硫代磷酸酯类农药通用抗原的合成与鉴定

Synthesis and identification of generic antigen for phosphorothioate pesticides

【目的】合成具有硫代磷酸酯类农药共性结构的系列半抗原,制备此类农药的通用人工抗原。【方法】以烷氧基硫代磷酸盐和卤代羧酸为原料合成半抗原,采用1 HNMR、13 CNMR及EIS-MS谱图对其结构进行表征。将合成的半抗原分别通过活性酯法、混合酸酐法与牛血清白蛋白(BSA)、卵清蛋白(OVA)偶联,制备免疫人工抗原和包被人工抗原。用紫外扫描法定性分析人工抗原的偶联情况,用2,4,6-三硝基苯磺酸(TNBS)法间接测定其偶联比。【结果】合成了H1～H6 6种具有硫代磷酸酯类农药共性结构的半抗原,其中H1、H2为包被半抗原,H3～H6为免疫半抗原。制备了H1-OVA、H2-OVA、H3-BSA、H4-BSA、H5-BSA、H6-BSA、H2-BSA、H4-OVA和H6-OVA 9种人工抗原,其偶联比分别为9∶1、10∶1、23∶1、26∶1、39∶1、43∶1、28∶1、14∶1和12∶1。【结论】成功合成硫代磷酸酯类农药的人工抗原,为下一步用其免疫动物制备抗体进而进行有机磷农药酶联免疫分析奠定了基础。

【Objective】 A series of generic haptens having the common thiophosphate group of thiophosphate pesticides were designed and synthesized,which were used to produce generic antigens.【Method】 Generic haptens were prepared by the reaction between Ammonium alkoxythiophosphate and halogenatedcarboxylicacid,and the structures were characterized by 1HNMR,13CNMR and EIS-MS.Generic immunizing haptens were conjugated to bovine serum albumin（BSA） and ovalbumin（OVA） by the methods of active ester and mixed-anhydride,respectively,to be used as immunogens and coating antigens.Spectral identification of artificial antigen was reported by UV absorbance spectrum,and then conjugating ratios were given indirectly by the method of 2,4,6-trinitrobenzene sulfonic acid（TNBS）.【Result】 Six generic haptens named H1-H6 which had the common thiophosphate group of thiophosphate pesticides were synthesized and determined by 1HNMR,13CNMR and MS.Also nine generic antigens were acquired.The conjugating ratios of antigens were 9∶1（H1-OVA）,10∶1（H2-OVA）,23∶1（H3-BSA）,26∶1（H4-BSA）,39∶1（H5-BSA）,43∶1（H6-BSA）,28∶1（H2-BSA）,14∶1（H4-OVA） and 12∶1（H6-OVA）.【Conclusion】 The artificial antigens have been synthesized successfully and could be used for further immunization to develop the enzyme-linked immunosorbent assay（ELISA） of organophosphorous pesticides.