摘要
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 develop a quantitative ELISA for monitoring the manufacturing process of enterovirus 71 vaccine.Methods A double antibody sandwich ELISA was developed by using a monoclonal antibody against EV71 as capture antibody and a HRP conjugated rabbit against EV71 antibody as detecting antibody. The ELISA kit was tested for linear range and validated for accuracy,precision,specificity,robustness and stability. The EV71 antigen yields were determined by the ELISA in tested samples in preparation of bulk of EV71 vaccine（ 2BS） and EV71 vaccine（ Vero）. Results The linear range of the developed ELISA was 3. 125- 50. 000 U / m L,with a value of r2 more than 0. 99. The recovery rate of samples was 92. 0%- 110. 0%,with a CV less than 8. 8%. All the A values of antigens in harvest of CVA16,CVA10,CVA6 and so on were less than the cu-toff value. The recovery rates were in a range between 100. 8% and 113. 8% while parameters such as incubation time and temperature varied within the certain range. After the ELISA kit was stored at 37 ℃ for three days,the kit＇s reagents were used in detection and the recovery rate of samples was 101. 4%- 112. 4%. In a applicable test,the recovery rate of antigens were between 92. 1% and 114. 9% in spiked antigen-buffers used in preparation of EV71 vaccine bulk by 2BS or Vero cell substrate. Conclusion A double antibody sandwich ELISA in quantitative determination of EV71 antigen was developed,which showed high accuracy,precision,specificity,robustness and stability,and laid a foundation for development of EV71 vaccine and multivalent HFMD vaccine.
出处
《微生物学免疫学进展》
2016年第1期1-6,共6页
Progress In Microbiology and Immunology
基金
国家科技重大专项"重大新药创制"课题(2013ZX09402-302)
武汉市2013年高新技术成果转化及产业化项目(2013060803010394)
卫计委的重大新药创制课题(2015ZX09102021)
