无细胞百日咳疫苗中百日咳毒素活性检测方法的建立  

Development of a method for determination of activity of pertussis toxin in acellular pertussis vaccine

作　　者：夏德菊 潘殊男 杨云凯 周第 金萧 王珣 张萍 肖詹蓉 

机构地区：[1]北京天坛生物制品股份有限公司,北京100024

出　　处：《中国生物制品学杂志》2014年第12期1601-1606,共6页Chinese Journal of Biologicals

基　　金：国家十二五重大新药创制生物药大品种技术升级项目(2012ZX09201301-001)

摘　　要：目的建立检测无细胞百日咳疫苗（acellular pertussis vaccine,APV）中百日咳毒素（pertussis toxin,PT）活性的胎球蛋白结合试验ELISA方法。方法优化APV中PT残余含量的胎球蛋白结合试验ELISA检测方法,分析不同抗原稀释液对该方法的影响,并对该方法进行线性、精密度、灵敏度、特异性验证。分析丝状血凝素（filamentous hemagglutinin,FHA）、百日咳黏着素（pertactin,Prn）、白喉类毒素（diphtheria toxoid,DT）、破伤风类毒素（tetanus toxoid,TT）在成品疫苗剂量时（即50μg/ml、16μg/ml、25 Lf/ml、7 Lf/ml）对PT与胎球蛋白结合的影响,确定疫苗解吸附条件,并对不同厂家的8批吸附无细胞百白破联合疫苗进行解析附后,检测PT含量,计算CV。结果在胎球蛋白包被浓度为5μg/ml时,使用p H 8.5含1%胎牛血清白蛋白（BSA）Tris缓冲液对样品进行稀释后,直接用酶标抗PT抗体进行检测,建立的胎球蛋白结合试验ELISA检测方法的剂量反应曲线线性良好（r均〉0.99）;试验内CV为13.74%-5.85%,试验间CV为2.75%-10.39%,灵敏度可达4 ng/ml,精密度好,灵敏度高;降低了PTd的干扰作用,特异性较好。FHA、Prn、DT、TT在成品疫苗剂量时对PT与胎球蛋白的结合无影响;确定疫苗解吸附条件为Tris-柠檬酸钠溶液（4%,w/v）（p H 8.5）;各批疫苗3次检测结果的CV值均符合要求,重复性较好。结论优化后的胎球蛋白结合试验ELISA检测方法可用于APV中残余PT含量的检测。Objective To develop fetuin binding ELISA for determination of the activity of pertussis toxin（PT）in acellular pertussis vaccine（APV）. Methods Fetuin binding ELISA for determination of residual PT content in APV was optimized,on which the influence of various antigen diluents was analyzed. The method was verified for linearity,precision,sensitivity and specificity. The effects of filamentous hemagglutinin（FHA）,pertactin（Prn）,diphtheria toxoid（DT）,tetanus toxoid（TT）at dosages of final products（50 μg / ml,16 μg / ml,25 Lf / ml and 7 Lf / ml）on binding of PT to fetuin were analyzed,based on which the condition for vaccine desorption was determined. Eight batches of DTa P from various manufacturers were desorbed to detect the PT content and calculate the CV. Results When the fetuin concentration for coating was 5 μg / ml,and the samples were diluted with Tris buffer containing 1% fetal bovine serum albumin（BSA）（p H 8. 5）,the dose-response curve of fetuin binding ELISA method developed with enzyme-labeled anti-PT antibody showed good linearity（each r 〉0. 99）. The CVs in intra- and inter-assays were 13. 74% - 5. 85% and2. 75% - 10. 39% respectively,while the sensitivity reached 4 ng / ml. The developed method showed high precision,sensitivity,and high specificity because of decrease of interference of PTd. FHA,Prn,DT and TT at dosages of final products showed no significant effect on binding of PT to fetuin. The optimal desorption agent was Tris-sodium citrate solution（4%,w / v）（p H 8. 5）. The CVs of three testing results of DTa P vaccine of various batches were promising,indicating high reproducibility of the developed method. Conclusion The developed fetuin binding ELISA method may be used for the determination of residual PT content in APV.

关 键 词：无细胞百日咳疫苗 毒素 胎球蛋白 活性 

分 类 号：R378.42[医药卫生—病原生物学] R392-33[医药卫生—基础医学]