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 ED50 of 20 fmol (2 x 10(-10) M in 100 microliters assay volume) is attained. The ED50'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 ED50's in the ecdysone assays (ecdysone 2 beta, 3 beta, 14 alpha, 22R, 25-pentahydroxy-5 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
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Arizona Research Laboratories, University of Arizona, Tucson 85721.
D000818:Animals; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004440:Ecdysone / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006041:Goats; D008970:Molecular Weight; D009712:Nucleotides, Cyclic / Q000032:analysis; D010455:Peptides / Q000032:analysis; D011817:Rabbits; D011863:Radioimmunoassay; D012844:Sincalide / Q000032:analysis; D013256:Steroids / Q000032:analysis; D013552:Swine
D009712:Nucleotides, Cyclic; D010455:Peptides; D013256:Steroids; D004440:Ecdysone; D000242:Cyclic AMP; D012844:Sincalide