Quantifying Influenza Neutralizing Antibodies

Pooled, heat-inactivated, and RDE-treated sera (starting concentration 1:50 and serially diluted 2-fold) and viruses (1000 PFUs) were preincubated at room temperature for 1 h to allow antibodies to bind to virions.^{42 (link)} After incubation, the mixture was added to monolayers of MDCK cells in 96-well tissue culture plates and incubated at 37 °C for 1 h to allow for attachment of virions to the cells. After washing with PBS three times to remove non-attached virions, the plates were re-incubated at 37 °C with infection medium containing the appropriate serum dilution. Eighteen hours later, the cells were fixed with 80% acetone in PBS and then stained for the NP protein using a primary biotinylated antibody (EMD Millipore; Cat. no. MAB8257B) (1:2000) and a secondary streptavidin conjugated to HRP (EMD Millipore; Cat. no. 21130) (1:5000). Wells were developed by incubating with SigmaFast OPD (Sigma-Aldrich) for 20 min. Reactions were stopped by adding 3 M HCl and absorbance at 490 nm was determined on a Synergy 4 plate reader (BioTek). Endpoint titers were defined as the reciprocal of the highest serum dilution that neutralized virus.