Comparative biochemistry and physiology

Abstract

The development of a sensitive and specific two-site, or sandwich, noncompetitive enzyme-linked immunosorbent assay (ELISA) for oncorhynchid growth hormone (GH) using monoclonal antibodies (MCAs) is reported. The MCAs were generated by the fusion of myeloma cells with spleen cells from mice that had been immunized with chum salmon (Oncorhynchus keta) recombinant GH. The MCAs specifically recognized the GH-secreting acidophils in the proximal pars distalis of immature male rainbow trout (Oncorhynchus mykiss) pituitaries. Affinity chromatography using one of the MCAs isolated a single protein with a molecular weight of 22,500 from a rainbow trout pituitary extract. The ELISA recognized recombinant chum salmon GH and the affinity-purified protein but did not recognize chum salmon prolactin, gonadotropin I or II, nor several mammalian hormone preparations. The ELISA recognized GH in rainbow trout, coho salmon (Oncorhynchus kisutch), and chinook salmon (Oncorhynchus tshawytscha) pituitary extracts, but not in goldfish (Carassius auratus) extracts, and recognized GH in rainbow trout, coho salmon, lake sturgeon (Acipenser fulvescens), and bowfin (Amia calva) plasma, but not in goldfish, yellow bullhead (Ictalurus natalis), or lamprey (Petromyzon marinus) plasma. The sensitivity of the ELISA was less than 1.56 ng/ml and circulating levels of GH in the plasma of coho salmon and rainbow trout plasma were measured as 75 and 35 ng equivalents/ml, respectively.