摘要
目的建立前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)单克隆抗体抗原结合活性的间接ELISA测定方法。方法利用链酶亲和素-生物素系统捕获包被PCSK9蛋白,采用间接ELISA法检测PCSK9单抗参比品和供试品,通过拟合四参数方程Y=(A-D)/[1+(X/C)~B]+D,得出两者的半数有效浓度(concentration for 50%of maximal effect,EC_(50)),计算供试品的抗原结合活性。同时验证该方法的专属性和精密性。结果 PCSK9单抗参比品、供试品及Evolocumab(Amgen)的曲线均呈显著的剂量依赖关系,符合四参数方程,且R^2>0.99;抗CD115抗体及空白稀释液不存在类似的剂量效应曲线;重复性、人员及日间精密性的相对标准偏差(relative standard deviatin,RSD)分别为7.5%、11.3%和10.4%,均<15%。结论成功建立了抗PCSK9单抗抗原结合活性的间接ELISA检测方法,该方法专属性和精密度良好,可用于PCSK9单抗抗原结合活性的常规测定。
Objective To develop an indirect ELISA for determination of antigen binding activity of monoclonal antibody（McAb）against proprotein convertase subtilisin/kexin type 9（PCSK9）.Methods The references and samples of PCSK9 McAb were detected by indirect ELISA with the PCSK9 protein captured by streptavidin-biotin system.By fitting the four-parameter equation,the median effective concentrations（EC_（50））of references and samples were calculated,based on which the antigen binding activities of samples were analyzed.The specificity and precision of the method were verified.Results Obvious dose-response modes were observed on the curves of reference and samples of PCSK9 McAb as well as Evolocumab（Amgen）,which complied with the following four-parameter equation：Y=（A-D）/[1＋（X/C）~B]＋D,with a R^（2 ）of more than 0.99.However,anti-CD115 antibody and blank diluent showed no similar doseresponse curve.The relative standard deviations（RSDs）of reproducibility,intermediate precision（different analysts）,and intermediate precision（different days）were 7.5%,11.3%and 10.4%respectively,all of which were less than 15%.Conclusion An indirect ELISA for determination of antigen binding activity of PCSK9 McAb was successfully developed,which showed high specificity and precision and might be used for routine test.
作者
杜向瑞
韩月然
陈妍
高扬
李剑
程绍辉
DU Xlang-rui , HAN Yue-ran, CHEN Yan, GAO Yang, LI Jian, CHENG Shao-hui(Tianjin Medical University, Tianjin 300000, China)
出处
《中国生物制品学杂志》
CAS
CSCD
2018年第3期311-314,共4页
Chinese Journal of Biologicals
关键词
前蛋白转化酶枯草溶菌素9
单克隆抗体
抗原结合活性
酶联免疫吸附试验
Proprotein convertase subtilisin-like kexin type 9 （PCSK9）
Monoclonal antibody （McAb）
Antigen bindingactivity
Enzyme-linked immunosorbent assay （ELISA）
