Goat anti human igg fc

Reacti-Bind 96-well polystyrene plates (Pierce) were coated with 100 μl of affinity purified goat anti-human IgG Fc (Rockland) at 1:20,000 in PBS, or 2 μg/ml SARS-CoV-2 recombinant protein in PBS overnight at 4 °C. Plates were washed in PBS-T (500 ml 10XPBS + 0.05% Tween-20 + 4.5 L H2O) and blocked for 1 h at 37 °C with 200 μl/well of B3T buffer: 8.8 g/liter NaCl, 7.87 g/liter Tris-HCl, 334.7 mg/liter EDTA, 20 g BSA Fraction V, 33.3 ml/liter fetal calf serum, 666 ml/liter Tween-20, and 0.02% Thimerosal, pH 7.4). Diluted antibody samples were applied and incubated 1 h at 37 °C followed by 6 washes with PBS-T; plates were then incubated with HRP-conjugated anti-human IgG (Jackson ImmunoResearch) diluted 1:10,000 in B3T buffer for 1 h at 37 °C. After 6 washes with PBS-T, SureBlue TMB Substrate (KPL) was added, incubated for 10 min, and the reaction was stopped with 1 N H2SO4 before measuring optical densities at 450 nm (Molecular Devices, SpectraMax using SoftMax Pro 5 software). For single-point assays, supernatants from transfected cells were diluted 1:10 in B3T and added to the blocked plates. ELISA signals were considered positive if they were greater than or equal to 2X the average of the blank wells of the plate.

Antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using goat anti-human IgG F(c) as well as goat anti-human kappa chain peroxidase conjugated antibody (Rockland, USA) and SeramunBlau^® (Seramun, Germany) as substrate. Absorption at 450 nm was measured by Infinite^® 200 PRO series microplate reader (Tecan, Switzerland). Amino acids and dipeptides were analyzed simultaneously by LC–MS after dansyl chloride derivatization as described below.

To test if there is a dose-dependent cytotoxicity-blocking effect with anti-human IgG antibodies, cells were treated with 5 µl of MS or HC A-FT, 5% NHS, plus various amounts of anti-human IgG antibodies. The following blocking antibodies were used: mouse anti-human IgG1 (Sigma #I2513), mouse anti-human IgG3 (Sigma #SAB4200759), goat anti-human IgG (H + L) (Vector Lab, #AI-3000), goat anti-human IgG-Fc (Rockland # 609-1103), and mouse anti-CD20 (R&D system #MAB4225). In addition, we tested MS drug Mitoxantrone (Cayman Chemical #14842) and pan-caspase inhibitor Z-VAD-FMK (Promega #G7231).