Circulating complexes of neutrophil myeloperoxidase with DNA were measured using ELISA as previously described (9 (link)). Briefly, 96-well ELISA plates were coated with anti-MPO antibody (5µg/mL, AbD Serotec, USA), and a Death plus EIA kit (DNA-Death plus, Roche Switzerland) was used as a source of all buffers and of a secondary, HRP-conjugated antibody. Raw results were presented as mean optical densities (OD).
Anti mpo antibody
Manufactured by Bio-Rad
Sourced in United States
The Anti-MPO antibody is a laboratory tool used for the detection and analysis of the myeloperoxidase (MPO) protein. MPO is an enzyme found in certain white blood cells and is a biomarker associated with various medical conditions. The Anti-MPO antibody can be utilized in techniques such as Western blotting, ELISA, and immunohistochemistry to identify and quantify the presence of MPO in biological samples.
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Lab products found in correlation
Cell death detection elisa kit, by Roche (3 mentions)
Abts substrate, by Merck Group (2 mentions)
Cell death detection elisa, by Merck Group (2 mentions)
Rhmpo, by R&D Systems (2 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Dcrp00, by R&D Systems (1 mentions)
Microvue cic c1q eia, by Quidel (1 mentions)
96 well microtitre plate, by Corning (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by BD (1 mentions)
Calprotectin, by R&D Systems (1 mentions)
96 well microtiter plate, by Corning (1 mentions)
Synergy plate reader, by Agilent Technologies (1 mentions)
Calf thymus dna, by R&D Systems (1 mentions)
Cell death elisa, by Roche (1 mentions)
10 protocols using anti mpo antibody
1
Neutrophil MPO-DNA Complexes Measurement
2
Quantifying MPO-DNA Complexes by ELISA
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MPO-DNA complexes were evaluated using a capture ELISA with the commercial cell death detection ELISA kit (11,774,425,001, Roche, Mannheim, Germany). 5 ug/ml anti-MPO antibody (0400–0002, ABD Serotec, Hercules, California, USA) was coated to 96-well microtiter plates overnight at 4 °C. The next day each well was blocked in 1% BSA, then 40 µL serum was added and incubated at RT on a shaking device (320 rpm) for 2 h according to the manufacturer´s instructions. After washing three times with 250 µL incubation buffer per well, 100 ul peroxidase substrate (ABTS) was added and incubated on a shaking device (200 rpm) in the dark for 40 min. The absorbance at 405 nm was measured using a microplate reader.
3
Detection of MPO-DNA complexes by ELISA
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MPO-DNA complexes were detected by capture ELISA [29 (link)]. Anti-MPO antibody (5 µg/ml, ABD Serotec, Cat-No. 0400-0002) was coated to 96-well microtiter plates overnight at 4 °C. After blocking with 1% BSA in PBS (30 min, 37 °C), patient’s sera was added to each well at a 1:20 dilution in peroxidase-labelled anti-DNA monoclonal antibody (component n° 2 of the cell death detection ELISA kit, Roche, Cat. No: 11774425001). The plate was incubated for 2 h at room temperature using a shaking device (320 rpm). The samples were subsequently washed three times with PBS and the peroxidase substrate (ABTS) contained in the kit (Roche, Cat. No: 11774425001) was added. After 40 min’ incubation at 37 °C in the dark, the absorbance was measured at 405 nm wavelength using VersaMax™ Microplate Reader (Molecular Devices, USA).
4
Serum Biomarkers in ANCA-Associated Vasculitis
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Concentration of CRP (high-sensitivity CRP, DCRP00, R&D Systems) was measured in participants sera by ELISA according to manufacturer's instructions. As methods for ANCA testing differ at participating sites, patient sera was re-analyzed using a standard ELISA for anti-PR3 antibody (ORG518, Orgentec) and anti-MPO antibody (425–2,380, BioRad) according to manufacturer's instructions. Concentration of S100A12 in sera from participants was measured with an in-house monoclonal antibody sandwich ELISA directed against a defined epitope on S100A12; samples from 32 healthy children and adolescents established that normal serum concentrations of S100A12 are < 75 ng/ml.
5
Quantification of MPO-DNA Complexes
6
ELISA for MPO-DNA Complexes
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An in-house ELISA was used to quantify MPO-DNA complexes. Briefly, after overnight coating with anti-MPO antibody (2 μg/mL; 0400-0002, Bio-Rad) at 4°C, a 96-well plate was blocked with 2.5% BSA in PBS for 2 hours at room temperature. The plate was subsequently washed before incubation for 90 minutes at room temperature with 20% human or mouse plasma in blocking buffer. The plate was washed 5 times, and then incubated for 90 minutes at room temperature with anti-DNA antibody (1:10; Cell Death Detection ELISA, 11544675001, Sigma-Aldrich). After 5 washes, the plate was developed with ABTS substrate (10102946001, Sigma-Aldrich).
