均相时间分辨荧光法测定抗PD-1/PD-L1单抗药物活性

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.

[1] 王志明,高健,李耿.治疗性单克隆抗体药物的现状及发展趋势[J].中国生物工程杂志,2013,33(6):117 WANG ZM,GAO J,LI G.Current status and development trend of the therapeutic monoclonal antibody drugs[J].China Biochnol,2013,33(6):117
[2] 王兰,朱磊,高凯,等.单克隆抗体生物治疗药物研究进展[J].中国药学杂志,2014,49(23):2058 WANG L,ZHU L,GAO K,et al.Progress of research and development of antibody -based therapeutics[J].Chin Pharm J,2014,49(23):2058
[3] 高凯,陶磊,王军志,等.重组抗体药物的质量控制[J].中国新药杂志,2011,20(19):1849 GAO K,TAO L,WANG JZ,et al.Quality control for recombinant therapeutic antibodies[J].Chin J New Drugs,2011,20(19):1849
[4] 解肖鹏,张雷.时间分辨荧光免疫分析技术的研究进展[J].食品与药品,2012,14(5):204 XIE XP,ZHANG L.Research development of time-resolved fluoroimmunoassay[J].Food Drug,2012,14(5):204
[5] ENOMOTO K,NAGASAKI T,YAMAUCHI A,et al.Development of high throughput spemidine synthase activity assay homogeneous time resolved fluorescence[J].Anal Biochem,2006,351(2):229
[6] ENOMOTO K,OKAMOTO H,NUMATA D,et al.A simple and rapid assay for eparanase activity using homogeneous time-resolved fluorescence[J].J Pharm Biomed Anal,2006,41(3):912
[7] DEGORCE F,CARD A,SOH S,et al.HTRF:a technology tailored for drug discovery-a review of theorrtical aspects and recent applications[J].Curr Chem Genomics,2009,28(3):22
[8] GERARD M.HTRF technology[J].J Biomol Screen,1999,4(6):309
[9] CLARKE E E,SHEARMAN MS.Quantitation of amyloid-beta peptides in biological milieu using a novel homogeneous time-resolved flurescence(HTRF) assay[J].J Neurosci Methods,2000,102(1):61
[10] 王慧洁,周子涵,徐嘉辰,等.基于均相时间分辨荧光技术(HTRF)的HSP90-HOP相互作用抑制剂活性测定方法的构建及应用[J].药学学报,2017,52(4):592 WANG HJ,ZHOU ZH,XU JC,et al.HTRF-based method for determination of HSP90-HOP inhibition activity and its application[J].Acta Pharm Sin,2017,52(4):592
[11] 王泽洲,吴俊清,张永宁,等.时间分辨荧光免疫分析技术的研究进展[J].四川畜牧兽医,2015,42(7):34 WANG ZZ,WU JQ,ZHANG YN,et al.Research progress of iime-resolved fluoroimmunoassay technology[J].Sichuan Anim Vet Sci,2015,42(7):34
[12] 梁菊,陈攀,吴文澜,等.荧光共振能量转移新技术及其在药物筛选中的应用[J].中国药学杂志,2009,44(13):964 LIANG J,CHEN P,WU WL,et al.The new technology fluorescence resonance energy transfer and its application in drug screening[J].Chin Pharm J,2009,44(13):964
[13] ROSSANT CJ,MATTHEWS C,NEAL F,et al.Versatility of homogeneous time-resolved fluorescence resonance energy transfer assays for biologics drug discovery[J].J Biomol Screen,2014,11(7):1
[14] 王兰,徐刚领,高凯,等.抗体类生物治疗药物活性测定方法[J].中国生物工程杂志,2015,35(6):101 WANG L,XU GL,GAO K,et al.Determination of biological activity of antibody-based biotherapeutics[J].China Biotechnol,2015,35(6):101
[15] 邓春平,秦玉敏,邓巧春,等.均相时间分辨荧光(HTRF)法测定重组单抗含量[J].中国生物工程杂志,2013,33(12):64 DENG CP,QIN YM,DENG QC,et al.Determination of the contents of recombinant monoclonal antibodies by homogeneous time-resolved fluorescence (HTRF) method[J].China Biotechnol,2013,33(12):67