Roquyya Gul, Farkhanda Ghafoor, Sumbal Mehmood, Rabail Alam and Muhammad Waheed Akhtar
Centre for Research in Molecular Medicine, the University of Lahore, Lahore, Pakistan
The study aims at establishing the biological assays for recombinant growth hormone (GH) of the local Beetal breed of bovidae specie; caprine. For this purpose, a simple and highly sensitive competitive enzyme immunoassay for recombinant caprine growth hormone (rcGH) was optimized. Antiserum was raised in two local breed rabbits and horseradish peroxidase was labeled with rcGH. 96-well microtitre plates were coated with primary antibody (rabbit anti-rcGH). The rcGH standards ranging from 0- 400ng/ml were prepared and standard curve was made. Assay parameters like pH, temperature, reaction time, antibody dilution and conjugate were optimized. The assay was simple to perform and was reliable. To determine the functional activity of rcGH, receptor binding assay was optimized. rcGH was iodinated and its biological activity was detected by using caprine liver receptor membrane and was dependent on receptor protein concentration, tracer counts, temperature, time of incubation and assay pH. In cross-reactive study, human GH competed and displaced the iodinated rcGH by binding effectively to the caprine receptor. Scatchard analysis of the rcGH binding suggested a single class of binding site to the caprine liver membrane with an affinity of 337.1 ± 82.94 × 109 M-1 and capacity of 57.61 ± 4.23 fmol mg-1.
Keywords: Caprine, Beetal, Recombinant growth hormone, EIA, Receptor binding assay.