Production of polyclonal antibodies specific to river catfish Hemibagrus nemurus vitellogenin

Hemibagrus nemurus, river catfish is a hardy fish with high nutritive value, good meat quality and excellent flavour that is commercially important in the fishery industry. However, due to asynchronous maturation in female and male, inability to reproduce in captivity, and low larvae survivability, this species is used as a model tropical fish for reproductive assay development in artificial propagation. Vitellogenin (Vtg) is a sex-specific protein that plays a direct role in oocyte development and is entirely responsible for egg maturation. This protein has proven to be a simple biomarker for determining reproductive status in female fish. However, due to the complexity of Vtg molecules and species specificity, no polyclonal antibodies (PAbs) for tropical fish, particularly river catfish, are available for the development of spawning prediction tools. Thus, the aims of this study are to produce and characterize PAbs specific to river catfish, in order to develop a reproductive assay. The purity of the river catfish Vtg revealed a high amount of 170 kDa and 120 kDa by SDS-PAGE, which was confirmed to be Vtg by Western blotting. Rabbits were then immunized to generate PAbs against the river catfish Vtg and their serum samples were purified using affinity chromatography. The purified PAbs were characterized, and a competitive ELISA was developed to quantify plasma Vtg. The sensitivity of the Vtg assay was 2.35 ng/mL, with a maximum of 92.5% antibody binding, corresponding to 31.2 ng/mL Vtg. The assay also has good intra-assay and inter-assay coefficients of variation, which are 6.4 and 12.5%, respectively. The findings indicate that PAbs enable specific Vtg recognition in blood plasma, making them more suitable for rapid and sensitive reproductive assay screening. Furthermore, Vtg antibodies successfully demonstrate for Vtg measurement and can be used for reproductive condition assessment and environmental monitoring.