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A sensitive and specific double-antibody enzyme immunoassay (EIA) for nociceptin (orphanin FQ)-like immu- noreactive substances (nociceptin-IS) was developed. In competitive reactions, the nociceptin-antibody was incu- bated with both a nociceptin standard (or plasma extract sample) and β-D-galactosidase (β-Gal)-labeled synthetic human nociceptin (delayed addition method). The free and antibody-bound enzyme haptens were separated using an anti-rabbit IgG-coated immunoplate. The enzyme activity on the immunoplate was determined fluorometrically. The present immunoassay allows the detection of 15-700 pg/ml (sensitivity: 1.5 pg, 0.6 pg/well) of nociceptin. Using this EIA, the nociceptin-IS levels in human plasma were determined, and found to be in the range of 5.0 to 16.0 pg/ml. No circadian rhythms in the daytime (9:00-19:00) or effects of eating meals on human plasma nociceptin levels were found. We have established an evaluation system for both nociceptin-IS and substance P-IS levels in 1 ml of human plasma. As for the evaluation of analgesic effects of drugs and pain, this sensitive and specific EIA system for endogenous nociceptin may be valuable for clinical use.

Application of an Enzyme Immunoassay for Nociceptin (Orphanin FQ)-like Immunoreactive Substances to Determination of the Human Plasma Levels