重金属铜的单抗的制备及免疫学检测方法的建立

Preparation of monoclonal antibody against copper and establishment of immunological detection methods

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4.5~196.7 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1.72~96.58 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。

Objective： Prepare monoclonal antibodies （McAbs）against copper, and establish indirect and direct competitive enzyme-linked immunoassay for detection of copper.Finally rapid quantitative determination of Cu^2＋ was achieved.Methods： Using double functional chelating agent p-SCN-Bn-NOTA to connect Cu^2＋ with carrier protein BSA and OVA,Immune Balb/c mice.After cell fusion, positive cells that stability secrete McAbs against Cu^2＋ were selected through indirect and indirect competitive ELLS＆ Preparation and identification of McAbs. Established indirect and direct competitive ELISA detection method.Results：4 hybridoma cell lines that stability secreting McAbs against Cu^2＋ were prepared successfully ,the titer of G8 was 5 × 10^5.The subtype and type of light chain were IgGI and K, specific detection showed that the cross-reactivity of McAb with Mn^2＋ ,Pb^2＋ , Cd^2＋ , Zn^2＋ and NOTA were less than 2% , except for Mg^2＋ which was less than 8% , this showed high specificity of this antibody.The detection sensitivity of established indirect competitive ELISA was 29.8 ng/mL, Detection of limit was 2.4 ng/mL and the detection rang was from 4.5 to 196.7 ng/mL. The detection sensitivity of established direct competitive ELISA was 12.89 ng/mL,detection of limit was 0.88 ng/mL and the detection rang was from 1.72 to 96.58 ng/mL. Conclusion：Successfully prepared McAbs against Cu^2＋ and established indirect competitive ELISA and direct competitive ELISA.