Human Myeloperoxidase DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Myeloperoxidase. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: Myeloperoxidase/MPO
Myeloperoxidase (MPO) is a heme-containing enzyme belonging to the XPO subfamily of peroxidases. It is an abundant neutrophil and monocyte glycoprotein that catalyzes the hydrogen peroxide dependent formation of hypochlorus acid (HOCl) and other reactive species. Enzymatically active MPO is a disulfide-linked tetramer that contains two heme groups and two copies each of the heavy and light chains. MPO binds Albumin, MMR, Cytokeratin 1 on vascular endothelial cells, HMW Kininogen, and Integrin CD11b/CD18 on neutrophils. These interactions promote MPO clearance, a reduction of nitric oxide and bradykinin levels, reduced vasodilation, and continued neutrophil activation. Neutrophil MPO is stored in cytoplasmic azurophilic granules. Upon cellular activation and degranulation, MPO is delivered into phagosomes where it is required for the killing of phagocytosed bacteria. Activated neutrophils also release granule contents extracellularly. Elevated plasma MPO levels have been associated with a variety of clinical conditions including systemic inflammation, eclampsia, risk of cardiovascular events, vascular endothelial dysfunction, severity of multiple sclerosis, and prospective mortality and oxidative stress during hemodialysis.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human Myeloperoxidase DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 10
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A lactobacilli-based inhaled live biotherapeutic product attenuates pulmonary neutrophilic inflammation
Authors: Nicola, T;Wenger, N;Xu, X;Evans, M;Qiao, L;Rezonzew, G;Yang, Y;Jilling, T;Margaroli, C;Genschmer, K;Willis, K;Ambalavanan, N;Blalock, JE;Gaggar, A;Lal, CV;
Nature communications
Species: Human
Sample Types: Endotracheal Tube Aspirates
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The assembly of neutrophil inflammasomes during COVID-19 is mediated by type I interferons
Authors: Cabrera, LE;Jokiranta, ST;Mäki, S;Miettinen, S;Kant, R;Kareinen, L;Sironen, T;Pietilä, JP;Kantele, A;Kekäläinen, E;Lindgren, H;Mattila, P;Kipar, A;Vapalahti, O;Strandin, T;
PLoS pathogens
Species: Human
Sample Types: Cell Culture Supernates
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Liraglutide and not lifestyle intervention reduces soluble CD163 after comparable weight loss in obese participants with prediabetes or type 2 diabetes mellitus
Authors: Grannes, H;Ueland, T;Simeone, P;Liani, R;Guagnano, MT;Aukrust, P;Michelsen, AE;Birkeland, K;di Castelnuovo, A;Cipollone, F;Consoli, A;Halvorsen, B;Gregersen, I;Santilli, F;
Cardiovascular diabetology
Species: Human
Sample Types: Plasma
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Staphylococcus aureus suppresses the pentose phosphate pathway in human neutrophils via the adenosine receptor A2aR to enhance intracellular survival
Authors: Vozza, EG;Daly, CM;O'Rourke, SA;Fitzgerald, HK;Dunne, A;McLoughlin, RM;
mBio
Species: Human
Sample Types: Cell Culture Supernates
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Myeloperoxidase enhances the migration and invasion of human choriocarcinoma JEG-3 cells
Authors: Mihalic, ZN;Kloimböck, T;Cosic-Mujkanovic, N;Valadez-Cosmes, P;Maitz, K;Kindler, O;Wadsack, C;Heinemann, A;Marsche, G;Gauster, M;Pollheimer, J;Kargl, J;
Redox biology
Species: Human
Sample Types: Cell Culture Supernates
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Glucosinolate-rich broccoli sprouts protect against oxidative stress and improve adaptations to intense exercise training
Authors: Flockhart, M;Nilsson, LC;Tillqvist, EN;Vinge, F;Millbert, F;Lännerström, J;Nilsson, PH;Samyn, D;Apró, W;Sundqvist, ML;Larsen, FJ;
Redox biology
Species: Human
Sample Types: Plasma
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Mitochondria-induced formation of neutrophil extracellular traps is enhanced in the elderly via Toll-like receptor 9
Authors: Pastorek, M;Kone?ná, B;Janko, J;Janovi?ová, ?;Podracká, ?;Záhumenský, J;te?ová, E;Dúbrava, M;Hodosy, J;Vlková, B;Celec, P;
Journal of leukocyte biology
Species: Human
Sample Types: Plasma
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Extracellular DNA concentrations in various aetiologies of acute kidney injury
Authors: A Gaál Koval, ? Janovi?ová, J Hodosy, J Bábí?ková, D Vavrincová, P Vavrinec, P Boor, ? Podracká, K Šebeková, P Celec, ? Tóthová
Scientific Reports, 2022-10-07;12(1):16812.
