A biotin-avidin-based enzyme immunoassay for beta h-endorphin.

An avidin-biotin enzyme-linked immunosorbent assay (ELISA) is described for beta h-endorphin (beta h-EP). Microtiter plates coated with commercially available antibodies were used together with beta h-EP tracer derivatives that were biotinylated in positions 24, 28, and 29 via a C6 spacer arm. Nonspecific binding of biotinylated derivatives to the microtiter plates was blocked with a mixture of 1% casein and 10% ethanolamine in 0.1 M NaHCO3. A sequential saturation procedure using a high-affinity antiserum in combination with an avidin-alkaline phosphatase complex matched the sensitivity of reported radioimmunoassays (RIAs), with a detection limit of 0.5 fmol/assay. The intra- and interassay coefficients of variation were 5 and 12%, respectively. Results obtained by ELISA and RIA showed good correlations (r = 0.95). The beta-EP concentration in extracted rat plasma after high-performance liquid chromatographic (HPLC) fractionation was determined by this method to be 1600 fmol/mol.