GENTAUR - Research solutions for the academic, biotechnology and pharmaceutical industries
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Fluoro MPOHOCL™
Fluorescent Myeloperoxidase Chlorination and peroxidation 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 - Fluorescent Plate Reader.
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| 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 |
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).
| 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 |
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