1 step turbo tmb elisa reagent

For detection of HA cross-reactivity, ELISA assays were performed as previously described (Wilson et al., 2014 ). Briefly, Costar Hi Bind plates (Corning Inc., Tewksbury, MA) were coated overnight with the appropriate virus isolate (25 HA units/well) at 4 °C. Plates were blocked for 1 h with PBS/0.1% Tween-20 (PBST) containing 1.5% BSA (blocking buffer) at room temperature. All rmAbs were serially titrated three-fold in blocking buffer and allowed to incubate with antigen-coated plates for 1 h at room temperature. After three PBST washes, wells were probed with goat anti-human (H&L)–HRP (Thermo Scientific) for 1 h at room temperature. Plates were washed three times with PBST and signal was developed with 1 step™ Turbo TMB-ELISA reagent (Thermo Scientific). Reactions were stopped with 1 N sulfuric acid and absorbance was read at 450 nm.

High-binding microtiter plates (Corning, Sigma Aldrich, USA) were used to coat rhPRG4 (M_r=240 KDa), HMW HA (M_r=1,500 KDa) (R & D System, USA), MMW HA (M_r=300KDa) (R & D System) and vitronectin (M_r=75 KDa) (Sigma Aldrich) at 400μg/ml in PBS buffer (100μl per well) overnight at 4°C. rhPRG4 is a full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [21 ]. Following washing with PBS+0.1% tween 20, wells were blocked with 2% bovine serum albumin (BSA; 300μl per well) for 2 hours at room temperature. CD44-IgG₁Fc (R & D systems) or IgG₁Fc (R & D systems), each at 1μg/ml (100μl per well), were added to the plate and incubated for 60 min at room temperature. Following washing with PBS+0.1% tween 20, anti-IgG₁Fc-HRP (Sigma Aldrich) was added at 1:10,000 dilution (100μl per well) and incubated for 60 min at room temp. Following washing with PBS+0.1% tween 20, the assay was developed using 1-step Turbo TMB ELISA reagent (ThermoScientific, USA) and absorbance was measured at 450 nm. Data represents the average of 4 independent experiments, each with triplicate wells per group.