ESTABLISHED OF ELISA METHOD OF VITELLIN FROM FRESHWATER CRAB (SINOPOTAMON HENANENSE) AND EFFECT OF CADMIUM ON VITELLIN ACCUMULATION IN OVARY

To explore the effect of cadmium (Cd) on oogenesis of freshwater crab (Sinopotamon henanense), vitellin (Vn) and the method of Vn-ELISA was studied and established. Vn was purified from mature ovaries of S. henanense by gel filtration chromatography. Using SDS- polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight and the quantity of Vn subunit were determined. Based on the purified Vn and Vn anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) was developed. To explore the effect of cadmium on the Vn in the ovaries, freshwater crab S. henanense were treated with 5.8 mg/L Cd for 10, 15, and 20d. The results showed that Vn was composed of three subunits (116, 66 and 45 kD). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal Vn anti-serum. The optimal dilution rates of Vn anti-serum were shown to be 1:100000. According to these optimal parameters, the standard linear equation was established for the determination of Vn concentration with a valid range of 1252000 ng/mL, and qualitative detection can be 15.62 ng/mL. In verification experiments, Vn content in the ovaries showed no significant differences, and the mean coefficients of variation of inter-assay were 7.81%, suggesting that the developed ELISA of this study was precise, stable and repeatable. Moreover, Cd caused a time-dependend down-regulation of Vn level, and showed significant effects of Cd on vitellogenesis, which suggest that Cd slows down oocyte maturation and vitellogenesis in S. henanense.