Improved high-throughput virus neutralisation assay for antibody estimation against pandemic and seasonal influenza strains from 2009 to 2011

• We developed a focus-reduction neutralisation test for influenza viruses.
• This assay uses automated plaque counting of foci by ViruSpot software.
• Pre-treatment of the human sera samples with RDE reduced the neutralising titres.
• The HI titre of 1:40 was equivalent to a neutralising titre of 1:100–1:200.

An automatable focus-reduction neutralisation test (AFRNT) for detecting influenza neutralising antibodies in serum was developed. The assay used immunoperoxidase staining and automated foci counting with AID Diagnostika ViruSpot software. Human serum samples (n = 108) were collected before and after vaccination with Pandemrix or Begrivac and were tested by AFRNT and a haemagglutination inhibition assay (HI) using seasonal and pandemic influenza vaccine strains from 2009 to 2011.Much attention has been given to the factors that influence detection of neutralising titre, such as viral quantification and the use of receptor destroying enzyme (RDE) for serum treatment. Foci counting enabled precise virus quantification and the development of a highly sensitive assay. Pre-treatment of the human sera with RDE significantly reduced the neutralising titres against all strains, with the exception of the seasonal H1N1 (2009/2010) strain. An HI titre of 1:40, which is associated with a 50% clinical protection against influenza, was equivalent to an AFRNT titre of 1:100–1:200.In conclusion, the AFRNT is rapid, highly sensitive, and fully automatable; therefore, this test is perfectly suitable for the high-throughput detection of influenza-neutralising antibodies.