抗噻菌灵单克隆抗体的制备及异源IC-ELISA检测方法的建立

为建立噻菌灵酶免疫检测方法,制备3种噻菌灵的半抗原(H1、H2和H3)。利用活泼酯法制备免疫抗原H1-BSA、H3-BSA和包被抗原H1-OVA、H2-OVA、H3-OVA。采用杂交瘤细胞融合方法获得4株稳定分泌抗噻菌灵单克隆抗体的细胞株,分别命名为1-4G9、1-6D3、3-1F9和3-5D4。采用间接竞争酶联免疫法(IC-ELISA),将上述4株抗体分别与3种包被抗原进行组合,从中选择出一个最优组合:H3-OVA/1-6D3。据此建立的异源IC-ELISA检测方法对噻菌灵的IC50为10.9μg.L-1,检测限(IC10)为2.2μg.L-1,与3种噻菌灵类似物的交叉反应率均小于0.1%;在自来水、苹果、梨的添加回收试验中,IC-ELISA法测定的回收率依次为95.9%～118.1%、90.0%～117.6%、74.9%～95.6%;HPLC法测定的回收率依次为92.7%～97.6%、98.9%～100.9%、91.6%～105.4%,IC-ELISA法与HPLC法测定结果基本一致。上述结果表明:抗噻菌灵单克隆抗体酶免疫检测具有很强的特异性和准确性。

Production of monoclonal antibodies against thiabendazole and development of heterologous IC-ELISA

In order to establish the immunoassay method of thiabendazole,three haptens had been prepared(H1,H2,H3).The immunogen haptens(H1-BSA,H3-BSA)and coating antigens(H1-OVA,H2-OVA,H3-OVA)were prepared by using the active ester method.Four hybridoma cell lines which secreted monoclonal antibody(McAb)were obtained by cell fusion method.They were designated as 1-4G9,1-6D3,3-1F9 and 3-5D4,respectively.H1-OVA,H2-OVA,H3-OVA as coating antigens were combined with the mentioned four hybridoma cell lines by indirect competitive enzyme-linked immunosorbent assay(IC-ELISA).Among the combinations,the best one was selected for detailed study:H3-OVA/1-6D3.Based on it,a heterologous IC-ELISA was developed for the determination of thiabendazole,and the detection limits(IC10)and IC50 of thiabendazole were 2.2 μg·L-1 and 10.9 μg·L-1,respectively.The cross-reactivities(CRs)were all less than 0.1% with some analogues of thiabendazole.In recovery test of tap water,apple and pear,the results of IC-ELISA were in the range of 95.9%-118.1%,90.0%-117.6%,74.9%-95.6%,respectively;the results of HPLC,in the range of 92.7%-97.6%,98.9%-100.9%,91.6%-105.4%,respectively.The results of IC-ELISA and HPLC had good consistency.The results showed that the immunoassay of thiabendazole had strong accuracy and specificity.