Idiometric assay: noncompetitive immunoassay for small molecules typified by the measurement of estradiol in serum.

We report here a novel noncompetitive immunoassay applicable to the measurement of small-molecular-mass compounds and typified by the direct measurement of estradiol in serum. Two types of anti-idiotypic antibody recognize different epitopes within the hypervariable region of the specific primary antibody (e.g., anti-estradiol). The first anti-idiotype (betatype) recognizes an epitope at the unoccupied binding site, which is masked in the presence of the analyte (e.g., estradiol). The second (alphatype) recognizes an epitope close to the binding site and is unaffected by the presence or absence of the analyte, but is sterically hindered from binding to the primary antibody in the presence of the betatype. The use of these matched antibodies (primary antibody, alphatype, and betatype) has enabled the development of a method for determining antibody occupancy that is not based on a conventional two-site assay. An excess amount of purified monoclonal antibody against estradiol is immobilized onto the walls of microtiter wells. After the capture of analyte, the unoccupied antibody sites are blocked by the addition of an excess amount of betatype. Subsequently, analyte occupancy is determined by the addition of excess europium-labeled alphatype, incubation, washing, and time-resolved fluorometry. The method demonstrates good sensitivity, precision, and comparability with alternative competitive immunoassays.