Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Republic of Korea; Vaccine Translational Research Center, Yonsei University, Republic of Korea.
Department of Integrated OMICS for Biomedical Science, College of World Class University, Yonsei University, Republic of Korea; Vaccine Translational Research Center, Yonsei University, Republic of Korea.
Vaccine. 2019 Mar 7;37(11):1457-1466. doi: 10.1016/j.vaccine.2019.01.068. Epub 2019 Feb 11.
Seasonal and pandemic influenza infections remain a serious public health concern. Many health authorities recommend annual vaccination as the most effective way to control influenza infection. Accordingly, regulatory guidelines ask vaccine manufacturers to determine vaccine potency at the time of release and throughout shelf-life to ensure vaccine quality. The potency of inactivated influenza vaccine is related to the quantity of hemagglutinin (HA). Since 1970s, single radial immunodiffusion (SRID) assay has been standardly used for the quantitation of HA in influenza vaccine. However, SRID is labor-intensive, inaccurate, and requires standard reference reagents that should be updated annually. Therefore, there have been extensive efforts to develop alternative potency assays. In this study, we developed and tested a new HA quantitative enzyme-linked immunosorbent assay (ELISA) using a universal monoclonal antibody that can bind to HAs from various subtypes in group 1 influenza A virus (IAV). We analyzed the conserved stalk domain of HA via a library approach to design a consensus HA antigen for group 1 IAV. The antigens were expressed as a soluble form in E. coli and were purified by Ni-affinity chromatography. When tested with variety of HAs from IAVs or influenza B viruses (IBVs), the mAbs exhibited specific binding to group 1 HAs, with potential exception to H9 subtype. Among various conditions of pH, urea, and reducing agents, pretreatment of HA at low pH exposing the conserved stalk domain was crucially important for optimal ELISA performance. Calibration curves for various HAs were generated to determine accuracy, specificity, sensitivity, and linear dynamic range. The ELISA method shows high sensitivity and accuracy compared with the SRID assay. The HA group specific universal mAbs against the consensus stalk domain of HA are conducive to establishing an ELISA-based standard procedure for the quantitation of HA antigens for annual vaccination against influenza infection.
季节性和大流行性流感感染仍然是一个严重的公共卫生问题。许多卫生当局建议每年接种疫苗，这是控制流感感染的最有效方法。因此，监管指南要求疫苗制造商在发布时以及整个保质期内确定疫苗效力，以确保疫苗质量。灭活流感疫苗的效力与血凝素（HA）的数量有关。自 20 世纪 70 年代以来，单扩散免疫沉淀（SRID）测定法已被常规用于流感疫苗中 HA 的定量。然而，SRID 劳动强度大，不准确，并且需要每年更新的标准参考试剂。因此，人们一直在努力开发替代效力测定法。在这项研究中，我们使用能够结合甲型流感病毒（IAV）组 1 中各种亚型的 HA 的通用单克隆抗体开发并测试了一种新的 HA 定量酶联免疫吸附测定（ELISA）。我们通过文库方法分析 HA 的保守茎结构域，以设计针对组 1 IAV 的共识 HA 抗原。抗原以可溶形式在大肠杆菌中表达，并通过 Ni 亲和力层析进行纯化。用来自 IAV 或流感 B 病毒（IBV）的各种 HA 进行测试时，该 mAb 表现出对组 1 HA 的特异性结合，除 H9 亚型外，可能还有其他例外。在各种 pH、尿素和还原剂条件下，HA 在低 pH 下预处理以暴露保守的茎结构域对于最佳 ELISA 性能至关重要。针对各种 HA 生成校准曲线以确定准确性、特异性、灵敏度和线性动态范围。与 SRID 测定法相比，ELISA 方法显示出更高的灵敏度和准确性。针对 HA 保守茎结构域的通用 mAb 有利于建立基于 ELISA 的 HA 抗原定量标准程序，用于每年预防流感感染的疫苗接种。
