目的：建立灵敏度高、特异性好且快速的ELISA 方法,用以测定食蟹猴血清中的抗PD-1 单克隆抗体（代号BGB-A317）浓度。方法：建立间接ELISA 方法对BGB-A317 进行定量分析。测定原理：包被重组人PD-1,加入稀释的血清样品,之后加入稀释好的辣根过氧化物酶标记的羊抗人IgG（酶标抗体）,再加入TMB 底物进行显色和2 mol·L^-1 硫酸作为终止液,在酶标仪上用双波长读取D_450/630nm 值,以标准曲线样品D_450/630nm 为纵坐标,以样品浓度为横坐标,进行非线性五参回归运算,得到标准曲线方程,将待测样品D_450/630nm 代入方程即可得到待测样品浓度。结果：建立并验证了该ELISA 方法,定量范围为3~180 ng·mL^-1。精密度（板内和板间）均在±15% 之内,准确度介于75%~125%;食蟹猴空白血清、样品稀释液和干扰单克隆抗体对BGB-A317 在食蟹猴血清中的浓度测定无影响;稀释线性样品的精密度小于20%,准确度介于80%~120%;平行性样品的精密度为小于20%;BGB-A317 在食蟹猴血清中室温放置4 h、-70 ℃以下放置70 d 及反复冻融3 次后均保持稳定,样品准确度均介于80%~120%。结论：方法学验证表明该间接ELISA 方法具有良好的灵敏度、准确度、特异性和可重复性,可用于定量分析食蟹猴血清中BGB-A317的浓度。

Objective: To establish a sensitive,specific and rapid enzyme linked immunosorbent assay（ELISA） method for the determination of anti-PD-1 monoclonal antibody（code: BGB-A317） in Cynomolgus monkey serum. Methods: A indirect ELISA method was established for quantitative analysis of BGB-A317. For the determination of the BGB-A317,the recombinant human PD-1 was coated on the solid phase carrier,and diluted serum samples were added,following by HRP labeled goat anti-human IgG 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 under 450/630 nm. The calibration curve was generated by the D_450/630nm value versus the concentration. Fit the calibration curves using a 5-parameter logistics algorithm to get the standard curve equation,and substitute the sample D_450/630nm to be tested into the equation to obtain the sample concentration to be tested. Results: An ELISA assay was developed and validated with the quantitative range of concentrations from 3 to 180 ng·mL^-1. Precision（within-plate and between plates） was within ±15% and the accuracy ranged from 75% to 125%. The blank Cynomolgus monkey serum,sample diluent and interfering monoclonal antibody had no effect on the concentration determination of BGB-A317 in Cynomolgus monkey serum,the precision of dilution linearity samples was less than 20%,and the accuracy ranged from 80% to 120%. The precision of parallelism samples was less than 20%. BGB-A317 in Cynomolgus monkey serum was stable after stored at room temperature for 4 hours,below -70 ℃ for 70 days and repeatedly frozen and thawed for 3 times,and the sample accuracy ranged from 80% to 120%. Conclusion: This method is highly sensitive,accurate, specific,and reproducible. It is proven to be feasible for the quantitative analysis of anti-PD-1 monoclonal antibody in Cynomolgus monkey serum.