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.
Hochhaus
G
G
Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143.
Sadée
W
W
D000818:Animals; D001360:Avidin; D001667:Binding, Competitive; D001710:Biotin; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay; D008297:Male; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D001615:beta-Endorphin / Q000032:analysis*
D002021:Buffers; D001360:Avidin; D001615:beta-Endorphin; D001710:Biotin