基于NOTA的重金属铅人工抗原的合成与鉴定

为研究杂环类双功能螯合剂在重金属人工抗原制备方面的效果,选取2-S-(4-氨基苯)-1,4,7三氮杂环壬烷-1,4,7-三乙酸(P-NH2-Bn-NOTA)和重金属铅为研究对象,以NOTA为双功能螯合剂,采用戊二醛法将重金属铅分别与载体蛋白BSA和OVA偶联制备人工抗原,经测定其结合比分别为9∶1和7∶1。以免疫原免疫兔子制备高效价的多克隆抗体并建立ic-ELISA,其回归方程为y=12.127ln(x)-11.386(R2=0.9961),IC50和IC20分别为157.89,13.30 ng/mL,说明制备的多抗具有较高的检测灵敏度。交叉反应试验显示:除了与镉交叉反应率为12.3%外,该抗体与重金属铁、铜和锌的交叉反应率均小于5.3%,具有较好的特异性。本研究成功制备了重金属铅人工抗原及多克隆抗体,采用杂环类双功能螯合剂制备的重金属人工抗原效果较好,为下一步开展杂环类双功能螯合剂在重金属人工抗原中的应用提供依据。

Synthesis and Identification of Heavy Metal Lead Artificial Antigen Based on Bifunctional Chelators NOTA

In order to evaluate the effect of heavy metal artificial antigen,heterocyclic bifunctional chelators(BFCs),2-S-(4-Aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid(P-NH2-Bn-NOTA)and heavy metal Pb2+were selected.The Pb2+was conjugated to bovine serum albumin(BSA)and ovalbumin(OVA)with glutaraldehyde method to produce the artificial antigen based on the bifunctional chelators NOTA,respectively.The conjugation ratio was 9 and 7 for the Pb2+-NOTA-BSA conjugate and the Pb2+-NOTA-OVA conjugate,respectively.The polyclonal antibody(PcAb)was obtained after immune rabbits.Based on the PcAb,an indirect ELISA was established and its linear regression equation was y=12.127ln(x)-11.386(R2=0.9961).According to the ELISA,the value of detection limit(IC20)and IC50 were 13.30 and 157.89 ng/mL,respectively.The specificity analysis showed that the cross-reactivities(CR)were all less 5.3%except Cd2+was 12.3%,which indicated that the PcAb was specific for Pb2+.The produced Pb2+artificial antigen and the PcAb could lay the foundation for rapid analysis of heavy metal residue using the BFCs.