Localization and quantification of the chicken gonadotropins using monoclonal antibodies

Abstract

Specific monoclonal antibodies (mabs) against chicken luteinizing hormone (LH) and follicle-stimulating hormone (FSH) have been used to develop novel tools for the study of the chicken gonadotropins. Earlier studies have identified separate LH- and FSH-producing gonadotrophs in the young and adult chicken. Our new data, obtained by dual immunofluorescence staining, on 9- to 18-day old embryos indicate the presence of single-stained FSH-containing and LH-containing cells, respectively, with virtually no double staining. At day 9 of embryogenesis, only LH immunoreactive cells seem to be present in both male and female embryos. No FSH- immunoreactive cells were found at day 9 but were identified from day 10 onward in both sexes, strongly supporting the hypothesis that both gonadotrophs originate as separate cell types. In addition, a mab-based cLH sandwich ELISA has been refined and validated for measurement in plasma. Its detection limit was on the order of 80 pg LH/mL (USDA-cLH-K-3). Surprisingly, however, final concentrations of up to 50% of laying hen plasma did not produce a measurable signal, suggesting circulating LH levels at least ten-fold lower than suggested by classic RIA. In contrast, the assay performed excellently in juvenile chicken and rooster plasma. This can likely be explained by the pronounced isoform preferences of this new assay, in combination with the assumption that the isoform composition of circulating LH is different in the adult hen as compared to juvenile chickens and adult males. Finally, a new mab-based cFSH sandwich ELISA was developed. Its detection limit was on the order of 0.3 ng/mL, which is in the range of physiological blood samples. No significant cross-reactivity with cLH was observed. However, dilution series of rooster plasma samples clearly indicated that interference by elevated LH, especially at high final concentrations of the plasma sample, was a concern. This problem was solved by using the smallest sample possible (1:20 dilution). Further research will be needed before this new ELISA can begin to replace the currently used RIA.