百日咳毒素、丝状血凝素和百日咳黏附素精制抗原纯度的SDS-PAGE检测方法的验证  

Validation of an SDS-PAGE assay for the purity detection of pertussis toxin,filamentous hemagglutinin,and pertactin

作　　者：陈丝丝 夏芙蓉 於洋 何鹏 王玲 张娜 赵芳圆 范天一 李世慧 CHEN Sisi;XIA Furong;YU Yang;HE Peng;WANG Ling;ZHANG Na;ZHAO Fangyuan;FAN Tianyi;LI Shihui(Research and Development Ofice of Beijing Institute of Biological Products Co.,Ltd.,Beijing 100176,China)

机构地区：[1]北京生物制品研究所有限责任公司研究开发室,北京100176

出　　处：《微生物学免疫学进展》2024年第4期23-29,共7页Progress In Microbiology and Immunology

基　　金：国家科技重大专项重大新药创制(2018ZX09738003-001)。

摘　　要：目的采用SDS-聚丙烯酰胺凝胶电泳(SDS polyacrylamide gel electrophoresis,SDS-PAGE)方法检测百日咳毒素(pertussis toxin,PT)、丝状血凝素(filamentous hemagglutinin,FHA)和百日咳黏附素(pertactin,PRN)精制抗原纯度,并进行验证。方法对样品进行SDS-PAGE电泳、考马斯亮蓝染色后,使用凝胶成像系统拍照并用Image Lab软件进行分析,获得样品各蛋白条带的光密度值。对建立的方法进行系统适用性、线性、准确度、重复性、中间精密度、灵敏度和耐用性验证。结果对应2套分子量标准蛋白,PT、FHA和PRN精制抗原分别在10.00~50.00μg/mL的低质量浓度范围和50.00~600.00μg/mL的高质量浓度范围内,蛋白浓度和对应条带光密度值之间具有良好的线性关系,相关系数(coefficient of correlation,r)≥0.99。3种精制抗原杂质水平准确度和重复性回收率均在80%~120%,且重复性验证相对标准差(relative standard deviation,RSD)≤10%;抗原水平准确度和重复性回收率均在90%~110%,重复性验证RSD≤10%;灵敏度12.50μg/mL的回收率均在80%~120%,RSD≤10%;中间精密度验证RSD≤10%。结论建立的测定PT、FHA和PRN精制抗原纯度的SDS-PAGE法,其系统适用性、准确度、重复性、中间精密度和灵敏度均良好,可用于以上精制抗原的纯度检测。Objective To verify a SDS-PAGE method for determination of purity of pertussis toxin(PT),filamentous hemagglutinin(FHA)and pertactin(PRN).Methods After SDS-PAGE electrophoresis and Coomassie brilliant blue staining,the samples were photographed by a gel imaging system and analyzed by Image Lab software to obtain the optical density values of each protein band in the sample.The established methods were validated for system suitability,linearity,accuracy,repeatability,intermediate precision,sensitivity,and robustness.Results Corresponding to two sets of molecular weight standards,PT,FHA and PRN refined antigens had a good linear relationship between the protein concentration and the optical density of the corresponding bands in the low concentration range of 10.00-50.00μg/mL and the high concentration range of 50.00-600.00μg/mL respectively,with a coefficient of correlation(r)≥0.99.The accuracy of impurity levels and repeatability recovery rates for the three refined antigens ranged between 80%and 120%,and the relative standard deviation(RSD)for repeatability validation was≤10%.The accuracy of antigen levels and repeatability recovery rates were between 90%and 110%,and the RSD for repeatability validation was≤10%.The recovery rates of 12.50μg/mL for sensitivity were between 80%and 120%,with RSD≤10%.The RSD for intermediate precision was≤10%.Conclusion The established SDS-PAGE methods for determining the purity of PT,FHA and PRN refined antigens were validated and the system applicability,accuracy,repeatability,intermediate precision and sensitivity of this method were good.

关 键 词：百日咳毒素 丝状血凝素 百日咳黏附素 SDS-聚丙烯酰胺凝胶电泳 验证 

分 类 号：R392-33[医药卫生—免疫学]