Hemagglutination Inhibition and Plaque Reduction Neutralization Assays

The hemagglutination inhibition assay (HI) was performed in 96-well plates (U-bottom) by a standard method, essentially as described previously [16 ] using 0.5% chicken red blood cells suspended in PBS (pH 7.2). For the plaque reduction neutralization assay, virus was titrated to approximately 250 pfu/ml and incubated with concentrations of mAb from 20 to 0.032 μg/ml for 1 hour at room temperature before infection of MDCK cells. After 1.5 hour adsorption, virus inoculum was removed and the cells overlaid with a 50:50 mixture of 2X EMEM plaque media (BioWhittaker, Walkersville, MD) and 2.4% Avicel RC-581 (FMC BioPolymer, Philadelphia, PA) [17 (link)] with TPCK-trypsin (Sigma-Aldrich Corp. St. Louis, MO) at a final concentration of 2 μg/ml. At 96 hours, the plaque media was removed and the cells fixed and stained with 0.3% crystal violet/20% methanol. Percent neutralization was calculated for each mAb concentration versus no mAb controls.