Development of a homologous enzyme-linked immunosorbent assay for Senegalese sole fsh using a recombinant chimeric gonadotropin

Póster presentado en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014
[Introduction] The Senegalese sole (Solea senegalensis) is a highly valued flatfish in Southern European aquaculture, but the industrial production of this species is still impaired due to poor knowledge of its reproductive endocrinology. The main endocrine regulators of fish gametogenesis are the pituitary gonadotropins (GTHs), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), which are composed by a specific beta chain (Fshb, Lhb) and a common alpha chain (Cga). Although cDNAs encoding both GTHs have been isolated in some flatfishes, no specific assay has been developed in these species to measure their physiological levels. Since immunoassays for GTH dimer proteins may be more sensitive than those using isolated subunits, in this study we developed a specific enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh using a chimeric protein containing homologous Fshb coupled with chicken (Gallus gallus) Cga.
[Methods] The methylotrophic yeast Pichia pastoris was used to produce a chimeric recombinant single-chain Fsh containing the coding sequence of the sole mature Fshb (SsFshb), followed by six His residues, the carboxyl-terminal peptide sequence of human chorionic gonadotropin b subunit as a linker, and the mature sequence of the chicken Cga (GgCga). Homologous recombinant single-chains of Fsh (SsFsh) and Lh (SsLh) were produced in CHO cells. Polyclonal antibodies against the chimeric construct were generated in rabbits, and specific SsFshb antibodies were further purified by affinity chromatography on GgCga-bound columns. Preadsorbed and GgCga adsorbed antibodies were tested in ELISA using SsFshb-GgCga for coating and standard curve generation.
[Results and Discussion] ELISA using the preadsorbed antiserum showed a crossreactivity with GgCGa and SsLh of 46.0% and 10.1%, respectively, which was respectively reduced to 0.5% and 0.05% after purification. Using the purified antiserum, the ELISA standard curves for SsFshb-GgCga paralleled those of serially diluted plasma and pituitary extracts, as well as that of SsFsh. The sensitivity of the assay for Fsh was