Mab8258

Virus neutralizing activity was determined using a WHO-recommended neutralization assay [24 (link)]. After mAb samples were serial diluted in 96-well microplates, 100 TCID50 of influenza virus was added and incubated at 37C with 5% CO₂ for 2 h before adding MDCK cells to assay plates. After 18–22 h, MDCK cells were fixed with 80% cold acetone and incubated with murine monoclonal anti-influenza A nucleoprotein (1:1 mixture of MAB8257 and MAB8258, Millipore Billerica, MA, USA) followed by peroxidase-conjugated goat anti-mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). Optical density following development with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Sigma Aldrich, Saint Louis, MO, USA) was used to calculate the 50% neutralization titer (IC50) for each sample.

MDCK cells were grown as a 50% confluent monolayer in 96-well black imaging plates with clear bottom (Costar). Wild type (WT) or mutant A/Perth/16/2009 virus was diluted in Flu Media to an MOI of 1 and incubated for 1 hour at 37°C with varying concentrations of 39.29. The antibody/virus cocktail was allowed to infect the MDCK cells for 16 hours at 37°C in a 5% CO₂ incubator prior to fixation with cold 100% ethanol. The fixed cells were then stained with Hoechst 33342 (Invitrogen Cat# H3570) to visualize cell nuclei and determine total cell number. The cells were also stained with a monoclonal antibody specific for IAV nucleoprotein (NP) (Millipore Cat# MAB8258) to determine the number of infected cells. Cells were imaged using an Image Express Micro apparatus (Molecular Devices) and data images were analyzed using the software MetaXpress 3.1. The percentages of infected cells (normalized to the lowest antibody concentration) were determined and plotted on the Y-axis versus the Log10 antibody concentrations on the X-axis. The data were fit with a nonlinear regression dose response curve using the GraphPad Prism v.6.0 software.

An in vitro virus micro-neutralisation assay, based on the concept of plaque reduction, was conducted. Briefly, after 50 μl of twofold serial dilutions of the ferret antisera in DMEM containing 200 nM oseltamivir was added to a monolayer of MDCK-SIAT1 cells in a 96-well plate, 1,000 focus-forming units of the indicated virus (50 µl) was added to each well. After a 1 hour incubation, 1.6% Avicel RC-581 (FMC BioPolymer) in DMEM was overlaid. Oseltamivir was used to prevent receptor binding via NA, and trypsin was not added to avoid virus multiplication. At 18‒20 hours after infection, the cells were stained with mouse anti-nucleoprotein (NP) monoclonal antibodies (MAB8257 and MAB8258; Millipore, Consett, US), followed by an anti-mouse IgG antibody conjugated with horseradish peroxidase. NP-positive cells were visualised with KPL TrueBlue substrate (Kirkegaard and Perry Laboratories) to count foci. The reciprocal number of the minimum dilution of sera needed to achieve 50% focus reduction was used as the neutralisation titre.