This procedure was performed to check the suitability of capture antibodies to use in enzyme-linked immunosorbent assay (ELISA). On a membrane (Hybond C; GE Healthcare, Buckinghamshire, UK), 10-μL aliquots of 1.25 μg/mL α-elastin from human aorta (Sigma-Aldrich, Poole, UK) in 0.1 M sodium carbonate (pH 9) were placed in dots in grid formation and allowed to dry. The membrane was blocked with blocking solution consisting of 5% (wt/vol) low-fat milk powder (Marvel; Premier Foods, St. Albans, UK) in tris-buffered saline (TBS) for 1 hour on an orbital shaker. The capture antibodies tested included a mouse monoclonal antibody to α-elastin (Abcam Ltd., Cambridge, UK) and a rabbit polyclonal antibody to α-elastin (Elastin Products Co., Pacific, MO). Each capture antibody was added in a range of dilutions to the blocking solution described earlier, and a strip of membrane was dotted with an α-elastin for 1 hour at room temperature. The membrane was then washed with TBS containing 0.05% (vol/vol) Tween 20 (TBST) five times, for 5 minutes each. The strips were then incubated with appropriate detection antibody (both horseradish peroxidase [HRP]–conjugated antibodies purchased from Santa Cruz Technology/Autogen Bioclear, Wiltshire, UK). Donkey anti-mouse detection antibody was used for the monoclonal capture antibody (1:1000 in 5% wt/vol milk in TBS) and goat anti-rabbit detection antibody for the polyclonal capture antibody (1:1000 in 5% wt/vol low-fat milk powder in TBS), both incubated for 1 hour at room temperature. Membranes were then washed again in TBST for 5 minutes five times. Chemiluminescence detection was then performed with a kit (Enhanced Chemiluminescence; GE Healthcare) and the membrane strips were placed in a cassette with a high-performance chemiluminescence hyperfilm (GE Healthcare) before the film was developed for analysis.