均相时间分辨荧光法测定抗PD-1/PD-L1单抗药物活性

Determination of biological activity of anti PD-1/PD-L1 monoclonal antibodies by homogeneous time resolved fluorescence method

目的:探索并建立一种均相时间分辨荧光(homogeneous time-resolved fluorescence,HTRF)检测方法,用于快速、灵敏、稳定检测单克隆抗体的活性。方法:将检测试剂、待检样品加入96半孔板,室温振荡孵育3 h后在酶标仪上读取荧光信号值,用软件对信号值进行四参数曲线拟和,根据四参数方程计算单抗的相对活性;同时对该方法进行验证。结果:本文构建的检测方法呈典型的反S型剂量效应曲线,且反应缓冲液中添加0.4 mol·L^(-1)氟化钾可提高荧光响应值。对该方法进行验证,专属性强,只与抗PD-1/PD-L1单抗发生反应;2名不同分析人员重复检测6次,相对效价范围为85%～117%,总RSD为13%;准确性良好,不同浓度供试品溶液回收率均在80%～120%范围内;理论相对活性与实际测得相对活性线性良好,相关系数在0.99以上,斜率接近1.0,符合方法验证要求。结论:通过对HTRF检测条件的摸索及验证,建立了一种新型测定单抗活性的方法,且该方法快速简便,可用于单抗的前期筛选或后期活性检测。

Objective:To explore and establish a homogeneous time resolved fluorescence(HTRF) method for rapid,sensitive and stable determination biological activity of monoclonal antibodies. Methods:Test reagents and samples were added to a 96-well plate,the fluorescence signal value was read on the microplate reader after the plate had been shaken at room temperature for 3 hours,and the signal value was subjected to four-parameter curve simulation by software,and the relative activity of monoclonal antibodies was calculated according to the four-parameter equation. Meanwhile,the method was validated. Results:The established assay was a typical inverse "S" dose-response curve,and the assay window could be improved by adding 0.4 mol·L-1 KF into the reaction buffer. It had excellent specificity and only reacted with anti PD-1/PD-L1 monoclonal antibodies. Two different analysts repeated the test 6 times and the result showed that the relative titer ranged from 85% to 117%,and the total RSD was 13%. Our study showed the established assay had excellent accuracy,the recovery rate of the sample solution at different concentrations was in the range of 80%-120%. There was good linear relationship between the theoretical relative activity and the actual measured relative activity,the correlation coefficient was above 0.99,the slope was close to 1.0,which met the requirements for method validation. Conclusion:A new method for determination of the biological activity of monoclonal antibodies was established by exploring and validating the conditions of HTRF. In addition,the method is rapid and simple,and can be used for early screening and late biological activity detection of monoclonal antibodies.