重金属铜离子单克隆抗体的制备及其间接竞争ELISA检测方法的建立

Preparation of Monoclonal Antibody against Heavy Metal Cupper Ion and Development of Indirect Competitive ELISA Method

目的制备重金属铜离子单克隆抗体,并建立间接竞争ELISA检测方法。方法利用双功能螯合剂p-SCN-Bn-DTPA将Cu(Ⅱ)与载体蛋白BSA和OVA偶联,制备免疫抗原Cu(Ⅱ)-DTPA-BSA和检测抗原Cu(Ⅱ)-DTPA-OVA,免疫BALB/c小鼠,通过细胞融合筛选稳定分泌抗重金属Cu(Ⅱ)-DTPA单克隆抗体的杂交瘤细胞株;制备腹水,测定抗体效价,鉴定抗体亚型,并检测其特异性;建立间接竞争ELISA检测方法,并与ICP-AES检测结果进行比较。结果共筛选出3株稳定分泌抗重金属Cu(Ⅱ)-DTPA单克隆抗体的杂交瘤细胞,选择其中的4C2E7株制备腹水。腹水抗体效价为1×106,抗体亚类为IgG1,该抗体除与Co(Ⅱ)、Zn(Ⅱ)和Hg(Ⅱ)存在17.5%、8.75%和1.46%的交叉反应外,与其他7种金属离子的交叉反应率均较低。该法最低检测限为2.0 ng/ml,与ICP-AES法检测结果的总符合率超过94%。结论已成功制备了重金属铜离子单克隆抗体,并建立了间接竞争ELISA检测方法。

Objective To prepare monoclonal antibody（McAb） against heavy metal cupper ion and develop an indirect competitive ELISA method.Methods Copper ion was coupled to vector proteins bovine serum albumin（BSA） and ovalbumin（OVA） via a bifunctional chelating agent p-SCN-Bn-DTPA respectively.BALB/c mice were immunized with the prepared antigens Cu（Ⅱ）-DTPA-BSA for immunization and Cu（Ⅱ）-DTPA-OVA for detection respectively,and the hybridoma cell strains stably secreting McAbs against Cu（Ⅱ）-DTPA were screened.The ascites of mice were collected,in which the McAbs were determined for titer,identified for subclass and tested for specificity.An indirect competitive ELISA method was developed,by which the determination result was compared with that by ICP-AES method.Results Three hybridoma cell strains stably secreting McAbs against Cu（Ⅱ）-DTPA were screened,of which 4C2E7 strain was selected for preparation of ascites.The titer and subclass of McAb in ascites were 1 × 106 and IgG1 respectively.The McAb showed cross reaction rates of 17.5%,8.75% and 1.46% with Co（Ⅱ）,Zn（Ⅱ） and Hg（Ⅱ） respectively,while showed low cross reaction rates with other seven kinds of metal ions.The minimum detection limit of the developed ELISA method was 2.0 ng/ml.The total coincidence rate of the developed ELISA method and ICP-AES method was more than 94%.Conclusion The McAb against Cu（Ⅱ） was successfully prepared,and an indirect competitive ELISA method was developed.