Development and validation of an enzyme-linked immunosorbent assay for vitellogenin in Chinese loach ( Misgurnus angaillicaudatus)

Abstract

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of vitellogenin (Vtg) in the plasma of Chinese loach ( Misgurnus angaillicaudatus). Vtg was isolated by anion exchange membrane chromatography from the plasma of 17β-estradiol (E 2)-treated loach. Polyclonal antibody against Vtg was raised in rabbits, and the specificity of the antibody was assessed by Western blotting analysis. No cross-reactivity was observed with plasma from male loach. Plasma samples from vitellogenic females and E 2-treated males were diluted parallel with the purified Vtg standard curve in the ELISA. The detection limit of the assay was 5.7 ng ml − 1 and a working range of the standard curve was between 15.6 and 1000 ng ml − 1. The intra- and inter-assay coefficients of variations were 6.9% and 10.4%, respectively. The recovery was 104.4%. The assay was applied for measuring vitellogenin concentration in the plasma from 24 wild Chinese loach from Lianyungang, Jiangsu Province, in October 2003. It was found that, in addition to females (plasma Vtg concentration ranging from 518.4 μg ml − 1 to 1922.3 μg ml − 1), male loach showed low plasma Vtg—from undetectable (two fish) to 2.9 ± 0.7 μg ml − 1 (mean ± S.D.). The results indicate that this method provides a valuable tool for the study of estrogenic effect in Chinese loach.