Vitellogenin in Asian catfish, Clarias batrachus: Purification, partial characterization and quantification during the reproductive cycle by ELISA

A study on vitellogenin (Vtg), the egg yolk precursor protein from Asian catfish, Clarias batrachus was undertaken. Vtg synthesis was induced in male C. batrachus by three intraperitoneal injections of 17β-estradiol (E2). It was purified from E2-induced plasma by precipitation with EDTA and MgCl2 followed by gel filtration chromatography on Sephacryl S-300-HR. The purified protein was run in native gradient PAGE that appeared as a single band with apparent molecular weight of ~ 482 kDa. Further, the protein in electrophoresed gels was stained separately and found positive for carbohydrate, lipid and phosphorus; and thus was confirmed as vitellogenin. In SDS-PAGE, two major polypeptides corresponding to ~ 97 and ~ 67 kDa could be observed. Polyclonal antiserum to C. batrachus vitellogenin (Vtg) was raised in a rabbit. It was found to be specific for C. batrachus Vtg in western blot, after adsorption with male C. batrachus liver homogenate. Using this antiserum, Vtg was found to be localized in liver and ovary of vitellogenic female C. batrachus by immunohistochemistry. Further, a competitive ELISA was developed for the quantification of Vtg in plasma samples of C. batrachus. The developed ELISA showed a detection range between 7.8 and 500 ng/ml. The intra- and inter-assay coefficients of variation of the assay were 7.7% (n = 10) and 12.6% (n = 6), respectively. The ELISA was used to measure the Vtg concentration during the annual vitellogenic cycle of female C. batrachus. The average maximum plasma Vtg concentration (2187.51 ± 228.8 μg/ml) was observed approximately 1 month before spawning (May) and the concentration was found maintained at significantly higher level during the majority of the spawning period (June and July).

► C. batrachus vitellogenin was purified to homogeneity.
► Molecular wt of native Vtg was determined to be ~ 482 kDa.
► SDS-PAGE revealed 2 polypeptide subunits of ~ 97 and ~ 67 kDa.
► A competitive ELISA was developed for the quantification of plasma Vtg.
► Plasma Vtg level was determined during annual reproductive cycle.