人tau蛋白N端结构域特异性单克隆抗体筛选及在血液检测中的应用

Human tau N-terminal domain-specific monoclonal antibodies: screening and application in blood detection

摘要:

微管相关蛋白tau抗体在阿尔茨海默病(Alzheimer’s disease, AD)及其他tau蛋白病的基础和临床研究中发挥重要作用。采用重组人tau441作为免疫原，通过细胞融合及有限稀释法筛选并获得分泌抗人tau蛋白N端结构域(tau N-terminal domain, NTD-tau)单克隆抗体杂交瘤细胞株，通过制备小鼠腹水及亲和层析获得纯化的单克隆抗体；分别采用间接ELISA检测其灵敏度，蛋白质印迹检测其特异性；建立并优化人tau蛋白双抗体夹心ELISA检测方法。结果显示，杂交瘤细胞克隆阳性率为83.6%，建立分泌ZD8F7单克隆抗体的稳定细胞株，细胞上清中抗体效价为1:16 000；腹水中抗体效价高于1:256 000；纯化后单克隆抗体效价高于1:128 000；表位分析显示，ZD8F7单克隆抗体识别tau21?37位氨基酸，位于N端结构域；蛋白质印迹分析显示，ZD8F7单克隆抗体识别50?70 kDa重组人tau蛋白及转基因AD模型小鼠(APP/PS1/tau)脑组织中50 kDa的人tau蛋白；以ZD8F7单克隆抗体作为捕获抗体建立的人tau蛋白定量检测方法，线性范围在7.8?500.0 pg/mL，可以识别AD转基因小鼠脑组织以及血浆中的人tau蛋白，而不识别小鼠tau蛋白。综上，本研究制备的人NTD-tau特异性单抗和双抗体夹心ELISA检测方法灵敏度高，为神经退行性疾病中tau蛋白检测提供了有力工具。

Abstract:

The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer’s disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21–37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50–70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8–500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.