An enzyme immunoassay for rat prolactin: application to the determination of plasma levels

Pure acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus has been covalently coupled to rat prolactin using the heterobifunctional reagent: N-succinimidyl-4 (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). This conjugate was used as a tracer in a competitive enzyme immunoassay using a rabbit antiserum, raised against rat prolactin, as first antibody. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody. This second antibody solid phase ensured separation of bound and free moieties of the tracer during the specific immunoreaction. The total reaction volume was 150 microliters. Each component (tracer, antiserum and standard) was added in a volume of 50 microliters. The sensitivity of the assay was good since calculation indicated a detection threshold of 25 pg (0.5 ng/ml) and a B/Bo 50% value of 220 pg (4.4 ng/ml). Intra-assay variation was better than 10% over a wide range (135 to 2500 pg) with an optimum of 4% at 300 pg. The inter-assay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 8 to 1000 ng/ml. The good parallelism observed between the standard curve and sample dilution curves, and recovery experiments, indicated that direct assay is possible. This was confirmed by molecular sieve fractionation of plasma samples. The reliability of the assay was confirmed by good correlation with conventional radioimmunoassay (r = 0.996, slope = 0.978).