Preparation of monoclonal antibody against copper and establishment of immunological detection methods
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摘要: 目的:制备针对重金属铜离子的单克隆抗体,建立Cu2+的间接和直接竞争ELISA检测法,实现对Cu2+的快速定量测定。方法:利用双功能螯合剂p-SCN-Bn-NOTA将Cu2+分别与载体蛋白BSA和OVA偶联,制备免疫原和检测原;用免疫原免疫Balb/c小鼠;细胞融合后,通过间接及间接竞争ELISA筛选稳定分泌抗重金属铜的单克隆抗体的杂交瘤细胞株;制备腹水型单抗并进行鉴定;建立间接竞争ELISA和直接竞争ELISA。结果:成功筛选出4株稳定分泌抗重金属铜的单克隆抗体的杂交瘤细胞株,其中G8制备出腹水型单抗效价为5×105倍,抗体亚型为Ig G1型,轻链类型为κ型,特异性检测结果表明该单抗对NOTA及Mn2+、Pb2+、Cd2+、Zn2+等其金属离子交叉反应率远低于2%,与Mg2+交叉反应率小于8%,表明单克隆抗体G8对重金属Cu2+具有很强的特异性,不会干扰结果的准确性;建立间接竞争ELISA的检测灵敏度为29.8 ng/m L,检测限为2.4 ng/m L,线性范围为4.5196.7 ng/m L;直接竞争ELISA检测法的检测灵敏度为12.89 ng/m L,检测限为0.88 ng/m L,检测范围为1.7296.58 ng/m L。结论:成功制备出抗重金属铜离子的单克隆抗体,并建立了间接竞争ELISA和直接竞争ELISA。Abstract: Objective: Prepare monoclonal antibodies ( Mc Abs) against copper, and establish indirect and direct competitive enzyme-linked immunoassay for detection of copper. Finally rapid quantitative determination of Cu2 +was achieved. Methods:Using double functional chelating agent p-SCN-Bn-NOTA to connect Cu2 +with carrier protein BSA and OVA, Immune Balb/c mice.After cell fusion, positive cells that stability secrete Mc Abs against Cu2 +were selected through indirect and indirect competitive ELISA. Preparation and identification of Mc Abs. Established indirect and direct competitive ELISA detection method.Results: 4 hybridoma cell lines that stability secreting Mc Abs against Cu2 +were prepared successfully, the titer of G8 was 5 × 105.The subtype and type of light chain were Ig G1 and κ, specific detection showed that the cross-reactivity of Mc Ab with Mn2 +, Pb2 +, Cd2 +, Zn2 +and NOTA were less than 2%, except for Mg2 +which was less than 8%, this showed high specificity of this antibody. The detection sensitivity of established indirect competitive ELISA was 29.8 ng/m L, Detection of limit was 2.4 ng/m L and the detection rang was from 4.5 to 196.7 ng/m L. The detection sensitivity of established direct competitive ELISA was 12.89 ng/m L, detection of limit was 0.88 ng/m L and the detection rang was from 1.72 to 96.58 ng/m L.Conclusion: Successfully prepared Mc Abs against Cu2 +and established indirect competitive ELISA and direct competitive ELISA.
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