Immulon 4hbx microtiter plates

An enzyme-linked immunosorbent assay (ELISA) was performed to further confirm FnBPA-fibrinogen binding as previously described (56 (link)). In brief, 96-well Immulon 4HBX microtiter plates (Thermo Scientific, Waltham, MA) were coated overnight at 4°C and with gentle rotation with 0.5 μg per well of FnBPA isotypes, diluted in PBS. After blocking the wells with 2% BSA in PBS, plates were washed once with Tris-buffered saline (TBS) and coated isotypes were preincubated with a concentration range of purified IgG, diluted in TBS, for 1 h at room temperature. Subsequently, after 3 additional wash steps with TBS plus 0.1% Tween, plates were incubated for another hour with a fixed concentration of 40 nM fibrinogen (diluted in TBS). After another 3 wash steps, the bound fibrinogen was detected through incubation with horseradish peroxidase (HRP)-conjugated antifibrinogen antibodies (1:10,000 dilution) (Thermo Scientific) for 1 h and quantified after adding the substrate o-phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich). The resulting absorbance at 450 nm (optical density [OD] at 450 nm) was measured in an ELISA microplate reader (Thermomax).
In the case of the ClfA competition assay, 40 nM fibrinogen was preincubated for 1 h with a 2-fold concentration range of recombinant ClfA (40 to 0.625 µM) and then incubated with FnBPA isotype-coated plates as described above.

The experiments were performed using a modified version of the protocol described previously (14 (link)). Immulon 4HBX microtiter plates (ThermoFisher Scientific, Waltham, MA) were coated with 2 µg/ml of peptides overnight at 4°C and blocked with 1x PBS containing 0.1% Tween 20 with 3% BSA (blocking buffer) for 1 hour at room temperature. Mouse sera diluted 1 to 100 in blocking buffer were added to the plate and incubated for 2 hours. Secondary anti-mouse IgG in blocking buffer was added and incubated for 1hour. Then the substrate o-phenylenediamine (0.05%) (Sigma-Aldrich, St. Louis, MO) and 0.06% hydrogen peroxide in citrate-phosphate buffer, pH 5.0 (100 µl/well) was applied. Reaction was stopped after 15 minutes by the addition of 50 µl/well of 2.5 N sulfuric acid. Absorbance was read at 490 nm with a reference wavelength of 560 nm.

To determine the IC₅₀ of each MAb, flat-bottom Immulon 4HBX microtiter plates (Thermo Scientific) were again coated with 100 μl/well of 25 μg/ml fetuin (Sigma) diluted in 1× PBS, and incubated overnight at 4°C. Antibodies were diluted to 120 μg/ml in sample dilutant buffer and then serially diluted 1:3 in a sterile 96-well plate. Virus was diluted to its calculated effective concentration, added to the MAb dilutions at a 1:1 ratio, and incubated for 1 h at room temperature, shaking. fetuin-coated plates were washed three times with PBS-T, and the virus-MAb dilutions were added. The assay was then performed according to the ELLA procedure above. IC₅₀ values were determined using Prism 7.0. NAI assays were completed in duplicates.

Sema4D concentration in the plasma was determined using direct ELISA. Immulon 4 HBX microtiter plates (Thermo Scientific, Waltham, MA) were coated with 50 microliters of undiluted plasma, washed, then incubated with anti-human CD100 antibody (clone: 133-1C6; Novus Biologicals). Goat anti-mouse IgM-HRP (catalog no. M31507; Life Technologies) was added followed by detection with TMB (Pierce). The plasma concentrations of Sema4D were calculated using the standard curve established for recombinant Sema4D (catalog no. 310-29) (Peprotech, RockyHill, NJ). The detection limit was 3.1 ng/mL. Mouse IgM isotype control was used for the direct ELISA assay (Catalog no. ab91546) (Abcam). MYBioSourceHuman Semaphorin sandwitch ELISA kit (Catalog no. MBS763518) was used to confirm the direct ELISA results. For detection of TGF-β1 by WSU-WSU-HN6 tumor cells, Human ELISA TGF-β1 total kit was used following manufacturer recommendations (catalog no.436707) (BioLegend, San Diego, CA). Conditioned medium was run in triplicates and plates were read using BioTek Epoch microplate spectrophotometer at 450nm wavelength.

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.