Abstract

A competitive ELISA was developed for quantifying LH and FSH in grass carp. • High sensitivity (1.27 ng/mL LH; 1.18 ng/mL FSH) and good reproducibility (CV 4.1%, 4.2%). • Immunofluorescence confirmed LH and FSH are from distinct pituitary cells. • In vivo and in vitro sGnRH induced hormonal responses, validating assay applicability. Grass carp ( Ctenopharyngodon idella ) is an important aquaculture species, yet no specific immunoassay is available for quantifying its pituitary gonadotropins—luteinizing hormone (LH) and follicle-stimulating hormone (FSH)—which are key regulators of reproduction in vertebrates. Here, we developed and validated competitive enzyme-linked immunosorbent assays (ELISAs) for grass carp LH and FSH using recombinant proteins and specific polyclonal antibodies. The recombinant proteins were expressed in E. coli based on the native hormone sequences, and polyclonal antibodies were generated in rabbits. Antibody specificity was validated by immunohistochemistry and western blotting, which confirmed that LH and FSH are produced by distinct cell populations in the grass carp pituitary, consistent with the organizational pattern observed in other teleosts. The standard curves showed excellent correlation (mean R 2  = 0.994). Assay sensitivities were 1.27 ng/mL for LH and 1.18 ng/mL for FSH, with IC 50 values of 9.19 ng/mL and 9.29 ng/mL, respectively. Intra-assay CVs ranged from 0.68% to 4.10% (LH) and 0.44% to 4.27% (FSH), while inter-assay CVs averaged 5.43% and 1.92%, demonstrating good reproducibility. Using this assay, we quantified LH and FSH levels in grass carp across different developmental stages, revealing distinct age- and sex-specific secretion patterns that provide insights into the differential regulation of gonadotropins during sexual maturation in cyprinids. The assay also successfully detected hormone responses to salmon gonadotropin-releasing hormone (sGnRH) stimulation both in vivo and in vitro . This ELISA system pro