A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.

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.

Influenza vaccination of dialysis patients: cross-reactivity of induced haemagglutination-inhibiting antibodies to H3N2 subtype antigenic variants is comparable with the response of naturally infected young healthy adults.

BACKGROUND: Annual influenza vaccination is recommended for patients with chronic renal failure, although vaccination responses in haemodialysis (HD) patients may be suboptimal. Typically, the seroreactivity has been analysed against the vaccine virus or the corresponding year's epidemic virus. No studies analysing cross-reactivity against subsequent years' viruses have been presented. METHODS: Twenty-three chronic HD patients and 26 cardiac patients were, in autumn 1995, vaccinated with a trivalent influenza vaccine. The cross-reacting haemagglutination-inhibiting antibodies to five consecutive years' (the last season 1999-2000) drift variants of H3N2 subtype influenza A virus were measured and compared with those of vaccinated cardiac patients and with those of 26 healthy military conscripts who suffered a serologically confirmed influenza A infection in the season 1995-1996. RESULTS: The influenza vaccination in HD patients resulted in comparable cross-reacting antibodies to the antibodies induced both by vaccination in cardiac patients and by natural infection in military conscripts. After a steady decline, the cross-reactivity to the latest epidemic virus improved in all the groups. This may be due to two reverted amino acid changes in the HA1 domain of the virus haemagglutinin. CONCLUSIONS: Influenza vaccination in HD patients is as effective as the vaccination of cardiac patients with normal kidney function. The cross-reactivity of vaccination-induced antibodies is even as good as that of antibodies induced by natural infection of young healthy males. Additionally, vaccination seems to prime the individual beneficially against subsequent years' influenza viruses.