title: A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.
abstract: A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum-virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies.
authors.affiliation: Viral Infectious Unit, Department of Vaccination and Immune Protection, National Institute for Health and Welfare, Mannerheimintie 166, 00300 Helsinki, Finland. liisa.lehtoranta@gmail.com
authors.forename: Liisa
authors.initials: L
authors.lastname: Lehtoranta
authors.forename: Anja
authors.initials: A
authors.lastname: Villberg
authors.forename: Riitta
authors.initials: R
authors.lastname: Santanen
authors.forename: Thedi
authors.initials: T
authors.lastname: Ziegler
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D002460:Cell Line; D002470:Cell Survival; D003124:Colorimetry / Q000379:methods; D004285:Dogs; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D009500:Neutralization Tests; D009975:Orthomyxoviridae / Q000276:immunology*; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D013778:Tetrazolium Salts