3 3 5 5 tetramethylbenzidine (tmb)

The binding solution (0.05 M carbonate buffer solution, pH 9.6) containing 100 μg/mL fetuin was added to a 96-well plate at 100 μL/well. After incubating at 4 °C for 16–18 h, the plate was washed with PBS containing 0.05% Tween-20 (referred to as PBST). PBS (200 μL) containing 1% bovine serum albumin (BSA) was then added for blocking at room temperature for 1 h to prevent non-specific binding. Next, the 96-well plate was washed with PBST, and serially diluted protein samples (twofold serial dilution prepared with PBS containing 1% BSA and 0.05% Tween-20 at a starting concentration of 10 μg/mL) were added to each well. The plate was incubated at room temperature for 1 h and washed with PBST. Then, the 96-well plate was treated with anti-H5 hemagglutinin antibody at 100 μL/well (1:10,000 dilution) for 1 h, washed with PBST, and treated with HRP-conjugated anti-rabbit IgG secondary antibody at 100 μL/well (1:5000 dilution) for 1 h at room temperature. Finally, HRP chemiluminescence substrate, 3,3’,5,5’-tetramethylbenzidine (TMB; Biolegend), was added to the 96-well plate at 100 μL/well. After color development in the dark for 15 min, 2 N sulfuric acid was added at 100 μL/well to stop the reaction, and the absorbance of each well was measured at 450 nm (O.D. 450) using an ELISA reader (TECAN SUNRISETM).

At the end of cell treatment, the medium was centrifuged at 300g for 5 min at 4 °C. Supernatant was frozen at − 80 °C until ELISA assays. TNFα and IL-1β ELISA kits were purchased from R&D systems and performed according to the manufacturer’s instructions. Briefly, plates were coated with a capture antibody and incubated overnight. Wells were blocked using 1% bovine serum albumin-PBS solution (Sigma), then exposed to cell supernatants at room temperature for two hours. Wells were washed and incubated with the detection antibody for an additional two hours. After thorough washing, Streptavidin-HRP solution was added for 20 min. Wells were washed, then incubated with TMB (Biolegend) solution for 20 min and the reaction was stopped using 1 M H₂SO₄. Absorbance was measured by a spectrometer (FLUOstar Omega; BMG Labtech) at 450 nm and the absorbance at 540 nm was subtracted.

The capacity of cultured B cells to secrete antibodies was assessed by ELISA. Plates were coated O/N at 4°C with anti-mouse Igκ (2ug/ml, Southern Biotech, 1050-01), washed 3x in PBS/0.05% Tween20, blocked for 1hr at RT with 0.5% BSA/PBS and washed again as before. Samples were diluted in PBS/0.05% Tween20 before incubation for 2hrs at RT or O/N at 4°C. Plates are washed as before, and the secondary HRP anti-mouse IgG antibody (Southern Biotech, 1033-05) was added at 1:2000 in 1x PBS/0.05% Tween20/0.5% BSA. Cells were washed as before, and plates were developed with TMB (Biolegend). Reactions were stopped with 50ul of Biolegend stop reagent and read on CLARIOstar at 450nm against a mouse IgG standard curve. Samples were scored as positive if they were > 4 standard deviations above the mean of the negative controls.

Sema4D concentration in the plasma was determined using direct ELISA as previously described (5 (link)). Briefly, Immulon 4 HBX microtiter plates (Thermo Scientific, Waltham, MA) were coated with undiluted plasma, washed with ELISA washing buffer, then incubated with anti-human CD100 antibody (clone: 133-1C6; Invitrogen, eBioscience, CA; cat # 14-1009-82) overnight, then followed by Goat anti-mouse IgM-Heavy chain, HRP conjugate secondary antibody (Invitrogen USA, IL; cat. # 62-6820), detection with TMB (Biolegend, CA; cat #421101) and Stop Solution (Biolegend, CA; cat #77316). The concentrations of Sema4D were calculated using the standard curve established using recombinant Sema4D (catalog no. 310- 29) (Peprotech, RockyHill, NJ). The detection limit was 3.1–1000 ng/ml. Plates were read at 450 nm wavelength using BioTek Epoch microplate spectrophotometer.