IMMUNOCHROMATOGRAPHIC ANALYSIS OF BOVINE GROWTH-HORMONE RELEASING-FACTOR INVOLVING REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY IMMUNODETECTION

We have developed high-performance immunoaffinity chromatography (HPIA C) methods for the detection and quantitation of bovine growth hormone releasing factor (GHRF), which could also be applicable to its metabo lites in biofluids, These approaches have involved a combination of IA C using immobilized antibody (Ab) to GHRF, together with reversed-phas e high-performance liquid chromatography (RP-HPLC) separations of init ially isolated and concentrated protein, followed by selective detecti on, involving on-line immunodetection (ID) schemes. ID methods involve d HPIAC supports of the Ab, together with synthesized Ab-fluorescein i sothiocyanate conjugates. We have demonstrated optimization methods fo r each step of the entire hyphenated technique (IAC-HPLC-LD), and then actually quantitated GHRF using this overall system. The minimum dete ctable concentration was about 1 ng/5 ml (200 ppt) with fluorescence d etection (excitation wavelength, 490 nm; emission wavelength, 510-650 nm). We have also tested a single blind, spiked biological sample (bov ine plasma), spiked with a known level of GHRF. Accuracy (7.4%) and pr ecision (S.D.=+/-22%) were quite acceptable for a double immunoassay m ethod.