Immunological and physiological validation of enzyme-linked immunosorbent assay for growth hormone of the Asian catfish, Clarias batrachus

Abstract

This study describes the development and validation of competitive antigen capture enzyme-linked immunosorbent assay (ELISA) for growth hormone (GH) of the catfish, Clarias batrachus. Isolated GH was characterized first through bioassay, amino acid sequencing, immunoblotting and immunocytochemistry, and then used to raise antibody to develop ELISA. The lowest detection limit of the assay system was 0.17 ng ml⁻¹, and the standard curve had an ED₅₀ value of 0.35 ng ml⁻¹. Repeated determination of GH in a plasma pool exhibited intra- and interassay co-efficient of variation of 8.1% and 7.09% (n=5), respectively. Dose–response inhibition curves resulting from dilutions of plasma and pituitary homogenate of C. batrachus were parallel to the standard curve, while such parallelism in the case of Cyprinus carpio and H. fossilis was absent revealing no cross reaction of them in the present ELISA. In vivo effects of sGnRH, testosterone, 5-HT and morphine on plasma GH were also examined to validate the ELISA physiologically. sGnRH, testosterone and morphine increased, while 5-HT decreased GH level in a dose-dependent manner.