7
Quantification of Circulating NETs by MPO-DNA ELISA
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Plasma levels of calprotectin (R&D Systems, Minneapolis, MN, USA) and immune complexes (ICs) (MicroVue CIC-C1q EIA, Quidel, Athens, OH, USA) were measured by ELISA, following the manufacturers’ instructions. The IC ELISA is based on the capacity of complement factor C1q, immobilized to the plate, to bind to circulating ICs. Quantification of circulating NETs was performed by utilizing myeloperoxidase (MPO)–DNA ELISA as described by us [10 (link)]. First, 96-well microtitre plates (Corning Inc., Corning, NY, USA) were coated with anti-MPO antibody (4 μg/ml; Bio-Rad Laboratories, Hercules, CA, USA) overnight at 4°C, and then blocked with 1% BSA in PBS for 2 h at room temperature (RT). Then, plasma samples diluted 1:100 (MPO–DNA ELISA) were added in 1% BSA in PBS with 2 mM EDTA, and incubated overnight at 4°C. Anti-DNA–HRP from the Cell Death Detection ELISA kit (clone MCA-33; Roche, Indianapolis, IN, USA) was added as detection antibody for 2 h at RT. The reaction was developed with 3,3′,5,5′-tetramethylbenzidine (BD Biosciences, San Jose, CA, USA) for 20 min and stopped by the addition of 2 M sulphuric acid. Known concentrations of MPO–DNA complexes (rhMPO, R&D Systems, Minneapolis, MN, USA; calf thymus DNA, Trevigen, Gaithersburg, MD, USA) were utilized to construct a standard curve. Absorbance was measured by a plate reader at 450 nm (Synergy 2, BioTek, Winooski, VT, USA).
8
Quantifying Circulating Calprotectin and NETs
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Levels of circulating calprotectin were analyzed using a commercial ELISA kit according to manufacturer’s instructions (R&D Systems, Minneapolis MN, USA). Circulating NETs were quantified using a myeloperoxidase (MPO)-DNA ELISA, as described previously (6 (link), 26 (link)). Briefly, 96-well microtiter plate (Corning) was coated with anti-MPO antibody (4 μg/ml; Bio-Rad Laboratories, Hercules, CA, USA) overnight at 4°C, followed by blocking with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 hours at RT. After blocking, plasma samples (1:1000 dilution in 1% BSA in PBS with 2mM EDTA) were added and incubated overnight at 4°C. Anti-DNA-HRP from Cell Death Detection ELISA kit (clone MCA-33; Roche) was added as secondary antibody for 1.5 hour at RT. The reaction was developed with 3,3′,5,5′ tetramethylbenzidine (TMB; BD Biosciences) and ended by the addition of 2N sulfuric acid. Known concentrations of MPO-DNA complexes (rhMPO, R&D Systems; Calf thymus DNA, Trevigon) were used to construct a standard curve. Plasma levels of human formyl methionine (fMet) were analyzed using a commercial ELISA kit according to manufacturer’s instructions (My BioSource Inc., San Diego, CA, USA). Absorbance for all ELISA assays were measured at 450 nm with a Synergy plate reader (BioTek).
9
Quantification of MPO-DNA Complexes
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An in-house ELISA was used to quantify MPO-DNA complexes. First, Plasma was collected from the whole blood of mice by centrifugation at 500 ×g for 20 min at 4 ℃; mouse cerebral cortex samples were homogenized in the lysis buffer (1:10 dilution) and centrifugated at 1500 rpm for 15 min at 4 ℃ to collect the supernatant. Briefly, after overnight coating with anti-MPO antibody (1:500, 0400–0002, Bio-Rad) at 4 ℃, 96-well plates were then blocked with 5% BSA for 2 h at room temperature. After washing three times (300 μL), 50 μL of plasma or 50 μL of tenfold dilution of the mouse brain homogenates was added into the wells with 80 μL of incubation buffer containing a peroxidase-labeled anti-DNA mAb (Cell Death ELISA, 11774425001, Roche, Basel, Switzerland). The plates were then incubated at room temperature for 2 h. After five washes, the plate was developed with 100 μL ABST substrate. The absorbance (OD) at 450 nm was measured after 30 min incubation in the dark. All samples for completion and analysis of ELISA were performed by two researchers blinded to experimental groups.
10
Quantification of MPO-DNA Complexes via ELISA
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An in-house enzyme-linked immunosorbent assay (ELISA) was used to quantify MPO-DNA complexes [16 (link)]. Briefly, after overnight coating with anti-MPO antibody (2 µg/mL; 0400-0002, Bio-Rad, Hercules, CA, USA) at 4 °C, a 96-well plate was blocked with 2.5% bovine serum albumin in phosphate-buffered solution for 2 h at room temperature. The plate was subsequently washed before incubating for 90 min at room temperature with 20% plasma isolated from the NASH in blocking buffer. The plate was washed five times and then incubated for 90 min at room temperature with an anti-DNA antibody (1:10; Cell Death Detection ELISA, 11544675001, Sigma, St. Louis, MO, USA). After five washes, the plate was developed with an ABTS substrate (Sigma).
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