2.2.5. Determination of CIV-DP

To measure CIV-DP concentrations, a sandwich ELISA was used. The assay was performed as follows: each well of the microtiter plate was sensitized with 100 μL of 10 μg/mL of mouse monoclonal antibody to collagen IV (COL-94) (Cat. No. ab6311, Abcam, Cambridge, UK) at room temperature for 3 h, followed by overnight incubation at 4 ◦C. The plate was washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 0.1% bovine serum albumin (BSA) (Cat. No. A2153, Sigma-Aldrich, St. Louis, MO, USA). Then, a 100-μL serum sample (diluted 1:5) was placed in each well of a microtiter plate and incubated for 1 h at 37 ◦C. After washing three times, 100 μL of rabbit anti-human CIV polyclonal antibody (Cat. No. ab6586, Abcam, Cambridge, UK; diluted 1:2000) was allowed to react in each well at 37 ◦C for 1 h. The wells were washed with PBS + Tween 20, and peroxidase-conjugated goat anti-rabbit IgG H&L (HRP) (Cat. No. ab205718, Abcam, Cambridge, UK), diluted 10,000 fold, was then added to each well. The plate was incubated for 1 h at 37 ◦C. Ortho-phenylenediamine (0.4 mg/mL) was added to citrate buffer, and 100 μL of this solution was added to each well and allowed to react for 30 min. The reaction was stopped by adding 50 μL 4M 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.

#### 2.2.6. Biochemical Assays

The analysis was performed using an automatic biochemistry analyzer. HbA1c and CRP were measured by a turbidimetric immunoassay. Enzymatic methods were used to measure total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG).
