Immulon 2hb 96 well plate

Collagens and peptides coatings were produced on the surface of Immulon 2HB 96-well plates (Thermo Scientific) by incubating 100 µl/well of 10 µg/ml solution or suspension (for insoluble collagen type I) in 10 mM acetic acid containing the appropriate proteins/peptides over night at 4 °C. Bovine serum albumin (BSA, Sigma) and triple-helical-like sequences GPP10 were plated in triplicate to act as nonspecific background adhesion controls.

The supernatant was tested by sandwich ELISA. Immulon 2HB 96 well plates (ThermoFisher Scientific Catalog #1424561) were coated with 4 μg/mL UC1MT anti-MT antibody (ThermoFisher custom production of endotoxin-free antibody from hybridoma cells generated in-house¹⁶ (link)) and incubated overnight at room temperature (RT). Plates were washed and blocked with 2% bovine serum albumin (ThermoFisher Scientific Catalog #AAJ6573122), followed by another wash step and incubation with purified MT1 standards (Enzo Life Sciences Catalog #ALX-202-072-M001) or cell supernatants for 2 h at RT. Following another wash step, plates were incubated with biotinylated UC1MT antibody (antibody biotinylated using ThermoFisher Scientific Catalog #90407) for 2 h at RT. After another wash, streptavidin-HRP (BioLegend Catalog #405103) was added to the wells and incubated in the dark at RT for 20 min. Following a final wash step, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (BioLegend Catalog #421501) was added, and color was allowed to develop in the dark at RT for 20 min 2N H₂SO₄ was added to stop the color reaction, and optical density (OD₄₅₀) was measured using a Spectramax plate reader (Molecular Devices).

Platelet adhesion onto immobilized collagen or the α2β1 integrin specific collagen-like peptide GFOGER was examined in static condition as described [7 (link)]. Briefly, Immulon 2HB 96-well plates (Thermo Scientific, MA, USA) were coated with Horm collagen (100 μL, 10 μg.mL^-1) or GFOGER (100 μL, 20 μg.mL^-1) overnight at 4°C. After saturation with 10 mg.mL^-1 BSA in PBS for 2 hours at RT, washed platelets (2×10⁸.mL^-1) in reaction buffer completed or not with Mg²⁺, treated or not with 22 μM losartan, were incubated into collagen- or GFOGER-coated wells for different times. After washing, the amount of adherent platelets was determined by quantifying alkaline phosphatase activity using p-nitrophenyl phosphate (pNPP).

Immulon 2 HB 96-well plates (Thermo Fisher) were coated overnight at RT with 100μl/well 0.25μg/ml recombinant SARS-CoV-2 Spike or Spike receptor binding domain protein (Sino biological, China) and washed thrice with PBS + 0.1% Tween-80 before use. Sera were first diluted 1:100 in PBS + 0.1% Tween-80 and then 2-fold serially diluted. Per well, 100μl of diluted sera was added and plates were incubated for 60 minutes at 37°C. After washing thrice with 0.1% Tween-80, plates were incubated for 60 minutes at 37°C with HRP-conjugated goat anti-ferret IgG (Alpha Diagnostic), diluted 1:5000 in PBS containing 0.1% Tween-80 and 0.5% Protivar (Nutricia). Plates were then washed trice with PBS + 0.1% Tween-80 and once with PBS, followed by development with 100μl SureBlue™ TMB (KPL) substrate. Development was stopped after 10 minutes by addition of 100μl 2M H₂SO₄ and OD₄₅₀-values were determined on the EL808 absorbance reader (Bio-Tek Instruments). Individual curves were visualized using local polynomial regression fitting with R software (42 ). Antibody titers were determined as the dilution at which antibody responses dropped below background. This background was calculated as the ‘mean + 3 * standard deviation’ of the OD₄₅₀ at a 400x serum-dilution of all animals tested before SARS-CoV-2 infection.

Blood was obtained from convalescent mice (four weeks after the initiation of antibiotics) via submandibular puncture using a 5 mm lancet (Goldenrod) and collected in a BD Microtainer serum separator tube (BD Biosciences). UTI89 and CFT073 were grown as described above and 1 ml of OD₆₀₀ = 1 bacteria was lysed by boiling at 100°C for 5 minutes. Lysed bacteria were diluted tenfold in PBS and applied to high-affinity Immulon 2 HB 96 well plates (Thermo Fisher) overnight at 4°C. Plates were washed with PBS containing 0.05% Tween 20 and a dilution series of serum was applied for 90 minutes at room temperature. After washing, a 1:10,000 dilution of HRP-conjugated goat anti-mouse IgG (Invitrogen) was applied for 90 minutes at room temperature. After a final wash step, the TMB substrate reagent set (BD Biosciences) was used to develop the reaction. Absorbance was measured on a VersaMax microplate reader (Molecular Devices).

For measurements of anti-pneumococcal antibody titers, pneumococcal strain P1121 was grown and resuspended to an OD₆₂₀ of 0.1 in coating buffer (0.015 M Na₂CO₃, 0.035 M NaHCO₃), then plated onto Immulon 2HB 96-well plates (Thermo) at 4°C overnight. Plates were washed with 0.05% Brij in PBS, and blocked for 1 hr at 37°C in 1% BSA in PBS. After additional washes, serum samples were added in serial 2-fold dilutions (made in 1% BSA in PBS) and incubated overnight at 4°C. Anti-pneumococcal antibodies were detected by incubating for 1.5 hrs at room temperature with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody, followed by developing for 1 hr at 37°C with p-nitrophenyl phosphatase. Absorbance was measured at 415 nm. The sample dilution at which the absorbance equaled 0.1 was used to calculate the geometric mean titer. For measurements of CCL2 protein levels in serum and nasal lavages, an ELISA kit was used according to the manufacturer’s protocol (eBioscience).

The production and isolation of α₂ I-domain as the glutathione S-transferase (GST) fusion protein has been described previously [42] (link). Immulon-2 HB 96-well plates (Thermo Scientific) were coated with 100 μl of GPC(GPP)₅GFOGER(GPP)₅GPC at 10 μg/ml in 0.01 M AcOH, or bovine serum albumin (BSA) overnight at 4 °C. Wells were blocked with 200 μl of 50 mg/ml BSA in tris-buffered saline (TBS; 50 mM Tris, 140 mM NaCl) for 1 h, then washed three times with adhesion buffer (TBS plus 1 mg/ml BSA and 5 mM MgCl₂). The α₂ I-domain GST was diluted to 0.5 μg/ml in adhesion buffer and incubated with 100 μg/ml of free peptide as indicated for 30 min at room temperature. 100 μl of the α₂ I-domain GST/peptide mixture was then added to the GPC(GPP)₅GFOGER(GPP)₅GPC-coated wells for 1 h at room temperature. After washing three times with 200 μl of adhesion buffer, bound α₂ I-domain GST was quantified by adding 200 μl of horseradish peroxidase-conjugated anti-GST antibody (Amersham Bioscience UK Ltd) diluted in adhesion buffer to 1:10,000 for 45 min at room temperature. Wells were washed four times with 200 μl of adhesion buffer and developed using 100 μl of TMB substrate (Thermo Scientific). The reaction was stopped with 100 μl of 2.5 M sulphuric acid and A₄₅₀ was measured [43] (link).