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Updated: Jun 23, 2026
Optimization of a Quantitative Micro-neutralization Assay
Published on: December 14, 2016
A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses
Liisa Lehtoranta1, Anja Villberg, Riitta Santanen
1Viral Infectious Unit, Department of Vaccination and Immune Protection, National Institute for Health and Welfare, Mannerheimintie 166, 00300 Helsinki, Finland. liisa.lehtoranta@gmail.com
Insights
A new colorimetric assay effectively measures neutralizing antibodies to influenza viruses in human sera. This method provides higher antibody titers than traditional tests, proving valuable for influenza vaccine studies.
Area of Science:
- Virology
- Immunology
- Assay Development
Background:
- Influenza viruses pose a significant public health threat, necessitating robust methods for evaluating vaccine efficacy.
- Accurate measurement of neutralizing antibodies is crucial for assessing immune responses to influenza vaccines.
Purpose of the Study:
- To describe a novel colorimetric cell proliferation assay for quantifying neutralizing antibodies against influenza viruses in human serum.
- To compare the performance of this new assay with the established hemagglutination inhibition assay.
Main Methods:
- Human sera were incubated with influenza viruses and then added to Madin-Darby canine kidney cells in 96-well plates.
- Cell viability was assessed using a tetrazolium salt, which is converted to formazan by mitochondrial dehydrogenases, producing a color change read spectrophotometrically.
- The assay was validated by studying antibody responses post-influenza vaccination and comparing titers with hemagglutination inhibition results.
Main Results:
- Absorbance values directly correlated with viable cell counts and neutralizing antibody activity.
- The colorimetric neutralization test yielded higher pre- and post-vaccination antibody titers compared to the hemagglutination inhibition assay.
- A greater number of significant increases in post-vaccination antibody titers were observed with the new assay.
Conclusions:
- The developed colorimetric cell proliferation assay is a sensitive and efficient method for measuring neutralizing antibodies to influenza viruses.
- This assay offers advantages in terms of throughput, automation potential, and suitability for biosafety level-3 conditions.
- The assay serves as a valuable laboratory tool for influenza vaccine research and development.
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.
