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Fluoro MPOHOCL™
Fluorescent Myeloperoxidase Chlorination and peroxidation Detection Kit
New!

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

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 powerful bactericidal produced by neutrophils ^{(4 review)}. Excessive production of these radicals can cause oxidative stress leading to oxidative tissue injury.

Chlorination Reaction:

H₂O₂ + MPO + Cl- ----> HOCl + APF (non fluorescent) ----> Fluorescent Dye

Excitation:488nm; Emission: 515-530nm

Peroxidation Reaction:

H₂O₂ + Detection reagent (non-fluorescent)+ MPO -----> fluorescent analog

Excitation 530-571nm Emission 590-600nm

Applications

Detection of MPO chlorination 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 chlorination tissue damage.
MPO chlorination activity in acute and chronic manifestations of cardiovascular disease.

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. Ken-ichi Setsukinai, Yasuteru Urano, Katsuko Kakinuma, Hideyuki J. Majima , and Tetsuo Nagano. Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 5, Issue of January 31, pp. 3170–3175, 2003

Kit contents (for 500 assays of Myeloperoxidase detection and 300 assays of Chlorination detection)

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
1 vial of APF

Not For Sale in Japan

Peroxidase and Chlorination Detection
Catalog #	Size	Price (US$)
MPOHOCL 100-3	500 + 300 Tests	$590

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

Product Data Sheet

Protocol

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

Figure 2. MPO standard was serially diluted in 1X reaction buffer. 50mL of the MPO standard and 50mL of the chlorination reaction cocktail was added to black clear bottom 96 well plates. The plate was incubated in the dark for 20 minutes and then read in a fluorescent plate reader (Ex: 488 nm Em: 515 nm).