To reduce endotoxin concentrations in proteins used for T cell assays, each of the Ags was subjected to affinity chromatography using a polymyxin matrix (Bio-Rad, Hercules, California). The endotoxin contents in the protein solutions were determined by the Limulus Amebocyte Lysate assay (Lonza, Basel, Switzerland) and were found to be in the range of 12.4–54 ng/mg of protein. Heparinized venous blood samples were collected from six patients with HDM allergy. PBMCs were isolated by Ficoll (Amersham Bioscience, Uppsala, Sweden) and incubated in control medium or 1.25 μM recombinant Der p 23, PreS-2XP4P5, PreS, peptide 4 (P4), or peptide 5 (P5) for 6 d (37°C). Proliferation assays were performed as previously described, and results were expressed as stimulation index (33 (link)). The cultured cell supernatants from the lymphocyte proliferation assays were used to determine the concentrations of cytokines with the Bio-Plex Pro Human Cytokine 17-Plex Panel (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions, as described (25 (link)).