96-well round bottom plates (medium binding; Greiner Bio-One) were coated with 400 ng/well of recombinant BTLA-Fc protein in PBS (50 μL/well) and incubated overnight at 4°C. Wells were washed twice with PBS-T (phosphate buffer saline with 0.1% Tween-20, pH 7.4) and blocked with 1% milk in PBS-T for 1 hour at 37°C. After 2 washes with PBS-T, the peptides were titrated down in triplicates from 10 to 0.1 mg/well in 1% milk/PBS-T and incubated for 2 hours at 37°C. Maximum binding of HVEM to BTLA was controlled by adding 1% milk/PBS-T to negative control wells, whereas maximum inhibition was monitored by adding anti-BTLA 7.1 mAb (0.1 μg/well). Next, the plate was washed and incubated with recombinant HVEM-Fc protein at 400 ng/well in 1% milk/PBS-T for 1 hour at 37°C. Biotinylated anti-HVEM 11.8 antibody was then added (biotinylated preparation diluted at 1/10 000 in 1% milk/PBS-T) and incubated for 1 hour at 37°C. Streptavidin horseradish peroxidase (Dako) was finally added at a 1/8000 dilution in 1% BSA/PBS-T for 30 min at 37°C and detected with TMB (Biorad). The substrate reaction was blocked with 0.2 M H2SO4 and the absorbance was read at 450 nm and 630 nm. The percent of inhibition was calculated assuming that the anti-BTLA 7.1 antibody inhibit the complex at 100% and milk/PBS-T in 0%. Statistical analysis of the results was performed using the one-way analysis of variance (one-way ANOVA).