十氯酮人工抗原的制备及鉴定

Preparation and identification of chlordecone artificial antigen

为制备及鉴定十氯酮(Chlordecone)的抗体,利用羰基二咪唑(CDI)法将十氯酮还原产物与血蓝蛋白(KLH)及牛血清白蛋白(BSA)的氨基偶联,制备出人工免疫原CD-KLH和人工检测抗原CDBSA,并采用傅里叶红外光谱对人工抗原进行鉴定。用CD-KLH以腹腔皮下多点免疫注射的方式免疫BALB/c小鼠,断尾采血制备鼠源多克隆抗体血清。ELISA测定血清效价,间接竞争ELISA测定血清敏感性及特异性。结果显示,十氯酮鼠源多克隆抗体效价达10 000。确定了ELISA方法包被抗原与十氯酮抗体的最佳质量浓度分别为10μg/mL和25μg/mL,十氯酮间接竞争ELISA试验结果表明,十氯酮最佳线性检测限为2.5μg/mL~40μg/mL,最低检测限达0.625μg/mL,与BSA、KLH交叉反应率均小于0.01%,低于常规气相色谱-电子捕获检测方法的检出限。

For developing antibodies against Chlordecone,the metabolite of Chlordecone was coupled with bovine serum albumin(BSA)and Keyhole Limpet Hemocyanin(KLH)by CDI as a coupling regent.These two conjugations were confirmed by infra-red spectroscopy.Polyclonal antibody was obtained after immunization of BALB/c mice with CD-KLH.tail-cut blood samples were collected after the immunization to prepare polyclonal antibodies.Serum titer was determined by ELISA,and serum sensitivity and specificity were determined by indirect competitive ELISA.The results showed that the artificial antigen of Chlordecone was prepared successfully.The serum titers of immunized mice were more than 10,000.The working concentration of coating antigen and antibody were optimized to set up an indirect competitive ELISA for Chlordecone,results showed that the curve has a favorable linearity relation within the concentration range of 2.5μg/mL~40μg/mL.The minimum detection reaches to 0.625μg/mL.the cross reaction rate with BSA and KLH was less than 0.01%.In conclusion,Chlordecone artificial antigen was successfully prepared,and mouse polyclonal antibodies with high sensitivity and specificity were obtained by animal immunization.