Monoclonal antibodies used in solid-phase and liquid-phase assays, as exemplified by progesterone assay.
Two immunoglobulins secreted by hybridoma cell lines have been systematically investigated to determine if they could be used in solid-phase assays to give results comparable with those obtained by conventional liquid-phase radioimmunoassay (RIA). The antibodies, BQ.1 and BQ.2, bind with high specificity to the steroid hormone progesterone. The affinity constants, Ka, to 125I-labeled progesterone derivatives are 1.1 X 10(11) L/mol and 9.1 X 10(9) L/mol, respectively. Progesterone inhibited the binding of radioiodinated derivatives (amides of tyramine, histamine, and tyrosine methylester with 11 alpha-progesterone hemisuccinate) equally well. For solid-phase assays, we immobilized antibody BQ.1 via Protein A to different polystyrene surfaces (about 30 pg per tube at 50% inhibition of radiolabeled tracer). Under these conditions, the performance of this antibody for the quantification of progesterone was equivalent to that obtained in RIA. For the immobilized antibody BQ.2, only 1/10 of the amount used for optimal results in RIA was required in solid-phase assays. Binding of either antibody to the antigen was undiminished after several freezing-thawing cycles. When immobilized on solid matrices, both antibodies retained up to 95% of their binding properties for one year. Thus high-affinity, high-specificity monoclonal antibodies can be obtained for haptens and, when suitably immobilized, can be used in solid-phase assays with results equal to or better than those obtained with liquid RIA.
D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000917:Antibody Formation; D000937:Antigen-Antibody Reactions; D002846:Chromatography, Affinity; D003429:Cross Reactions; D006801:Humans; D011374:Progesterone / Q000097:blood* / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods; D013696:Temperature
D000911:Antibodies, Monoclonal; D011374:Progesterone