Purification and polyclonal antibody preparation of Cd-MT from Pteria penguin

Heavy metals in three wastes are discharged into water by a variety of means with the industry development such as mine,smelting and electroplating,which lead to pollution of aquatic environments and threaten human health and biological survival seriously.Cadmium(Cd) is one of the main marine pollutants.As Cd can accumulate in different tissues of shellfish through food chains,which threaten the quality and safety of shellfish foods,it is extremely necessary to research and monitor conditions of shellfish contamination with Cd.Cadmium-metallothioneins(Cd-MTs) are important biomarker used for indicating cadmium pollution in aquatic environment.The research on these Cd-MTs not only is propitious to establish Cd-MT immunoassay method,but also provides the theoretical basis for technology development in monitoring heavy metal pollution.Cd-MT was induced to synthesize by injecting different concentrations of CdCl₂(0-0.8 mg/L)in Pteria penguin and was isolated and purified from whole tissues of P.penguin by tissue homogenization,freezing centrifugation,heat treatment and Sephadex G-50 gel column chromatography.The characteristic absorption of Cd-MT in crude extract by ultraviolet spectrophotometry was the strongest at 0.8 mg/L Cd²⁺.The results showed that the content of Cd-MT in P.penguin increased with concentration of Cd²⁺.The results from SDS-PAGE assay showed that molecular weight of purified Cd-MT and its dimer were 9 ku and 18 ku respectively.Then Cd-MT was coupled to bovine serum albumin(BSA) with glutaraldehyde and polyclonal antiserum by immunizing rabbits with Cd-MT-BSA was prepared.The titer of antiserum was measured by indirect ELISA assay and reached 1∶12 800.IgG(molecular weight is about 35 ku) in the antiserum was obtained by methods including saturated ammonium persulfate precipitation and DEAE-Sephadex A50 column chromatography.ELISA assay showed that the IgG from antiserum could react to Cd-MT of P.martensi as well as Cd-MT of P.penguin.The study will lay a foundation for establishing an indirect ELISA method of Cd-MT in shellfish and meanwhile,afford technological and theoretical support for exploitation of Cd-MT test kit used in shellfish.