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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 establish a method for the determination of pertussis toxin (PT) activity in acellular pertussis vaccine (APV). Methods The ELISA method for the determination of PT content in APV was optimized and the effect of different antigen dilutions on this method was analyzed. The linearity, precision, sensitivity and specificity of this method were also validated. Analysis of filamentous hematocrit, FHA, pertactin (Prn), diphtheria toxoid (DT), and tetanus toxoid (TT) at the finished vaccine dose (ie, 50 μg / ml, 16 μg / ml, 25 Lf / ml, 7 Lf / ml) on the binding of PT to fetuin. The desorption conditions of the vaccine were determined and 8 batches of adsorbed cell free diphtheria-binding vaccine from different manufacturers were analyzed Attached, the detection of PT content, calculate CV. Results The samples were diluted with pH 8.5 Tris buffer containing 1% fetal bovine serum albumin (BSA) when the fetuin coating concentration was 5μg / ml, and then directly detected by enzyme-labeled anti-PT antibody. Fetal globulin binding assay ELISA detection method of the dose response curve was good (r> 0.99); test CV was 13.74% ~ 5.85%, CV between the test 2.75% ~ 10.39%, the sensitivity of up to 4 ng / ml, precision Good degree of sensitivity and high; reduce PTd interference, specificity is better. FHA, Prn, DT, TT had no effect on the binding of PT to fetuin at the finished vaccine dose; the desorption conditions for the vaccine were determined to be Tris-sodium citrate solution (4%, w / v) The results of the three batches of vaccine CV values ?? are in line with the requirements, reproducibility is better. Conclusion The optimized test method of fetuin-binding ELISA can be used to detect the residual PT in APV.