Time-resolved fluoroimmunoassay for pituitary adenylate cyclase activating polypeptide 27 (PACAP27) using europium (III) ion chelate labeled streptavidin-biotin complex

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel peptide hormone and has a variety of biological action. In studies of the physiological behaviour of endogenous PACAP, the determination of PACAP levels in biological materials require a highly sensitive and specific method. Therefore, we developed a sensitive time-resolved fluoroimmunoassay (TR-FIA) for PACAP27 which is the biologically important fragment of PACAP. Accordingly, we developed TR-FIA using a biotinylated PACAP27 (b-PACAP27) as a tracer and europium (III) chelate labeled streptavidin-biotinylated bovine serum albumin complex as a detection of biotin on solid phase. A measurable range of PACAP27 was 7.8–1000 pg ml −1 by the proposed TR-FIA. For measurement of biological samples, the samples were purified to eliminate substances which interfered with the TR-FIA. The mean recovery of PACAP27 using commercially reversed phase column was 74.7% ( n = 12). The various tissues, extracts and plasma concentrations of rat could be measured by the proposed TR-FIA.