Optimisation of a micro-neutralisation assay and its application in antigenic characterisation of influenza viruses.
Optimisation of a micro-neutralisation assay and its application in antigenic characterisation of influenza viruses.
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DOI：
10.1111/irv.12333
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发表时间：
2015-11
影响因子：
4.4
通讯作者：
McCauley JW
中科院分区：
文献类型：
--
作者：
Lin Y;Gu Y;Wharton SA;Whittaker L;Gregory V;Li X;Metin S;Cattle N;Daniels RS;Hay AJ;McCauley JW
识别抗原变异体和选择流感病毒用于疫苗生产在很大程度上是基于使用血凝抑制(HI)试验对流行病毒的血凝素(HA)进行抗原性表征。然而，除了与逃避宿主免疫相关的进化外，在不同细胞底物中繁殖产生的变异可能会使对HI结果的解释复杂化。其目的是进一步发展微量中和(MN)试验，以补充HI试验在流感病毒抗原性鉴定中的作用，以评估新的抗原变异的出现，并加强疫苗病毒的选择。一种基于使用简单成像技术测量感染细胞数量的96孔板空斑减少MN检测方法。2004年至2013年期间分离到的具有代表性的甲型H1N1流感pdm09、甲型H3N2型和乙型流感病毒对空斑减少MN试验的改进包括根据病毒类型或亚型选择最合适的细胞系，以及优化实验设计和数据量化。对MN和HI检测结果的比较表明，补充数据在确定最近的人类甲型H1N1流感病毒pdm09、A(H3N2)和B型病毒之间的真实抗原关系方面具有重要意义。我们的研究表明，与HI法相比，改进的MN法具有一定的优势：它不受A(H3N2)病毒神经氨酸酶(NA)中细胞选择的氨基酸取代的显著影响，并且对于那些生长到低HA滴度和/或经历流产感染导致无法在培养细胞中形成斑块的病毒的抗原性特征特别有用。
The identification of antigenic variants and the selection of influenza viruses for vaccine production are based largely on antigenic characterisation of the haemagglutinin (HA) of circulating viruses using the haemagglutination inhibition (HI) assay. However, in addition to evolution related to escape from host immunity, variants emerging as a result of propagation in different cell substrates can complicate the interpretation of HI results. The objective was to develop further a micro-neutralisation (MN) assay to complement the HI assay in antigenic characterisation of influenza viruses to assess the emergence of new antigenic variants and reinforce the selection of vaccine viruses. A 96-well-plate plaque reduction MN assay based on the measurement of infected cell population using a simple imaging technique. Representative influenza A (H1N1) pdm09, A(H3N2) and B viruses isolated between 2004 and 2013 Improvements to the plaque reduction MN assay included selection of the most suitable cell line according to virus type or subtype, and optimisation of experimental design and data quantitation. Comparisons of the results of MN and HI assays showed the importance of complementary data in determining the true antigenic relationships among recent human influenza A(H1N1)pdm09, A(H3N2) and type B viruses. Our study demonstrates that the improved MN assay has certain advantages over the HI assay: it is not significantly influenced by the cell-selected amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses, and it is particularly useful for antigenic characterisation of viruses which either grow to low HA titre and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.