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Fluoro MPO™
Fluorescent Myeloperoxidase Detection Kit

Key Benefits:

Can monitor multiple time points to follow kinetics.
One-step, no wash assay.
Adaptable for High Throughput format
Highly Sensitive
Applications - Fluorescence (Plate Reader) or Absorbance

Assay Principle

Myeloperoxidase (MPO) is a highly cationic glycosolated hemoprotein that has a molecular weight of 144kD. The hemoprotein consists of two dimers linked via a disulfide bridge. Each dimmer is composed of a heavy (53kD) and light (15kD) subunit. Each heavy chain contains an independently acting protoporphyrin group containing a central iron ^(1-5). MPO is present in the azurophilic granules of polymorphonuclear leukocytes (PMNs) and is unique to neutrophils and monocytes. However, monocytes contain only one third of the MPO found in PMN’s. MPO utilizes H202 produced by the neutrophils to oxidize a varity of aromatic compounds to give substrate radicals for bactericidal activity ^(4
review). This enzyme is unique however in that it can oxidize chloride ions to produce a strong nonradical oxidant,HOCl. HOCl is the most powerfull bactericidal produced by neutrophils ^(4
review). Excessive production of these radicals can cause oxidative stress leading to oxidative tissue injury.

H₂O₂ + Detection reagent (non-fluorescent)+ MPO
---------> fluorescent analog
Excitation:530-571nm; Emission: 590-600nm

Applications

Detection of MPO activity in neutrophils and macrophages.
Detection of PMN infiltration in tissue samples (inflammation and innate host defense mechanisms).
Acute and chronic inflammatory disorders due to oxidative tissue damage.
MPO activity in acute and chronic manifestations of cardiovascular disease.

Citations

Use of a hanging-weight system for liver ischemic preconditioning in mice - Hart, Much, Kohler et.al - Am J Physiol Gastrointest Liver Physiol 294: G1431-G1440, 2008.

Extracellular Adenosine Production by Ecto-5′-Nucleotidase Protects During Murine Hepatic Ischemic Preconditioning - M. Hart, C. Much, I. Gorzolla, J. Schittenhelm, D. Kloor, G. Stahl, H. Eltzschig - Gastroenterology, Volume 135, Issue 5, Pages 1739-1750.e3

Inflammatory Biomarkers of Sulfur Mustard Analog 2-Chloroethyl Ethyl Sulfide–Induced Skin Injury in SKH-1 Hairless Mice - Neera Tewari-Singh, Sumeet Rana, Mallikarjuna Gu, Arttatrana Pal, David J. Orlicky, Carl W. White and Rajesh Agarwal - Toxicological Sciences 2009 108(1):194-206

References

1.Olsen, R. L. & Little, c. (1983) Biochem. J. 209, 781-787.

2.Nauseef, W. M., and Malech, H. L. (1986) Blood, 67, 1504-1507.

3. Andrews, P. C., Parnes, C., and Krinsky, N. I. (1984) Arch. Biochem. Biophys., 228, 439-442.

4. Mark B. Hampton, Anthony J. Kettle, and Christine C. Winterbourn. Inside the Neutrophil Phagosome: Oxidants, Myeloperoxidase, and Bacterial Killing. Blood, Vol. 92 No. 9 (November 1), 1998: pp. 3007-3017.

5. Andrews, P.C., Parnes, C. & Krinsky, N.I. (1984) Comparison of myeloperoxidase and hemi-myeloperoxidase with respect to catalysis, regulation, and bacterial activity. Arch. Biochem. Biophys. 228, 439–442.

6. Mingjie Zhou, Zhenjun Diwu, Nataliya Panchuk-Voloshina and Richard P. Haugland. A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases. Anal Biochem 253, 162 (1997).

7. J. G. Mohanty, Jonathan S. Jaffe, Edward S. Schulman and Donald G. Raible. A highly sensitive fluorescent micro-assay of H₂O₂ release from activated human leukocytes using a dihydroxyphenoxazine derivative. J. Immunol Methods 202, 133 (1997).

8. Tatyana V. Votyakova and Ian J. Reynolds. Membrane Potential dependent and -independent production of reactive oxygen species by rat brain mitochondria. J Neurochem 79, 266 (2001).

9. Chun Song, Abu B. Al-Mehdi, and Aron B. Fisher. An immediate endothelial cell signaling response to lung ischemia. Am J Physiol Lung Cell Mol Physiol 281, L993 (2001).

10. Samantha C. Richer and W.C.L. Ford. A critical investigation of NADPH oxidase activity in human spermatozoa. Mol Hum Reprod 7, 237 (2001).

11. William G. Gutheil, Miglena E. Stefanova and Robert A. Nicholas. Fluorescent Coupled Enzyme Assays for -Alanine: Application to Penicillin-Binding Protein and Vancomycin Activity Assays. Anal Biochem 287, 196 (2000).

12. Dominik Peus, Remus A. Vasa, Astrid Beyerle, Alexander Meves, Carsten Krautmacher and Mark R. Pittelkow. UVB Activates ERK1/2 and p38 Signaling Pathways via Reactive Oxygen Species in Cultured Keratinocytes. J Invest Dermatol 112, 751 (1999).

13. Tatyana V.Votyakova ,Ian J.Reynolds. Detection of hydrogen peroxide with Amplex Red:interference by NADH and reduced glutathione auto-oxidation. Archives of Biochemistry and Biophysics, 431: 138-144 (2004).

14. Elizabeth Forbes, Tosei Murase, Ming Yang, Klaus I. Matthaei,*James J. Lee, Nancy A. Lee, Paul S. Foster, and Simon P. Hogan. Immunopathogenesis of Experimental Ulcerative Colitis Is Mediated by Eosinophil Peroxidase. The Journal of Immunology, 2004, 172:5664–5675.

Kit contents (for 500 assays)

10X Assay Buffer: 60ml
Detection reagent: One vial for 500 assays.
Hydrogen Peroxide: 1000µL of a stabilized 3% solution.
Myeloperoxidase: 1 vial at 30 units/ml

The following kits are available:
Catalog #	Size	Price (US$)
FLMPO 100-3	500 Tests	$395

* Please call 888 7 ASSAYS (888-727-7297) or email info@celltechnology.com for volume pricing

Product Data Sheet

Protocol

MSDS

Figure 1. MPO standard curve was serially diluted in 1X Reaction Buffer. Reaction cocktail (RC) was prepared as described (without EPO inhibitor). Next 50mL of MPO standard and 50mL of RC was added to individual well of a 96 well black plates. The plate was incubated at room and temperature in the dark. Data collected Ex:530nm Em:590nm