柯萨奇病毒A组16型抗原的ELISA定量检测方法建立

摘要

Objective:To develop an a quantitative enzyme linked immunosorbent assay (Q-ELISA) to determine the concentration of Coxsackievirus A Group 16 Strain ( CA16) antigen. This method was used to determine CA16 antigen content at each stage of CA16 vaccine developing and manufacturing process. Methods:A double antibody sandwich Q-ELISA was developed to determine concentration of CA16 antigen,which was based on the high-affinity neutralizing monoclonal antibodies T26H12 as capture antibodies ,and NA14B9 as HRP-labeled antibody. The performance of reagent were evaluated. ResultS;The Q-ELISA for CA16 antigen content was successfully developed. The reagent had good performance. The quantitation scope was 8-128 ng/ml, the coefficient correlation was 0. 998, the limit of detection was 8 ng/ml,the recovery was between 87% and 113. 8% . The stability was up to 80% after reagent was heated for 6 days at 37℃ . The variation coefficient was lower than 15% ,and thereagent was no reaction with other sample except CA16 antigen. Conclusion;The Q-ELISA for CA16 antigen was developed with good specificity, accuracy, precision and stability. The method can be used to determine CA16 antigen content during development and production of CA16 vaccine.%目的:建立柯萨奇病毒A组16型(CA16)抗原的双抗体夹心ELISA定量检测方法,用于CA16灭活疫苗的研发和生产过程的抗原定量检测.方法:以CA16中和单抗T26H12为包被抗体、NA14B9为标酶抗体,构建定量检测CA16抗原的双抗体夹心ELISA方法,并对方法的特异性、灵敏度、精密度、准确性、线性和稳定性进行分析.结果:建立了双抗体夹心定量检测CA16抗原的ELISA方法.方法的线性相关系数R2=0.998,线性范围为8~128 ng/ml,定量限度为8 ng/ml;变异系数CV<15%;回收率介于87.0%~113.8%之间;37℃6天的回收率>80%;与CA16以外的其他样本没有交叉反应.结论:构建的CA16抗原ELISA定量检测方法的各项性能符合定量检测需要,可用于CA16疫苗的研发和生产过程的抗原活性的定量检测.