基于单克隆抗体的滴滴涕、硫丹和水胺硫磷酶联免疫检测方法研究  

Research on an Enzyme-linked Immunosorbent Assay for Dichlorodiphenyltrich-loroethane,Endosulfan and Isocarbophos Based on Monoclonal Antibodies

作　　者：丁园 秦爽 刘伟美 王鸣华[1] 华修德[1] DING Yuan;QIN Shuang;LIU Wei-mei;WANG Ming-hua;HUA Xiu-de(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学植物保护学院,江苏南京210095

出　　处：《分析测试学报》2021年第5期662-671,共10页Journal of Instrumental Analysis

基　　金：国家重点研发计划(2017YFF0210200)。

摘　　要：该研究制备了滴滴涕、硫丹和水胺硫磷的单克隆抗体。利用商业化的辣根过氧化物酶标记羊抗鼠抗体作为检测二抗,分别建立了3种农药的间接竞争酶联免疫吸附分析方法(Indirect competitive enzyme-linked immunosorbent assay,ic-ELISA)。在最优条件下,ic-ELISA对滴滴涕、硫丹和水胺硫磷检测的半数抑制浓度和检出限分别为10.72 ng·mL^(-1)和1.85 ng·mL^(-1)、9.69 ng·mL^(-1)和2.19 ng·mL^(-1)及13.26 ng·mL^(-1)和3.04 ng·mL^(-1)。特异性评价结果显示,滴滴涕对o,p-滴滴伊、p,p-滴滴伊和o,p-滴滴涕的交叉反应率(CR)为9.0%~33.4%;硫丹对艾氏剂、氯丹、狄氏剂、β-硫丹和α-硫丹的交叉反应率为17.4%~84.3%;水胺硫磷对其6种结构类似物均无明显交叉反应(CR<0.1%)。所建立的方法对稻田水、土壤、苹果和香蕉样品中滴滴涕、硫丹和水胺硫磷的平均加标回收率和相对标准偏差分别为71.4%~93.3%和3.8%~9.6%、74.0%~97.4%和3.0%~11%及70.8%~97.0%和4.2%~12%。同时,ic-ELISA对滴滴涕、硫丹和水胺硫磷的检测结果与色谱仪器具有较好的一致性。上述结果表明,该研究制备的滴滴涕、硫丹和水胺硫磷的单克隆抗体可用于建立ic-ELISA,且具有较好的灵敏度和准确性。Development of detection techniques for pesticide residues such as dichlorodiphenyltrichloroethane(DDT),endosulfan and isocarbophos is of great importance to ensure the safety of agricultural products and environment due to their high toxicity.Meanwhile,immunoassay has the advantages of economy and simplicity in the rapid detection of a large number of samples.In this paper,anti-DDT,anti-endosulfan and anti-isocarbophos monoclonal antibodies were prepared.An indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)for the pesticides was developed with a commercial horse radish peroxidase-labeled goat anti-mouse antibody as secondary antibody.Under optimal conditions,the 50%inhibitory concentrations and detection limits of the method for DDT,endosulfan and isocarbophos were 10.72 ng·mL^(-1)and 1.85 ng·mL^(-1),9.69 ng·mL^(-1)and 2.19 ng·mL^(-1),13.26 ng·mL^(-1)and 3.04 ng·mL^(-1),respectively.The ic-ELISA for DDT showed that the cross-reactivities(CRs)of DDT with o,p-2,2-bis(4-chlorophenyl)-1,1-dichloroethylene(DDE),p,p-DDE and o,p-DDT were in the range of 9.0%-33.4%.The ic-ELISA for endosulfan showed that CRs of endosulfan with aldrin,chlordane,dieldrin,β-endosulfan andα-endosulfan were in the range of 17.4%-84.3%.The ic-ELISA for isocarbophos showed that isocarbophos had no significant CR with its six analogues(CR<0.1%).The average recoveries of the ic-ELISA for DDT,endosulfan and isocarbophos in paddy water,soil,apple and banana samples were in the ranges of 71.4%-93.3%,74.0%-97.4%and 70.8%-97.0%,with relative standard deviations of 3.8%-9.6%,3.0%-11%and 4.2%-12%,respectively.Meanwhile,the results of ic-ELISA showed good correlations with those of chromatography in the detection of DDT,endosulfan and isocarbophos.The related results showed that the monoclonal antibodies of DDT,endosulfan and isocarbophos prepared in this study could be used to establish an ic-ELISA with good sensitivity and accuracy.

关 键 词：间接竞争酶联免疫吸附分析 滴滴涕 硫丹 水胺硫磷 农药残留 单克隆抗体 

分 类 号：O657.3[理学—分析化学]