抗H7N9流感病毒中和抗体快速检测方法的建立及应用

Establishment and Application of Rapid Detection Method for Neutralizing Antibody Against Influenza A(H7N9) Virus

目的:对建立的抗H7N9流感病毒中和抗体快速检测方法进行方法学验证及初步应用。方法:分别采用不同代次细胞对高、中、低不同滴度的阳性血清进行多次平行检测,考察细胞代次对检验结果的影响;采用NIBSC提供的参考品对方法学的特异性进行验证;同时应用抗H7N9的阳性血清检测进一步评估方法的准确性和精密性;采用ELISA-MNT法和血凝抑制(hemagglutination inhibition,HI)试验分别接种H7N9灭活流感疫苗免疫的小鼠血清样本20份,分析两种方法检测结果的相关性。结果:采用ELISA-MNT中和法,使用不同代次MDCK细胞(25、30和35代)检测相同的血清样本的中和抗体滴度结果相同;该方法只对羊抗H7N9的血清具有较高保护力,对其他血清基本没有交叉反应;该方法准确性良好;该方法组间、组内精密性的平均变异系数分别为4%和11%。该方法测定H7N9型流感疫苗免疫后的小鼠血清抗体效价,其结果与HI抗体的相关系数为0. 61,表明两种方法的检测结果之间呈良好的正相关性。结论:建立的微量病毒中和法能够满足H7N9流感病毒中和抗体效价检测的要求,可用于H7N9新型大流行流感疫苗的免疫评价。

Objective:To carry out methodological validation and preliminary application of the rapid detection method for neutralizing antibody against influenza A(H7N9)virus.Methods:Different generations of positive cells were used to perform multiple parallel tests on high,medium and low titers of positive serums,and the effects of cell generation on the test results were investigated.Specificity of the method was verified by the reference material provide by NIBSC.Accuracy and precision of the method were evaluated by anti-H7N9 virus positive serums.ELISA-MNT and hemagglutination inhibition(HI)assay were separately used to test the antibody titers from mouse serums immunized with inactivated influenza H7N9 vaccine and the correlation of the two assays was evaluated.Results:The neutralizing antibody titers of the same serum samples were identical by using the ELISA-MNT neutralization method and different generations of MDCK cells(the 25 th,30 th and 35 th generation).The method only had high protection against the serum of sheep anti-H7N9 and had no cross reaction with other serums.The method had good accuracy and average coefficient variations of intra-assay and inter-assay were 4%and 11.2%,respectively.The correlation coefficient between the antibody titers from mice immunized with H7N9 influenza vaccine measured by the ELISA-MNT assay that obtained from the HI assay was 0.61,indicating that there was a good positive correlation between the detection results of the two methods.Conclusion:The established micro-virus neutralization method can meet the requirement of H7N9 influenza virus neutralizing antibody titer detection,and can be used for immunological evaluation of vaccine for H7N9 new pandemic influenza.