Seasonal influenza is an acute respiratory infection causing around 500 000 global deaths annually. There is an unmet medical need to develop more effective antiviral drugs and vaccines against influenza infection. A rapid, accurate, high‐throughput titration assay for influenza virus particles or neutralizing antibodies would be extremely useful in these research fields. However, commonly used methods such as tissue culture infective dose and plaque‐forming units (PFU) for virus particle quantification, and the plaque reduction neutralization test (PRNT) for antibody determination are time‐consuming, laborious, and have limited accuracy. In this study, we developed an efficient assay based on the enzyme‐linked immunospot (ELISPOT) technique for the influenza virus and neutralizing antibody titration. Two broad‐spectrum antibodies recognizing the nucleoproteins of influenza A and B viruses were used in the assay to broadly and highly sensitively detect influenza virus‐infected cells at 16 hours postinfection. An optimized cell culture medium with no tosyl phenylalanyl chloromethyl ketone trypsin and high dose oseltamivir acid was used to improve quantitation accuracy. This ELISPOT assay displayed a good correlation (R² = 0.9851) with the PFU assay when used to titrate 30 influenza virus isolates. The assay was also applied to measure influenza‐neutralizing antibodies in 40 human sera samples, showing a good correlation (R² = 0.9965) with the PRNT assay. This ELISPOT titration assay is a rapid, accurate, high‐throughput assay for quantification of influenza virus and neutralizing antibodies, and provides a powerful tool for research into and development of drugs and vaccines against influenza.

中文翻译：

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流感病毒滴定和中和抗体检测快速ELISPOT检测方法的建立

季节性流感是一种急性呼吸道感染，每年导致全球约 50 万人死亡。开发更有效的抗病毒药物和针对流感感染的疫苗的医疗需求尚未得到满足。对流感病毒颗粒或中和抗体进行快速、准确、高通量的滴定测定在这些研究领域将非常有用。然而，常用的方法如用于病毒颗粒定量的组织培养感染剂量和空斑形成单位（PFU）以及用于抗体测定的空斑减少中和试验（PRNT）等方法耗时、费力且准确性有限。在本研究中，我们开发了一种基于酶联免疫斑点 (ELISPOT) 技术的流感病毒和中和抗体滴定的有效检测方法。该检测中使用了两种识别甲型和乙型流感病毒核蛋白的广谱抗体，可在感染后 16 小时广泛且高灵敏度地检测流感病毒感染的细胞。使用不含甲苯磺酰苯丙氨酰氯甲基酮胰蛋白酶和高剂量奥司他韦酸的优化细胞培养基来提高定量准确性。 当用于滴定 30 种流感病毒分离株时，该 ELISPOT 测定与 PFU 测定显示出良好的相关性 (R ^{2 = 0.9851)。}该测定还用于测量 40 份人类血清样本中的流感中和抗体，显示 与 PRNT 测定具有良好的相关性（R ^{2 = 0.9965）。}该ELISPOT滴定测定是一种快速、准确、高通量的流感病毒和中和抗体定量测定方法，为流感药物和疫苗的研究和开发提供了强大的工具。