Characterizing Influenza Antibody Inhibition

Assays to characterize the ability of mAbs to inhibit influenza virus hemagglutination or neutralization were performed as described previously [28 (link)]. The HI assay was performed in 96-well plates (U-bottom) by standard methods, using 0.5% chicken red blood cells suspended in PBS (pH 7.2). The plaque reduction neutralization assay was performed in MDCK cells with a 50:50 mixture of 2X EMEM plaque media (BioWhittaker, Walkersville, MD) and 2.4% Avicel RC-581 (FMC BioPolymer, Philadelphia, PA) in the presence of TPCK-trypsin (Sigma-Aldrich Corp. St. Louis, MO) at a final concentration of 2 μg/ml. Retroviral pseudotypes expressing the HA of influenza B were constructed and produced in 293T cells essentially as described previously [29 (link), 30 (link)]. The antibody dilution causing a 95% reduction of vector expressed luciferase compared to control was used as the neutralization endpoint titer (IC₉₅-neutralizing antibody titer).