Species: Human
Sample Types: Urine
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Lung Adenocarcinoma Cell Sensitivity to Chemotherapies: A Spotlight on Lipid Droplets and SREBF1 Gene
Authors: AR Gründing, MA Schneider, S Richtmann, M Kriegsmann, H Winter, B Martinez-D, S Varona, B Liu, DS DeLuca, J Held, S Wrenger, T Muley, M Meister, T Welte, S Janciauski
Cancers, 2022-09-14;14(18):.
Species: Human
Sample Types: Cell Culture Supernates
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Mycobacterium avium-intracellulare complex promote release of pro-inflammatory enzymes matrix metalloproteinases by inducing neutrophil extracellular trap formation
Authors: K Nakamura, H Nakayama, S Sasaki, K Takahashi, K Iwabuchi
Scientific Reports, 2022-04-11;12(1):5181.
Species: Human
Sample Types: Cell Culture Supernates
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Circulating Interleukin-6 and CD16 positive monocytes increase following angioplasty of an arteriovenous fistula
Authors: S Hakki, EJ Robinson, MG Robson
Scientific Reports, 2022-01-26;12(1):1427.
Species: Human
Sample Types: Plasma
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The Value of Fetuin-A as a Predictor to Identify Takotsubo Patients at Risk of Cardiovascular Events
Authors: A Topf, M Mirna, N Bacher, V Paar, C Edlinger, LJ Motloch, S Gharibeh, M Bannehr, UC Hoppe, M Lichtenaue
Journal of cardiovascular development and disease, 2021-10-05;8(10):.
Species: Human
Sample Types: Serum
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Characterization of low-density granulocytes in COVID-19
Authors: LE Cabrera, PT Pekkarinen, M Alander, KHA Nowlan, NA Nguyen, S Jokiranta, S Kuivanen, A Patjas, S Mero, SH Pakkanen, S Heinonen, A Kantele, O Vapalahti, E Kekäläinen, T Strandin
PloS Pathogens, 2021-07-06;17(7):e1009721.
Species: Human
Sample Types: Serum
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Reduced neutrophil granular proteins and post-treatment modulation in tuberculous lymphadenitis
Authors: GR Kathamuthu, K Moideen, R Sridhar, D Baskaran, S Babu
PLoS ONE, 2021-06-21;16(6):e0253534.
Species: Human
Sample Types: Plasma
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Neutrophil extracellular trap clearance by synovial macrophages in gout
Authors: JH Jeong, SJ Choi, SM Ahn, JS Oh, YG Kim, CK Lee, B Yoo, S Hong
Arthritis Research & Therapy, 2021-03-19;23(1):88.
Species: Human
Sample Types: Cell Culture Supernates
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Polymicrobial stimulation of human fetal membranes induce neutrophil activation and neutrophil extracellular trap release
Authors: M Tong, SE Hanna, VM Abrahams
Journal of reproductive immunology, 2021-03-09;145(0):103306.
Species: Human
Sample Types: Cell Culture Supernates
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The association between impairment of HDL cholesterol efflux capacity and atrial remodeling in atrial fibrillation
Authors: A Minami-Tak, H Iwata, K Miyosawa, T Shiozawa, H Hayashi, T Funamizu, K Ishii, Y Nozaki, H Tabuchi, G Sekita, K Shimada, M Sumiyoshi, Y Nakazato, H Daida, T Minamino
Scientific Reports, 2021-02-11;11(1):3547.
