High binding half area 96 well plates

Initial antibody binding screening against p62-E1 was performed by coating 250 ng/well diluted in PBS in half-area 96-well high binding plates (Costar). Wells were blocked with 3% BSA at 37°C for 2 h. Antibody dilutions at 300 nM and 30 nM were performed in PB-T (PBS pH 7.4, 0.5% BSA, 0.05% Tween) and incubated 1 h at 37°C. After antibody binding plates were washed with PBS-T (PBS pH 7.4, 0.005% Tween-20) five times. Horseradish peroxidase conjugated-(HRP)-Protein A (life technologies) diluted at 1:2000 in PB-T was added in for 1 h at 37°C. Plates were washed five times with PBS-T and developed using TMB (Thermo Fischer). Optical density at 450 nm was read on Synergy H4 Hybrid reader (BioTek). Procedures were similar for full (8-point) ELISA curves and serum ELISA, except that initial stock of mAb or serum were serially diluted. Experiments with E1’ as the target were similar.

To assess antibody reactivity by ELISA, POWV EDIII or LS-POWV-EDIII was coated on half-area 96-well high binding plates (Costar) at 200 ng/well. Wells were blocked with 1% BSA at 25°C for 2 h and washed 5 times with PBS-T (PBS pH 7.4, 0.05% Tween-20). Mouse sera or mAbs were diluted in PB-T (PBS pH 7.4, 0.2% BSA, 0.05% Tween) and incubated for 1 h at 37°C. Plates were washed and HRP-conjugated anti-mouse IgG (Sigma) for mouse sera or protein A (Life Technologies) for mAbs was added. For IgG subclass detection, HRP-conjugated anti-mouse IgG1, IgG2a, or IgG2b (Invitrogen) was used as secondary. After 1 h incubation at 37°C, plates were washed and developed using TMB (Thermo Fisher). Absorbance at 450 nm was measured on Synergy H4 Hybrid reader (BioTek).

VWF and recombinant AIM-A1 fragments (6 μg/mL in PBS) were coated to high-binding half-area 96-well plates (Corning). VHH81 binding to immobilized VWF and AIM-A1 fragments were detected with horse-radish peroxidase (HRP)-conjugated anti-VHH monoclonal antibody 96A3F5 (Genscript) (1:1000). Purified GPIb-IX complex was coated to the plate using 0.1% Triton X-100, 20 mM Na₂CO₃/NaHCO₃, pH 9.6. Fixed platelets, prepared from human washed platelets followed by two rounds of washing in 4% paraformaldehyde, were coated to the plate using 1% poly-l-lysine (Sigma) in PBS. Bound AIM-A1 fragments were detected with HRP-conjugated anti-HisTag antibody 4E3D10H2/E3 (1:2000). Monoclonal antibody binding to AIM-A1 fragments was detected using HRP-conjugated anti-mouse secondary antibody sc2005 (Santa Cruz Biotechnology) (1:2000). In all cases, plates were washed three times with HEPES buffered saline with 0.1% Tween-20 on a BioTek ELx405 plate washer. After binding and washing, 1-Step Ultra-TMB substrate (ThermoFisher) was added to each well, quenched with 2 M H₂SO₄. Absorbance was measured at 450 nm. Empty wells were used to subtract baseline absorbance.

HEK293/TLR4 cells were plated into 96-well plates. Next day, cells were pretreated with different concentrations of metformin (MP Biomedicals, CA) for 24 or 48 h and then incubated with or without 1 μg/ml lipopolysaccharide (LPS, Sigma-Aldrich, MO) for additional 24 h. The concentration of secreted CXCL8 in the cell culture media was determined using ELISA assay.
The high binding half-area 96-well plates (Corning, NY) were coated with 1 μg/ml antihuman IL-8 coating antibody (Invitrogen, MD) overnight. The plates were then washed with washing buffer (1.47 mM KH₂PO4 and 8.32 mM K₂HPO4, 0.05% Tween 20, pH 7.4) and blocked with assay buffer (13.69 mM NaCl, 7.69 mM Na₂HPO4, 1.15 mM K₂HPO4, and 2.68 mM KCl, 0.5% bovine serum albumin, 0.05% Tween 20, pH 7.4).
The cell culture media samples, IL-8 standards and antihuman IL-8 antibodies conjugated with biotin, were added to the ELISA plates and incubated for 2 h. Wells were washed and incubated with streptavidin-HRP solution for 30 min. The plates were then washed, and 1-Step™ Ultra TMB-ELISA (Thermo Scientific, MA) substrate was added to the plates and incubated in dark for 30 min. The HRP reaction was stopped by sulfuric acid, and absorbance was measured at 450 nm and 650 nm. CXCL8 concentration in samples was calculated and managed via Prism software (GraphPad Software, CA).