Peroxidase conjugated goat anti rabbit igg h l hrp

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 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), followed by an overnight incubation at 4 °C. Then, 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, 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. After washing, peroxidase-conjugated goat anti-rabbit IgG H&L (HRP) (Cat. No. ab205718, Abcam, Cambridge, UK) diluted 10,000 fold was added to each well. The plate was incubated for 1 h at 37 °C. 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.

For the determination of CIC-CIV, a method based on C3-binding glycoprotein was used: Complement-Inhibiting Factor (CIF)-Enzyme Linked Immunosorbent Assay (CIF-ELISA) [16 (link)]. The assay was performed as follows: microtiter 96-well polystyrene CIF-ELISA plates were prepared by incubation of the wells with CIF (20 μg/mL in 0.2 M carbonate-bicarbonate buffer, pH 9.6) overnight at 4 °C. After repeatedly washed in order to remove unbound CIF, human sera (100 μL) were added to the plates and incubated for 1 h at 37 °C. After washing, 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. After washing, peroxidase-conjugated goat anti-rabbit IgG H&L (HRP) (Cat. No. ab205718, Abcam, Cambridge, UK) diluted 10,000 fold was added to each well. The plate was incubated for 1 h at 37 °C. 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.