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Methods. 2015 Nov 15;90:95-100. doi: 10.1016/j.ymeth.2015.04.037. Epub 2015 May 6.

Measuring the neutralization potency of influenza A virus hemagglutinin stalk/stem-binding antibodies in polyclonal preparations by microneutralization assay.

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Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA; The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Department of Biochemistry and Biomedical Sciences, Institute for Infectious Diseases Research, McMaster Immunology Research Centre, McMaster University, Hamilton, Ontario, Canada. Electronic address:


The discovery of broadly-neutralizing antibodies that bind to the hemagglutinin stalk/stem domain has opened exciting new avenues for the development of "universal" influenza virus vaccines and therapeutics. Unlike strain-specific antibodies which bind to the hemagglutinin head domain and inhibit receptor binding, antibodies that bind to the stalk domain function to inhibit later stages of infection. The hemagglutination inhibition assay has long been the standard for evaluating titers of neutralizing hemagglutinin-specific antibodies in serum. The assay has the beneficial properties of being relatively rapid, easy-to-perform, and requires very little specialized equipment. Historically, hemagglutination inhibition titers of 40 or above against a given strain of influenza has been considered a correlate of protection on a population level. Unfortunately, this assay cannot be used to measure titers of hemagglutinin stalk-specific antibodies due to their lack of hemagglutination inhibiting activity. This has necessitated the development of novel reagents and assays capable of sensitive and specific detection of broadly-neutralizing HA stalk-binding antibodies in polyclonal mixtures. Here, we describe a novel microneutralization-based assay that utilizes recombinant influenza A viruses expressing chimeric hemagglutinin molecules and 'exotic' neuraminidase to measure titers of broadly-neutralizing antibodies in polyclonal preparations.


Broadly neutralizing antibodies; Hemagglutinin; Influenza A virus; Microneutralization; Polyclonal; Universal vaccine

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