Rockfish (Sebastes schlegeli) Vitellogenin: Purification, Characterization and Development of Sandwich ELISA System

Abstract

Vitellogenin (VTG) was purified from serum of estradiol-17β-treated rockfish (Sebastes schlegeli) by precipitation with EDTA-Mg2+and ammonium sulfate and two step chromatography (anion exchange chromatography and gel permeation chromatography) was performed on FPLC system. Rockfish VTG (rfVTG) was characterized and its properties were determined. The monomers have apparent, molecular mass of about 188kDa as indicated by SDS-PAGE. Amino acid composition analysis of rfVTG was similar to VTG from other oviparous teleosts. Cysteine and lysine were present at relatively high level. Leucine was present at relatively lower level than in other species. The N-terminal amino acid sequence was evaluated to identify rfVTG. Western blot analysis using an antibody against the purified VTG showed that the antibody reacted with both plasma of estradiol-17β treated male and purified VTG, whereas there was no reaction with male serum of the control. An ELISA was developed using monoclonal and polyclonal antibodies against rfVTG. The assay range was 3.2ng/mL and 1,000ng/mL and the value of the intra and inter assay variations were within 9.7% and 11.2%, respectively. Recovery rate was 96.8%. The sandwich ELISA could be useful for the detection of VTG and could be good for screening of estrogenic compounds.