Species: Human
Sample Types: Serum
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Experimental Human Challenge Defines Distinct Pneumococcal Kinetic Profiles and Mucosal Responses between Colonized and Non-Colonized Adults
Authors: E Nikolaou, SP Jochems, E Mitsi, S Pojar, A Blizard, J Reiné, C Solórzano, E Negera, B Carniel, A Soares-Sch, V Connor, H Adler, SR Zaidi, C Hales, H Hill, A Hyder-Wrig, SB Gordon, J Rylance, DM Ferreira
MBio, 2021-01-12;12(1):.
Species: Human
Sample Types: Nasal Lining Fluid
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Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae
Authors: C Thomas, H Nothaft, R Yadav, C Fodor, A Alemka, O Oni, M Bell, B Rada, CM Szymanski
PLoS ONE, 2020-12-30;15(12):e0244031.
Species: Human
Sample Types: Cell Culture Supernates
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Low-Avidity Autoantibodies Against Bactericidal/Permeability Increasing Protein Occur in Gram-Negative and Gram-Positive Bacteremia
Authors: J Theprungsi, JT Thaden, RM Wierzbicki, AS Burns, S Skopelja-G, VG Fowler, KL Winthrop, IW Martin, WFC Rigby
Infect. Immun., 2020-09-18;0(0):.
Species: Human
Sample Types: Serum
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Activation of a TLR9 mediated innate immune response in preeclampsia
Authors: RD Williamson, FP McCarthy, LC Kenny, CM McCarthy
Sci Rep, 2019-04-11;9(1):5920.
Species: Human
Sample Types: Plasma
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Role of NOD1/NOD2 receptors in Fusobacterium nucleatum mediated NETosis
Authors: HM Alyami, LS Finoti, HS Teixeira, A Aljefri, DF Kinane, MR Benakanake
Microb. Pathog., 2019-03-30;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Calcineurin inhibitor Tacrolimus impairs host immune response against urinary tract infection
Authors: D Emal, E Rampanelli, N Claessen, FJ Bemelman, JC Leemans, S Florquin, MC Dessing
Sci Rep, 2019-01-14;9(1):106.
Species: Human
Sample Types: Cell Culture Supernates
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Possible involvement of NETosis in inflammatory processes in the eye: Evidence from a small cohort of patients
Authors: T Barliya, R Dardik, Y Nisgav, M Dachbash, D Gaton, G Kenet, R Ehrlich, D Weinberger, T Livnat
Mol. Vis., 2017-12-13;23(0):922-932.
Species: Human
Sample Types: Vitreous Humor
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Myeloperoxidase in Hypertensive Disorders of Pregnancy and Its Relation With Nitric Oxide
Authors: L Rocha-Penh, M Caldeira-D, JE Tanus-Sant, R de Carvalh, VC Sandrim
Hypertension, 2017-05-01;0(0):.
Species: Human
Sample Types: Plasma
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Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa
PLoS Pathog., 2016-11-17;12(11):e1005987.
Species: Human
Sample Types: Cell Culture Supernates
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Therapy with plasma purified alpha1-antitrypsin (Prolastin(R)) induces time-dependent changes in plasma levels of MMP-9 and MPO.
Authors: Koepke J, Dresel M, Schmid S, Greulich T, Beutel B, Schmeck B, Vogelmeier C, Janciauskiene S, Koczulla A
PLoS ONE, 2015-01-30;10(0):.
Species: Human
Sample Types: Serum
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Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.
Authors: Yoo D, Winn M, Pang L, Moskowitz S, Malech H, Leto T, Rada B
J Immunol, 2014-04-16;192(10):4728-38.
Species: Human
Sample Types: Cell Culture Supernates
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Average Rating: 4.6 (Based on 5 Reviews)
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Very reliable. I ran human serum samples in 1:200 fold in duplicates. CV remain very low which is very nice. Background stayed low too.
Great kit - multiple plates, only need to run in singlicates due to low CV%
Very pleased with this kit. Very reliable. Used EDTA plasma diluted 1/200 with RD buffer on a 384-well plate format.
Serum samples require a 50-fold diluition
