Influenza Virus Titration, Antigenic Characterization, and Serological Methods for Antibody Detection
This chapter describes some commonly used methods of influenza virus titration, antigenic characterization, and serological methods by antibody detection. These methods are essential not only for virus characterization but also for identifying new antigenic variants, vaccine strain selection, and sero-epidemiologic studies of influenza virus transmission and prevalence. Virus titration methods such as the hemagglutination assay, 50% egg or tissue culture infectious dose, and plaque assay are employed to determine the amount of virus particles in a sample. The hemagglutination inhibition assay is a reliable, relatively simple and inexpensive technique to antigenically characterize isolates of influenza viruses. Serological methods such as virus neutralization and hemagglutination inhibition are the fundamental tools used in sero-epidemiologic studies of influenza virus transmission and prevalence and in the evaluation of vaccine immunogenicity. While serological methods rarely yield an early diagnosis of acute influenza virus infection, well-timed, paired acute, and convalescent serum samples may establish the diagnosis of a recent influenza infection even when attempts to detect the virus are negative.
Key wordsVirus titration Hemagglutination assay Plaque assay 50% Egg infectious dose Hemagglutination inhibition assay 50% Tissue culture infectious dose Microneutralization Virus neutralization Neutralizing antibodies Influenza
The microneutralization protocol presented here is based on the work of Thomas Rowe and others who developed the original microneutralization protocol for avian influenza H5N1 viruses. Thanks is expressed to Yaohui Bai and Li Cronin for their work determining the number of MDCK cells needed to obtain flasks of cells at the appropriate confluency for the microneutralization assay.
- 3.Miller, G.L. (1965) Improved measurement of influenza virus hemagglutinin titer. J. of Immuno. 95, 336–344.Google Scholar
- 4.Reed, L.J. and H. Muench. (1938) A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493–497.Google Scholar
- 7.Hirst, G.K. (1942) The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J. Exp. Med. 75, 47–64.Google Scholar
- 8.Salk, J. E. (1944) Simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J. Immun. 49, 87.Google Scholar
- 9.Rowe, T., Abernathy, R. A., Hu-Primmer, J., Thompson, W.W., Lu, X., Lim, W., Fukuda, K., Cox, N. J., and Katz, J. M. (1999) Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Micro. 37, 937–343.Google Scholar
- 11.Stephenson, I., Heath, A., Major, D., Newman, R. W., Hoschler, K., Junzi, W., Katz, J. M., Weir, J. P., Zambon, M. C., and Wood, J. M. (2009) Reproducibility of serologic assays for influenza virus A (H5N1). EID 15, 1250–1259.Google Scholar