酶联免疫吸附方法分析海水和贝类中的滴滴涕及代谢物

采用间接竞争酶联免疫吸附分析(Ind irect Competitive Enzym e-linked Immunosorbent Assay,ELISA)方法测定海水样品和海洋贝类中的有机氯农药滴滴涕及其主要代谢产物的含量。采用碳二亚胺法,将半抗原DDA分别与载体蛋白牛血清白蛋白(Bovine serum album in,BSA)和卵清蛋白(Ovalbu-m in,OVA)偶联,得到包被抗原和免疫抗原。以DDA-OVA作为免疫抗原,免疫雌性BALB/c小鼠,免疫数次后,测定血清中抗体效价。以DDA-BSA为包被抗原,免疫多克隆抗血清为多克隆抗体,建立间接竞争酶联免疫吸附方法分析检测海水样品和海洋贝类中的有机氯农药二氯二苯三氯乙烷(DDT)及其代谢产物2,2-双(4-氯苯基)乙酸(DDA)、2,2-双(4-氯苯基)-1,1-二氯乙烷(DDD)、2,2-双(4-氯苯基)-1,1-二氯乙烯(DDE)的方法。最低检出限分别为DDT:25 ng/m l,DDA:1.56 ng/m l,DDD:25 ng/m l,DDE:25 ng/m l。贝类样品DDA回收率为96.37%~100.88%,批内变异系数为2.28%~5.13%;贝类样品DDT回收率为93.86%~100.08%,批内变异系数为4.71%~7.68%;海水样品DDT回收率88.23%,批内变异系数6.60%。

Detection of DDT in Seawater and Shellfish Using Indirect Competitive ELISA

The content of DDT and its metabolites in seawater and shellfish were analyzed by indirect competitive ELISA.DDA was attached to Bovine serum albumin(BSA) and Ovalbumin(OVA) using N,N-dicyclohexylcarbodiimide,during which the synthesis of hapten DDA-BSA and DDA-OVA was gained.DDA-BSA was used as the coating antigen and DDA-OVA was used as immunogens.The coating antigen(Hapten DDA-BSA) was titrated against to measure the reactivity of antibodies serum,after female BALB/c white rabbits were immunized several times.The serum was used as the polyclonal antibodies to develop the indirect competitive ELISA to analyze DDT and its metabolites(including DDA,DDE and DDD) in seawater and shellfish.The lowest detection limits were: DDT 25 ng/ml;DDA 1.56 ng/ml;DDD 25 ng/ml;DDE 25 ng/ml.The recoveries of DDA added to the extracts from shellfish was 96.37%~100.88%.The mean within-assay of the extracts from shellfish was 2.28%~5.13%.The recoveries of DDT added to the extracts from shellfish was 93.86%~100.08%,while the mean within-assay of shellfish was 4.71%~7.68%.The recoveries of DDT in seawater was 88.23%,and the mean within-assay was less than 6.60%.