Ap114p

AGE-elastin was obtained via the incubation of human aortic α-elastin (1.33 mg/mL) with 100 mmol/L glucose for 30 days, as described by Baydanoff et al. [27 (link)]. A blocking ELISA was used for the detection of IgM and IgG autoantibodies to AGEs of vascular EL. The 96-well plates were coated with AGE-elastin (5 μg/mL) and incubated with 100 μL of human sera (diluted 1:20) for 1 h at 37 °C. Then, 100 µL of goat anti-human IgM Ab, Fc5µ, HRP conjugate (AP114P, Sigma-Aldrich, St. Louis, MO, USA) and goat anti-human IgG Ab, Fc, HRP conjugate (AP113P, SigmaAldrich, St. Louis, MO, USA), respectively, were added to each well. Immunoconjugates were diluted 1:10,000 and ortho-phenylenediamine was used as the chromogen. The reaction was stopped by adding 50 μL/well of sulfuric acid (4 M H₂SO₄), and the optical density was measured on a Coulter Microplate Reader UV Max (Molecular Devices Corp., Menlo Park, CA, USA) at a wavelength of 492 nm. All samples were tested in triplicate.

To measure the ACIVAbs IgM and ACIVAbs IgG concentrations, a sandwich ELISA was used. The assays were performed as follows: microtiter 96-well polystyrene plates were coated with 100 μL of 10 μg/mL of human CIV (Sigma-Aldrich, St. Louis, MO, USA), followed by an overnight incubation at 4 °C. Then, 100 μL serum sample (diluted 1:10) was placed in each well of the microtiter plates and incubated for 1 h at 37 °C. After washing, 100 μL of goat anti-human IgM Ab, Fc5µ, HRP conjugate (AP114P, Sigma-Aldrich, St. Louis, MO, USA), or goat anti-human IgG Ab, Fc, and HRP conjugate (AP113P, Sigma-Aldrich, St. Louis, MO, USA), respectively, were added to each well for 1 h at 37 °C. All immunoconjugates were diluted 1:10,000. Then, 100 μL of ortho-phenylenediamine (4 mg/mL in 0.05 M citrate buffer) was added as a colorimetric substrate for 30 min. 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, Molecular Devices Corp., Menlo Park, CA, USA) at a wavelength of 492 nm.