Endogenous Δ6-TNFR1 was measured in control and patient sera by ELISA. An anti-Δ6-TNFR1-specific monoclonal Fab antibody was generated using HuCAL® antibody technology (AbD Serotec) that employs a human combinatorial antibody library and the CysDisplay® method of phage display. The anti-Δ6-TNFR1-specific antibody was coated onto Nunc™ microtitre plates at a concentration of 2 μg/ml overnight. The anti-human TNFR1 mouse monoclonal antibody (clone 16805; R&D Systems) was used at 2 μg/ml for detection, in conjunction with Europium-conjugated anti-mouse IgG. The sensitivity limit of the assay was determined to be ~1.25 ng/ml.
