Development of a homologous radioimmunoassay for a gonadotropin of the gilthead seabream, Sparus aurata

Abstract A glycoproteic gonadotropin (GtH) was partially isolated from pituitaries of sexually mature Sparus aurata and used for the development of its radioimmunoassay (RIA). Two major chromatographic steps were used for the isolation of the GtH: affinity chromatography on Concanavalin A-Sepharose and gel filtration on Ultrogel ACA-54. The Sparus aurata GtH (SaGtH) was located using both a bioassay of in vitro production of 17 α -hydroxy,20 β -dihydroprogesterone (17 α , 20 β -OHP) by follicles of rainbow trout and a RIA for the β sub-unit of salmon GtH (sGtH β ). The isolated SaGtH was used for raising antibodies and as a standard. A highly purified preparation of SaGtH- 125 I was obtained by running the radiolabel through Sephadex G-50 and polyacrylamide gel electrophoresis (PAGE). The RIA was found to be sensitive (0.3 ng/ml), precise (intra-assay variability of 10–11%), and specific for GtH of Sparidae species ( Sparus aurata and Sparus latus ). It showed a poor cross-reactivity with pituitary extracts of several other marine fish. Using this RIA, a GnRH-induced SaGtH secretion was monitored both in vivo and in vitro.