(A) The effect of increasing concentrations (5, 1 and 0.1 mg/mL) of free amino acids were tested in BTLA/HVEM ELISA (three experiments in triplicate). The percentage of inhibition was calculated in relation to the negative control (PBS). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). Statistical analysis was performed by comparing each concentration of residue or peptide to the same concentration of Cys. (B) Assessment of free thiol groups over time in rhBTLA-Fc protein and HVEM(23–39) peptide alone or mixed together. **: p< 0.01 and ***: p< 0.001 following One-way ANOVA and Dunn’s (A) or Dunnett’s (B) post-test.