A Specific Homologous Radioimmunoassay for Human β-Endorphin: Direct Measurement in Biological Fluids*

Abstract

A highly sensitive and specific homologous RIA for direct measurement of human β-endorphin (βh-EP) has been developed. This RIA can detect as little as 5 pg/tube and cross-reacts with human β-lipotropin (βh-LPH) by only 5% on a molar basis and 2% by mass. Although the inhibition of binding of [125I]βh-EP in the RIA by βh-LPH was parallel to the inhibition curve produced by the addition of unlabeled βh-EP, the curves obtained by the addition of ovine and porcine β-endorphins and ovine β-LPH were not parallel to the βh-EP curve. This indicates that the antiserum against βh-EP recognizes different antigenic determinants in the βh-EP molecule compared to the corresponding animal peptides. Assay cross-reaction with ovine β-EP (10%), porcine β-EP (5%), and ovine β-LPH (16%) was small. No cross-reaction with 22 other peptides was found. Quantitative recovery of βh-EP was obtained after addition of the peptide to human plasma, cerebrospinal fluid, and amniotic fluid. Control experiments in which increasing quantities of βh-LPH up to 1.6 ng/ml were added to plasma revealed no significant alteration of β-EP immunoreactivity. The ability of the present RIA to discriminate between βh-EP and βh-LPH was further validated by column chromatography. The plasma concentrations of βh-EP in normal men and women determined by direct RIA were found to be 115 ± 9 pg/ml (n = 27) and 112 ± 5 pg/ml (n = 25), respectively. The βh-EP concentration in normal human plasma measured by direct RIA was comparable to that found after silicic acid extraction and gel chromatography (107 pg/ml). βh-EP concentrations in paired maternal (248 ± 28 pg/ml; n = 6) and cord (250 ± 54 pg/ml; n = 6) plasma were significantly higher (P < 0.05) than those in nonpregnant subjects. βh-EP concentration in term amniotic fluid was 104 ± 6 pg/ml (n = 6). The administration of metyrapone produced a rise in plasma βh-EP concentration of 37–155%. βh-EP was not detectable in cerebrospinal fluid samples obtained by lumbar puncture (n = 19). The present RIA system is sufficiently sensitive and specific to permit direct measurement of βh-EP in human biological fluids.