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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 antibodies against heavy metal copper ions and establish an indirect competitive ELISA assay. Methods Cu (Ⅱ) -DTPA-BSA and Cu (Ⅱ) -DTPA-OVA were prepared by coupling Cu (Ⅱ) with BSA and OVA using bimodal chelator p-SCN-Bn- The BALB / c mice were immunized, and the hybridoma cell lines stably secreting anti-heavy metal Cu (Ⅱ) -DTPA monoclonal antibodies were screened by cell fusion; the ascites was prepared, the antibody titers were determined, the antibody subtypes were identified, and the specificity was determined; Indirect competition ELISA detection methods, and ICP-AES test results were compared. Results Three hybridoma cells secreting McAbs against heavy metals Cu (Ⅱ) -DTPA were screened out and 4C4E7 strains were selected for the preparation of ascites. The antibody titer of ascites was 1 × 106 and the subclass of IgG1 was IgG1. The antibody was expressed in the presence of 17.5%, 8.75% and 1.46% cross-reactivity with Co (Ⅱ), Zn (Ⅱ) and Hg Cross-reaction rate of species of metal ions are low. The minimum detection limit of this method is 2.0 ng / ml, which is more than 94% consistent with ICP-AES test results. Conclusion The heavy metal copper ion monoclonal antibody has been successfully prepared and an indirect competitive ELISA was established.