Anti mpo monoclonal antibody

MPO–DNA complexes have been identified using a capture ELISA [60 (link),69 (link),70 (link)]. As the capturing antibody, 75 µL of anti-MPO monoclonal antibody (5 µg/mL, ABD Serotec, Oxford, UK) was applied to 96-well microtiter plates and was incubated overnight at 4 °C, sealed with a film cover. After blocking with 1% bovine serum albumin (BSA, Irving, TX, USA), 40 µL of patient serum was added per well in combination with peroxidase-labeled anti-DNA monoclonal antibody (component No. 2 of cell death detection ELISA kit; Roche, Basel, Switzerland) according to the manufacturer’s protocol. After 2 h of incubation at room temperature (RT) on a shaking device (320 rpm), the samples were washed three times with 200 µL of phosphate-buffered saline (PBS, Boston, MA, USA) per well, and the peroxidase substrate (ABTS of cell death detection ELISA kit; Roche) was added. The absorbance at a wavelength of 405 nm was measured using a GloMax Navigator System with DI and PS (Promega, Madison, WI, USA) after 40 min of incubation at 37 °C under light shielding.

To quantify NETs in mouse BALF, we created a capture ELISA on the strength of MPO associated with DNA. First, 5 μg/mL of anti-MPO monoclonal antibody (1:500; ABD Serotec, Cat-No. 0400-0002), a key capturing antibody, was used to coat 96-well plates (75 μL per well) overnight at 4°C. Second, non-specific bindings were blocked by 1% bovine serum albumin. BALF (20 μL) and the peroxidase-labeled anti-DNA monoclonal antibody (1:25; Roche) were added to each well. The plate was incubated for 2 hours in a shaking table at 300 rpm and was washed three times. Finally, 100 μL of peroxidase substrate were added. The absorbance at 405 nm wavelength was measured using Fluostar Optima (BMG Labtech) after 30 minutes of incubation at 37°C in the dark.

Cell-free DNA (cfDNA) in the serum and culture supernatant was detected using the Quant-It PicoGreen® dsDNA kit (Thermo Fisher) according to the manufacturer's instructions. The fluorescence intensity was measured at a 480 nm excitation wavelength and 530 nm emission wavelength to reflect the amount of DNA. Myeloperoxidase-DNA (MPO-DNA) complexes in the supernatant and plasma were detected using capture ELISA as previously described 22 (link). Briefly, 75 μL of 5 μg/ml anti-MPO monoclonal antibody (ABD Serotec; Cat-No. 0400-0002), as the capturing antibody, was coated onto 96-well microtiter plates overnight at 4 °C. After blocking in 1% BSA, 40 μL of patient serum was added to the wells with peroxidase-labeled anti-DNA monoclonal antibody (component No. 2 of the commercial cell death detection ELISA kit; Roche, Cat. No. 11774425001). The plate was incubated for two hours at room temperature and washed three times with 200 μL of PBS per well. Next, the peroxidase substrate (ABTS) (Roche, Cat. No. 11774425001) was added. After 40 mins of incubation at 37 °C in the dark, the optical density (OD) was measured at 405 nm using a microplate reader.