Development of a time-resolved fluoroimmunoassay for octopus gonadotropin-releasing hormone

Abstract

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for octopus gonadotropin-releasing hormone (oct-GnRH) to determine the profiles of oct-GnRH peptide levels in cephalopods. The sensitivity and the intra-assay and inter-assay coefficients of variation were 4.9 pg/well and 6.8 (n = 10) and 2.7% (n = 5), respectively. Anti-oct-GnRH antibody was tested on all known forms of GnRH and found to cross-react with lamprey GnRH-II (27.1%), annelid GnRH (3.36%), tunicate GnRH-I (0.92%), dogfish GnRH (0.51%), and scallop Patinopecten yessoensis GnRH (0.05%). The displacement curve obtained for serially diluted brain extracts of three cephalopods, the spear squid Loligo bleekeri, swordtip squid Loligo edulis, and North Pacific giant octopus Octopus dofleini, paralleled the oct-GnRH standard curve. The presence of oct-GnRH in the central nervous system (CNS) of these cephalopods was further examined by a combination of reverse-phase high performance liquid chromatography and oct-GnRH TR-FIA. CNS extracts from these cephalopods showed peaks with retention times similar to that of synthetic oct-GnRH. These results indicate that this novel oct-GnRH TR-FIA is widely applicable for oct-GnRH measurement in cephalopods.