The neutralisation activity of recombinant mAbs was examined using a modified microneutralisation assay52 . MDCK cells were seeded in 96-well plates at 1.5 × 105 per well. The next day, serial dilutions of recombinant mAbs were incubated in Flu-media with 100TCID50 of WT or mutant B/Phuket/3073/2013 and B/Brisbane/60/2008 viruses for one hour at 37oC, before addition to MDCK cells. After 18–24 h, supernatants were removed, cells were fixed and cellular cytopathicity was visualised by ELISA using mouse anti-influenza B nucleoprotein (1:1000;Abcam) primary and goat anti-mouse HRP-conjugated secondary antibodies. Plates were developed using TMB substrate and read at 450 nm. The concentration of mAb preventing 50% infectivity (IC50) was calculated.
The ability of recombinant mAbs to bind cell-surface HA on infected cells was examined by flow cytometry. MDCK cells were seeded into 6-well plates and infected with ~10,000 TCID50 WT or mutant B/Phuket/3073/2013 and B/Brisbane/60/2008 viruses and incubated at 37 °C for 18–24 h. Cells were resuspended by manual scraping and infectivity confirmed by staining with mouse anti-influenza B nucleoprotein (1:1000; Abcam) and goat anti-mouse Alexa647 (1:5000; ThermoFisher). The binding of human anti-IBV mAbs (5 μg/mL) or an anti-HIV negative control (VRC01) to surface expressed HA was detected using goat anti-human Alexa647 (1:5000; ThermoFisher).
The ability of recombinant mAbs to bind cell-surface HA on infected cells was examined by flow cytometry. MDCK cells were seeded into 6-well plates and infected with ~10,000 TCID50 WT or mutant B/Phuket/3073/2013 and B/Brisbane/60/2008 viruses and incubated at 37 °C for 18–24 h. Cells were resuspended by manual scraping and infectivity confirmed by staining with mouse anti-influenza B nucleoprotein (1:1000; Abcam) and goat anti-mouse Alexa647 (1:5000; ThermoFisher). The binding of human anti-IBV mAbs (5 μg/mL) or an anti-HIV negative control (VRC01) to surface expressed HA was detected using goat anti-human Alexa647 (1:5000; ThermoFisher).