目的: 建立酶联免疫吸附法（ELISA 法）用于测定食蟹猴血清中的抗CTLA-4 单克隆抗体（伊匹单抗,YERVOY®）的浓度。方法: 建立间接ELISA 方法对待测抗体药进行浓度测定,将Human CTLA-4 蛋白包被于酶标板中,封闭后加入稀释后样品,采用HRP 标记的羊抗人IgG 作为酶标抗体,用TMB 底物进行显色并用2 mol·L-1 硫酸终止,在酶标仪上用双波长读取D450/630 nm 值。最后以标准曲线样品D450/630 nm 为纵坐标（Y）,以样品浓度为横坐标（X）,进行非线性五参回归运算,得到标准曲线方程,将待测样品D450/630 nm 带入方程即可得到待测样品浓度。结果: 在优化的条件下,本方法的定量范围为0.625~30.000 μg·mL-1,定量下限为0.625 μg·mL-1。精密度（批内和批间）均在±15% 之内,准确度介于75%~125%。方法特异性、选择性、稀释线性均满足可接受标准,准确度介于80%~120%。同时抗CTLA-4 单克隆抗体在食蟹猴血清中经验证具有良好的室温稳定性、反复冻融稳定性及长期稳定性,样品准确度均介于80%~120%。结论: 本研究建立的方法满足《中华人民共和国药典》2020 年版中生物样品定量分析方法验证指导原则的要求,可用于定量分析食蟹猴血清中抗CTLA-4 单克隆抗体（YERVOY®）的浓度。
Objective： To establish an enzyme linked immunosorbent assay（ ELISA） method for determination of anti-CTLA-4 monoclonal antibody（ ipilimumab,YERVOY®） in Cynomolgus monkey serum. Methods： Anindirect ELISA method was established for the determination of the monoclonal antibody drug. Human CTLA- 4 protein was coated in microplate. After plate blocking,diluted samples were added. Then HRP labeled Goat Anti-human IgG antibody was used as enzyme-labeled antibody. TMB substrate was added for color development and the reaction was terminated by 2 mol·L-1 of sulfuric acid. Finally,the absorbance of each well was measured by a microplate reader at 450/630 nm. The calibration curve was generated by the D450/630 nm value（ Y） versus the concentration（ X）.A 5-parameter logistics algorithm was used to get the standard curve equation,and the sample D450/630 nm to be tested was substituted into the equation to obtain the sample concentration to be tested. Results： Under the optimized conditions,the method dynamic range was 0.625-30.000 μg·mL-1,with the lowest quantification limit of 0.625 μg·mL-1. Precision（ within-plate and between plate） was within ±15% and the accuracy ranged from 75% to 125%. The specificity,selectivity and dilution linearity of the method all met the acceptable standard,and the accuracy ranged from 80% to 120%. At the same time,the anti-CTLA-4 monoclonal antibody was stable in room temperature,freeze-thaw,and long-term stability tests in Cynomolgus monkey serum meanwhile,and the sample accuracy ranged from 80% to 120%. Conclusion： The method established in this study meets the requirements of the guidelines for verification of quantitative analysis methods for biological samples in the Pharmacopoeia of the People’s Republic of China 2020 edition. It is proven to be feasible for the quantitative analysis of anti-CTLA-4 monoclonal antibody（ YERVOY®） in Cynomolgus monkey serum.
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