A competitive enzyme-linked immunosorbent assay: Applications in the assay of peptides, steroids, and cyclic nucleotides

Indirect competitive enzyme-linked immunosorbent assays (ELISAs) that can be used to quantify several types of small, bioactive molecules, including peptides, steroids, and cyclic nucleotides, are described. The assays require no special expertise to perform, and the sensitivities are very high, equally or exceeding what is commonly achieved in radioimmunoassay (RIA). The molecule to be assayed or a synthetic derivative is coupled to a protein carrier (=conjugate). The conjugate is adsorbed to the wells of a microtiter plate where it is bound by antibody in inverse proportion to free hapten in a sample or standard. Bound antibody is then quantified with enzyme-labeled anti-immunoglobulin and appropriate substrate. The assay of peptides is illustrated for the sulfated cholecystokinin octapeptide, in which an ED 50 of 20 fmol (2 × 10 −10 m in 100 μl assay volume) is attained. The ED 50's and slopes of the dose-response curves in the steroid and cyclic nucleotide ELISAs are compared with those parameters obtained earlier by RIA using the same antisera. This comparison indicates that a steroid, ecdysone, can be quantified with no apparent participation of the bridging group of the conjugate in the competitive assay. Furthermore, the ED 50's in the ecdysone assays (ecdysone 2β, 3β, 14α, 22 R, 25-pentahydroxy-5β-cholest-7-en-6-one, 7.7 fmol; 20-hydroxyecdysone, 16 fmol) are 19- to 38-fold lower for ELISA than for RIA. In the cyclic nucleotide assay, the bridge of a cAMP conjugate (homologous with the bridge of the immunogen) decreases the slope of the dose-response curve. This effect is minimized by the use of short incubations with anti-cAMP. Under these conditions the specificity of the antiserum (previously determined by RIA) is retained, and the ED 50 is 14 fmol. Our approach to ELISA is validated by showing agreement in levels, obtained in parallel by ELISA and RIA, of cAMP content in extracts of forskolin-stimulated AtT-20 cells.