Enzyme-linked lectin assay

Enzyme-linked lectin assays were performed as previously described (59). Ninety-six-well plates (Nunc) were coated with 25 µg/µL fetuin in PBS and stored at 4°C overnight. In a separate dilution plate, heat-inactivated ferret serum (starting dilution of 1:160) was serially diluted and mixed with an inactivated recombinant H6N1 virus containing the HA from A/turkey/Massachusetts/3740/1965 (H6N2), NA from A/California/07/2009 (H1N1pdm09), and the remaining six genes from the A/Puerto Rico/8/1934 (H1N1) virus diluted (1:5000) in PBS with calcium and magnesium, bovine serum albumin (BSA, 1.0%), and Tween-20 (0.05%). Fetuin-coated plates were washed with PBST (0.05% tween), and the serum-virus mixture was transferred from the dilution plate to the fetuin-coated plates. Plates were incubated overnight at 37°C. The following day, plates were washed with PBST and incubated with biotinylated lectin from Arachis hypogaea (PNA-Bio, Sigma) diluted 1:500 in PBS with calcium and magnesium and BSA (1.0%) for 2 hours at room temperature in the dark. After washing, streptavidin-HRP (1:500, Millipore) in PBS with calcium and magnesium and BSA (1.0%) was added and incubated for 1 hour at room temperature in the dark. OPD substrate solution (Sigma) was added to all wells, and 1 N sulfuric acid was added to stop the reaction after 10 minutes. Optical density was measured at 490 nm on a SpectraMax iD3 plate reader (Molecular Devices), and end point titers were calculated by determining the serum dilution that resulted in at least 50% inhibition of the maximum signal.