Microplate reader uv max

To measure MMP-2 concentrations, an ELISA kit from R&D Systems (Cat. No. DMP2F0) (Minneapolis, MN, USA) was used. According to the manufacturer’s instructions, 100 μL of assay diluent RD1-74 was added to each well-plate, then 50 μL tested sera, diluted 1:10 with calibrator diluent RD5-32 (20 μL serum + 180 μL calibrator diluent) or standards, was added at various concentrations to construct a calibration curve. After 2 h downtime at room temperature on a shaker, plates were washed three times with 400 μL wash buffer per well. After the last wash, 200 μL of the conjugate was added to each well and incubated for 2 h at room temperature on a shaker. The plate was washed again three times, and in each well, 200 μL substrate solution was added. This was incubated for 30 min at room temperature in the dark. The reaction was stopped by adding 50 μL of stop solution to each well. Within 30 min, the serum samples were assayed at 450 nm on an automatic micro-ELISA plate reader (Coulter Microplate Reader UV Max).

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.

To measure ACIVAbs IgM concentrations, a sandwich ELISA was used. The assay was performed as follows: a microtiter 96-well polystyrene plate was coated with 100 μL of 10 μg/mL of human CIV (Sigma-Aldrich, St. Louis, MO, USA) 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 1% bovine serum albumin (BSA; Cat. No. A2153, Sigma-Aldrich, St. Louis, MO, USA). Then, a 100-μL serum sample (diluted 1:10) was placed in each well of a microtiter plate and incubated for 1 h at 37 °C. After washing three times, 100 μL of goat anti-human IgM Ab, Fc5µ, HRP conjugate (AP114P, Sigma-Aldrich, St. Louis, MO, USA) were added to each well for 1 h at 37 °C. All immunoconjugates were diluted 1:10,000 with PBS containing 1% BSA and 0.05% Tween 20. The plate was incubated for 1 h at 37 °C. Ortho-phenylenediamine (4 mg/mL in 0.05 M citrate buffer, pH 5.0 with H₂O₂) was used as a colorimetric substrate. 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.