Anti human mpo antibody

Inflammatory cytokines (TNF-α and IL-8) in plasma were measured by enzyme-linked immunosorbent assays (ELISA) (Invitrogen, Waltham, MA, USA). The levels of IL-6, hs-CRP and procalcitonin were quantified by an automated immunoturbidimetric assay (cobas c502, Roche Diagnostics). Neutrophil elastase (NE) level was measured by sandwich ELISA (Abcam270204, Cambridge, United Kingdom). Levels of Histone–DNA complexes were measured by the Cell Death Detection ELISA kit (Roche, Mannheim, Germany).
Levels of MPO–DNA complexes were measured using an adapted protocol from previous studies [47 (link),48 (link)]. Briefly, Costar 96 flat-well plates were coated with 1 µg/mL antihuman MPO antibody (Bio-Rad 0400-0002, Oxford, United Kingdom) in coating buffer from the Cell Death ELISA kit, overnight at 4 °C. After washing and blocking, sera or supernatants were added and incubated at room temperature for 90 min. The plate was washed and incubated with HRP-conjugated anti-DNA antibody (from the Cell Death kit), and color was developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Invitrogen, Waltham, MA, USA) followed by 2N H₂SO₄ to stop the reaction. The absorbance was measured at 450 nm.

MPO-DNA complexes were quantified similarly to what has been previously described (86 (link)). This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human MPO antibody (Bio-Rad 0400-0002), diluted to a concentration of 1 μg/ml in coating buffer (Cell Death kit). The plate was washed two times with wash buffer [0.05% Tween 20 in phosphate-buffered saline (PBS)], and then blocked with 4% bovine serum albumin in PBS (supplemented with 0.05% Tween 20) for 2 hours at room temperature. The plate was again washed five times, before incubating for 90 min at room temperature with 10% serum or plasma in the aforementioned blocking buffer (without Tween 20). The plate was washed five times and then incubated for 90 min at room temperature with 10× anti-DNA antibody [horseradish peroxidase (HRP) conjugated; from the Cell Death kit] diluted 1:100 in blocking buffer. After five more washes, the plate was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Invitrogen) followed by a 2N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Data were normalized to in vitro–prepared NET standards included on every plate, which were quantified on the basis of their DNA content.

cfDNA was quantified in plasma samples using the Quanti-iT PicoGreen kit (Invitrogen) according to the manufacture’s instructions, and concentrations were calculated based on a standard curve of λ DNA (Invitrogen). MPO-DNA complex levels were quantified by ELISA using a protocol modified from (37 (link)). Briefly, a 96-well flat-bottom plate was coated with anti-human MPO antibody (Bio-Rad) overnight at 4°C and then washed 4 times with PBS-tween buffer before blocking with PBS/4% BSA. The samples were then incubated with patient plasma for 2 hours at room temperature. After 4 washes with PBS-tween, the quantity of DNA bound to captured MPO was quantified using the Quant-iT PicoGreen kit (Invitrogen) as per the manufacturer’s instructions.

MPO-DNA complexes were quantified according to the protocol by Zuo and colleagues (26 (link)). Most of the reagents were taken from the Cell Death Detection ELISA kit (11544675001, Roche, Manheim, Germany); and all incubations except for antibody coating were done at room temperature (RT). In short, a high-binding 96-well plate (Costar, Corning, NY, USA) was coated overnight at 4°C with 1 µg/ml of anti-human MPO antibody (0400-0002, Bio-Rad, Hercules, CA, USA) and then blocked with 4% bovine serum albumin (Merck, Saint-Louis, MO, USA) in PBS with 0.05% Tween-20 (AppliChem GmbH, Darmstadt, Germany) for 2 hours at RT. The plate was washed 5 times and incubated for 90 minutes with isolated NETs that were quantified according to their DNA content, washed again and incubated for another 90 minutes with 10× HRP conjugated anti-DNA antibody diluted 100 times. Plate was washed 5 times at the end of the last incubation and developed with 3,3′,5,5′-Tetramethylbenzidine substrate Single Solution (Life Technologies Corporation, Carlsbad, CA, USA) followed by a 2N sulfuric acid Stop Solution (ThermoFisher Scientific, Waltham, MA, USA). Absorbance was measured at 450 nm using a Synergy H4 Hybrid Reader (BioTek, Santa Clara, CA, USA).

MPO-DNA complexes were quantified similarly to what has been previously described (38 (link)). This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human MPO antibody (Bio-Rad 0400-0002), diluted to a concentration of 0.5 μg/mL in coating buffer (Cell Death kit). Next, the plate was washed 3 times with wash buffer (0.05% Tween 20 in PBS) and then blocked with 1% BSA in PBS for 1 hour at room temperature. Next, the plate was washed 3 times before incubating for 1 hour at room temperature with 1:500 mouse serum in the blocking buffer. Next, the plate was washed 5 times and then incubated for 1 hour at room temperature with 1× anti-DNA antibody (HRP conjugated; Cell Death kit) diluted 1:100 in blocking buffer. After 5 more washes, the plate was developed with 3,3′,5,5′-TMB substrate followed by a 2N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm with a Synergy HT Multi-Mode Microplate Reader (BioTek). Data were normalized to an in vitro–prepared NET standard and included on every plate.