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.
Bibliographical noteFunding Information:
This research was supported by grants from the Ministry of Food and Drug Safety, Republic of Korea ( 16172MFDS199 and 18172MFDS252 ) and the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea ( HI13C0826 ). We thank Dr. Dong-wook Kim (Department of Statistics, Sungkyunkwan University, Seoul, Korea) for statistical analyses of data. The authors declare no conflict of interest.
National Research Council Committee on Methods of Producing Monoclonal A. The National Academies Collection: Reports funded by National Institutes of Health. Monoclonal Antibody Production. Washington (DC): National Academies Press (US) National Academy of Sciences; 1999.
All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Immunology and Microbiology(all)
- Public Health, Environmental and Occupational Health
- Infectious Diseases