Enzyme-Linked Lectin Assay (ELLA)

Neuraminidase inhibiting Ab titers were determined in serum and BAL fluid using an Enzyme-linked lectin assay (ELLA). Ninety six-well microtiter plates (Maxi Sorp, Nunc, Sigma-Aldrich, UK) were coated with 50 μl/well of 25 μg/ml fetuin and incubated at 4°C overnight. Heat inactivated sera samples were serially diluted in a separate 96-well plate. An equal volume of (H7(Net219) N1(Eng195) S-FLU (H7N1 S-FLU) (kindly provided by Professor Alain Townsend, University of Oxford) was added to each well and incubated at room temperature on a rocking platform for 20 min. The H7N1 S-FLU was titered beforehand in the absence of serum to determine optimal concentration for the assay. Fetuin plates were washed with PBS four times before 100 μl/well of the serum/virus mix was transferred and incubated overnight at 37°C. The serum/virus mix was removed, and the plate washed four times with PBS before adding 50 μl/well of Peanut Agglutinin-HRP at 1 μg/ml and incubating for 90 min at room temperature on a rocking platform. Plates were washed and 50 μl/well of TMB High Sensitivity substrate solution (BioLegend, UK) was added. Plates were developed for 6 min, the reaction stopped with 50 μl of 1 M H₂SO₄ and the plates were read at 450 and 630 nm using a Biotek Elx808 reader. Samples were measured as end titer representing the highest dilution with signal greater than cut-off. The cut off value was defined as the average of all blank wells plus three times the standard deviation of the blank wells.