2.2.3. Determination of AEAbs (IgM, IgG, and IgA)

To measure AEAbs IgM, AEAbs IgG, and AEAbs IgA concentrations, a sandwich ELISA was used. The assay was performed as follows: a microtiter 96-well polystyrene plate was coated with human aortic α-elastin, prepared as described by Baydanoff et al. [23] (1 μg of elastin in 100 μL of 0.05 M carbonate buffer, pH 9.6). Then the remaining "active" centers of the polystyrene wells were blocked by the plate incubation for 24 h with 1% solution of bovine serum albumin (BSA) (Cat. No. A2153, Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS), pH 7.4, containing 0.05% Tween 20. The next step was the addition of 100 μL of tested patient serum (diluted 1:10 with PBS) in each well of the microtiter plate, incubated for 1 h at 37 ◦C. After washing three times, incubation with anti-human immunoglobulin peroxidase conjugates to the heavy chain of IgM, IgG, and IgA, respectively (Sigma-Aldrich, St. Louis, MO, USA) followed. All immunoconjugates were diluted 1:10,000 with PBS containing 1% BSA and 0.05% Tween 20. Next, samples were incubated with substrate solution (ortho-phenylene diamine, 4 mg/mL in 10 mL 0.05 M citrate buffer, pH 5.0 with H2O2) for 1 h at room temperature in a dark chamber. The reaction was stopped by adding 50 μL of 4 M H2SO4 to each well, and the optical density was measured with a micro-ELISA plate reader (Coulter Microplate Reader UV Max) at a wavelength of 492 nm.
