Quantifying CR9114-Competing Antibodies

To determine stem-binding antibody responses, we measured CR9114-competing antibody responses as described before (44 (link)). Briefly, Maxisorp 96-well plates (Merck) were coated with purified polyclonal rabbit anti His-Tag IgG (Genscript, NJ, USA) O/N at 4°C followed by washing. After blocking and washing, plates were incubated with a titrated amount of His-Tagged HA of A/California/07/09 (in house produced and purified) for 2 h at RT. Plates were washed, and plasma was added to the plate in duplicate, serially diluted in block buffer, and incubated for 1 h at RT, followed by addition of a titrated amount of biotinylated human IgG1 CR9114 (produced and purified in house) and incubation for another hour at RT. After washing, streptavidin–HRP was added and incubated for 1 h at RT, followed by washing and OPD (Thermo Scientific, Bremen, Germany) development. The colorimetric reaction was stopped after 10 min by adding 1M H₂SO₄. The optical density (OD) was measured at 492 nm, and standard curves were created using a four-parameter logistic curve. The CR9114 competition of each sample was quantified as the slope of the linear regression of OD value on the log 10 dilution for the duplicate series.