A novel serological assay for influenza based on DiD fluorescence dequenching that is free from observer bias and potentially automatable - A proof of concept study.

BACKGROUND: Serum hemagglutination inhibition (HAI) and microneutralization (MN) antibodies are often used as a correlate of protection for influenza. However, these manual assays are labor-intensive and difficult to standardize due to variability in biologic reagents used and subjective interpretation of the results.

METHODS: Sera with known HAI and MN titers were used to assess a novel test based on the inhibition of fluorescence 'dequenching'. Whole influenza virions (A/California/07/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2) and B/Brisbane/60/2008) labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) were exposed to serial dilutions of serum and mixed with turkey red blood cells followed by acidification of the media (pH 5.0-5.5). The H1N1 and B/Brisbane strains were high hemagglutinating while the H3N2 strain had low hemagglutinating activity. In some experiments, labelled virions were subjected to repetitive freeze-thaw cycles prior to use in the assay.

RESULTS: In the absence of detectable HAI/MN antibodies, there were consistent and substantial increases from baseline DiD fluorescence upon acidification. Sera with known high titer HAI/MN antibodies reduced or completely prevented DiD dequenching at low dilutions with progressive increases in fluorescence at higher dilutions, which permitted a reproducible assignment of an antibody 'titer' based on baseline and acidified DiD fluorescence values. The 'titers' measured by the DiD dequenching assay were highly correlated with HAI/MN results for the H1N1 and B strains (Spearman's correlation coefficients (rs ) 0.874 to 0.946, p < 10-7 to 10-35 ). Correlations with HAI/MN titres for the low-hemagglutinating H3N2 strain tested were lower but remained statistically significant (rs 0.547-0.551, p < 0.004). Freeze-thawing of the DiD pre-stained virus stocks had no significant impact on the results of the assay.

CONCLUSIONS: The DiD dequenching assay may be a labour-saving and more objective alternative to the classic serologies. This novel assay could theoretically be standardized across laboratories using pre-stained virions and has the potential to be fully automated.