无细胞百日咳疫苗中百日咳毒素活性检测方法的建立

发布时间：2018-01-13 05:19

本文关键词：无细胞百日咳疫苗中百日咳毒素活性检测方法的建立　出处：《中国生物制品学杂志》2014年12期 　论文类型：期刊论文

【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。
[Abstract]:Objective to establish a method for the detection of pertussis toxin in acellular pertussis vaccine. Methods the ELISA method for the determination of PT residual content in APV was optimized by using the method of fetal globulin binding test (ELISA). The influence of different antigen diluents on the method was analyzed, and the method was linear, precise and sensitive. Specificity verification. The filamentous hemagglutininine (FHAA) and pertussis glutin (Prn) were analyzed. Diphtheria toxoidus, tetanus toxoid. The effect of finished vaccine dosage (50 渭 g / ml ~ (16 渭 g / ml) ~ (16 渭 g / ml ~ (-1))) on the binding of PT to fetal globulin (FG) was determined, and the conditions of vaccine desorption were determined. Eight batches of adsorbed acellular tetanus tetanus vaccine from different manufacturers were analytically attached, PT content was detected, and CV was calculated. The results showed that the concentration of fetal globulin was 5 渭 g / ml. The samples were diluted with pH 8.5 containing 1% fetal bovine serum albumin (BSA) Tris buffer solution and detected directly with enzyme-labeled anti-PT antibody. The dose response curve of the established ELISA assay for fetal globulin binding test was linear to 0.99g. The CV in the test was 13.74 and 5.85, the CV in the test room was 2.75 and 10.39, the sensitivity was up to 4 ng / ml, the precision was good and the sensitivity was high. The interference effect of PTd was reduced, and the specificity of PTd was better. There was no effect on the binding of PT to fetal globulin in the dosage of finished vaccine. The conditions of vaccine desorption were determined as follows: Tris-sodium citrate solution (Tris-sodium citrate solution) = 4w / v / v ~ (-1) H ~ (8.5); The CV values of each batch of vaccines met the requirements and the repeatability was good. Conclusion the optimized ELISA assay for fetal globulin binding test can be used to detect the residual PT content in APV.
【作者单位】：北京天坛生物制品股份有限公司;
【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)
【分类号】：R392
【正文快照】： 百日咳是由鲍特百日咳杆菌引起的一种儿童易感的急性呼吸道传染病,近年来随着百日咳疫苗进入免疫规划[1-2],百日咳在很多国家已得到控制。无细胞百日咳疫苗(acellular pertussis vaccine,APV)逐渐取代全细胞百日咳疫苗(whole pertussis vaccine,WPV),不仅改善了免疫程序,且显

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