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目的建立肠道病毒71型(EV71)抗原ELISA定量检测方法,用于EV71疫苗生产工艺中抗原定量检测。方法选取抗EV71单克隆抗体作为包被抗体,HRP标记抗EV71多克隆抗体作为酶标二抗,建立EV71抗原ELISA定量检测方法。该方法经系统验证后,应用于定量检测2BS及Vero细胞基质EV71疫苗生产工艺过程样品的抗原含量。结果验证结果显示,该方法的线性范围为3.125~50.000 U/m L,样品回收率为92.0%~110.0%,变异系数小于8.8%;孵育时间及温度在一定范围内偏差的样品回收率为100.8%~113.8%;检测其他肠道病毒中抗原其A值均小于cut-off值;试剂于37℃孵育3 d后的检测样品回收率为101.4%~112.4%;2BS及Vero细胞基质EV71疫苗生产工艺过程样品的适用性检测中,抗原回收率为92.1%~114.9%。结论建立并验证EV71抗原ELISA定量检测方法,其准确度、精密度、耐用性均优良,特异性强、稳定性好,适用于不同细胞基质的EV71疫苗及多价手足口病疫苗生产工艺过程的质量控制。
Objective To establish a quantitative ELISA method for the detection of enterovirus 71 (EV71) antigen for the quantitative detection of antigen in the EV71 vaccine production process. Methods Anti-EV71 monoclonal antibody was selected as coating antibody and HRP-labeled anti-EV71 polyclonal antibody as ELISA secondary antibody to establish quantitative detection method of EV71 antigen ELISA. The method is validated by the system and applied to quantitatively detect the antigenic content of the samples in the production process of 2BS and Vero cell matrix EV71 vaccine. The results showed that the linear range of this method was 3.125 ~ 50.000 U / m L, the recovery rate was 92.0% ~ 110.0%, the coefficient of variation was less than 8.8%. The recovery rate of samples with deviation of incubation time and temperature was 100.8 % ~ 113.8%. The A value of antigens in other enteroviruses was less than cut-off value. The recovery rate of test samples after incubation for 3 days at 37 ℃ was 101.4% ~ 112.4%. The production of 2BS and Vero cell matrix EV71 vaccine The applicability of the process sample testing, the recovery rate of the antigen was 92.1% ~ 114.9%. Conclusion The method for the quantitative detection of EV71 antigen was established and validated. The accuracy, precision and durability of EV71 antigen ELISA were both excellent, specific and stable. The EV71 vaccine and multi-valent hand-foot-and-mouth disease vaccine production process QC.