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

Abstract

This RIA [radioimmunoassay] can detect as little as 5 pg/tube and cross-reacts with human .beta.-lipotropin (.beta.h-LPH) by only 5% on a molar basis and 2% by mass. Although the inhibition of binding of [125I].beta.h-EP [human .beta.-endorphin] in the RIA by .beta.h-LPH was parallel to the inhibition curve produced by the addition of unlabeled .beta.h-EP, the curves obtained by the addition of ovine and porcine .beta.-endorphins and ovine .beta.-LPH were not parallel to the .beta.h-EP curve. The antiserum against .beta.h-EP recognizes different antigenic determinants in the .beta.h-EP molecule compared to the corresponding animal peptides. Assay cross-reaction with ovine .beta.-EP (10%), porcine .beta.-EP (5%), and ovine .beta.-LPH (16%) was small. No cross-reaction with 22 other peptides was found. Quantitative recovery of .beta.h-EP was obtained after addition of the peptide to human plasma, CSF and amniotic fluid. Control experiments in which increasing quantities of .beta.h-LPH up to 1.6 ng/ml were added to plasma revealed no significant alteration of .beta.-EP immunoreactivity. The ability of the present RIA to discriminate between .beta.h-EP and .beta.h-LPH was further validated by column chromatography. The plasma concentrations of .beta.h-EP in normal men and women determined by direct RIA were 115 .+-. 9 pg/ml (n = 27) and 112 .+-. 5 pg/ml (n = 25), respectively. The .beta.h-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). .beta.h-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. .beta.h-EP concentration in term amniotic fluid was 104 .+-. 6 pg/ml (n = 6). The administration of metyrapone produced a rise in plasma .beta.h-EP concentration of 37-155%. .beta.h-EP was not detectable in CSF samples obtained by lumbar puncture (n = 19). The present RIA system is sufficiently sensitive and specific to permit direct measurement of .beta.h-EP in human biological fluids.