 3.5 to 12.4%, and inter-assay variation from 6 to 13.4%. The results obtained by the colorimetric idiometric immunoassay correlated well with those obtained by a direct radioimmunoassay (n = 85, r = 0.97). This non-competitive immunoassay, termed idiometric assay, for haptens permits the development of sensitive immunoassays with a wide working range, and a variety of end-point determinations depending on the label used (e.g., enzyme, chemiluminescent or fluorogenic compound).