ELISA was used to assess the binding of mAbs to the endothelial cell monolayers. Confluent HUVEC monolayers prepared in 96-well plates were coincubated with interleukin (IL)-1P (20 U/mL), Escherichia coli lipopolysaccharide (LPS) (0.74 EU/mL, Sigma Chemical Co, USA), or HPE (10%) for 6 h at 37°C in a CO2 incubator. Immediately after pretreatment, the cells were fixed by the addition of 1% paraformaldehyde in PBS for 15 min at room temperature.
After washing with the buffer (Hanks’ balanced salt solution with 1% bovine serum albumin and 0.01% sodium azide), mAbs directed against ICAM-1, VCAM-1 or E-selectin were added and incubated for 30 min. After another wash, the cells were incubated for 15 min with biotinylated anti-rabbit and anti-mouse immunoglobulin in PBS containing carrier protein and 15 mM sodium azide (Dako Co, USA). Then the wells were washed, incubated in horseradish peroxidase-conjugated streptavidin (Dako Co, USA) (in PBS containing carrier protein and antimicrobial agents for 15 min, and were washed again in PBS. Finally, o-phenylene-diamine dihydrochloride (0.4 mg/mL in citrate buffer, pH 5) with 0.012% hydrogen peroxide was added and incubated for 10 min, the reaction was stopped by the addition of 1.5 M sulfuric acid. The plates were then read at 492 nm in a Micro plate reader (Tosoh, Japan) to quantitate the amount of bound antibody. Data were expressed as a ratio of each sample optical density (OD) to control OD (IL-1P).