Abstract
A sensitive and specific double-antibody enzyme immunoassay (EIA) for nociceptin (orphanin FQ)-like immunoreactive substances (nociceptin-IS) was developed. In competitive reactions, the nociceptin-antibody was incubated 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.
