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Amplite® Fluorimetric Myeloperoxidase Assay Kit *Red Fluorescence*

Myeloperoxidase dose response was measured with Amplite® Fluorimetric Myeloperoxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Myeloperoxidase dose response was measured with Amplite® Fluorimetric Myeloperoxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Myeloperoxidase dose response was measured with Amplite® Fluorimetric Myeloperoxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
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Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Excitation (nm)
Emission (nm)
Myeloperoxidase (MPO), most abundantly present in neutrophils and monocytes, is a green hemoprotein having peroxidase activity. It catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates; and plays an important role in the oxygen-dependent killing of tumor cells and microorganisms. MPO deficiency is a hereditary deficiency of the enzyme, which predisposes to immune deficiency. There are considerable interests in the development of therapeutic MPO inhibitors. Our Amplite® Myeloperoxidase Assay Kit provides a quick and sensitive method for the measurement of myeloperoxidase in solution and in cell lysates. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. The kit uses our Amplite® Red substrate which enables a dual recordable mode. The signal can be easily read by either a fluorescence microplate reader or an absorbance microplate reader. With the Amplite® Myeloperoxidase Assay Kit, we have detected as little as 0.1 mU/ml myeloperoxidase in a 100 µL reaction volume. The kit can be automated for high throughput screenings of MPO inhibitors.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. MPO standards or test samples (50 µL)
  2. Add MPO working solution (50 µL)
  3. Incubate at room temperature for 30 - 60 min
  4. Read fluorescence intensity at Ex/Em = 540/590 nm (cut off 570 nm)

Important notes
Thaw all the kit components to room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Red stock solution (250X):
Add 40 µL of DMSO (Component E) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Note: The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH 7 – 8. The provided assay buffer, pH 7.4, is recommended.

2. H2O2 stock solution (500X, 10 mM):
Add 10 µL of 3% H2O2 (0.88M, Component C) into 870 µL of Assay Buffer (Component B). Note: The diluted H2O2 solution is not stable. The unused portion should be discarded.

3. Myeloperoxidase (MPO) standard solution (200 mU/mL):
Add 50 µL of Assay Buffer (Component B) into the vial of Myeloperoxidase Standard (Component D). Note: One vial contains approximately 5 - 10 mU myeloperoxidase.


MPO standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11301

Add 20 µL of 200 mU/mL MPO standard solution into 380 µL of Assay Buffer (Component B) to get 10 mU/mL MPO standard solution (MPO7). Take 10 mU/mL MPO standard solution to perform 1:3 serial dilutions to get remaining serially diluted MPO standards (MPO6 - MPO1).


Add 20 μL of Amplite™ Red Stock Solution (250X) and 10 μL of H2O2 (500X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.03 mL MPO working solution. Protect from light.


Table 1. Layout of MPO standards and test samples in a 96-well solid black microplate. MPO= myeloperoxidase standards (MPO1 - MPO7, 0.01 to 10 mU/mL); BL=blank control; TS = test samples.


Table 2. Reagent composition for each well. Note that high concentration of MPO may cause reduced fluorescence signal due to the over oxidation of Amplite™ Red substrate (to a non-fluorescent product).

MPO1 - MPO750 µLSerial Dilution (0.01 to 10 mU/mL)
BL50 µLAssay Buffer (Component B)
TS50 µLtest sample
  1. Prepare myeloperoxidase standards (MPO), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of MPO working solution to each well of myeloperoxidase standard, blank control, and test samples to make the total MPO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of MPO working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.

  4. Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off = 570 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to that of the fluorescence reading.


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Spectral properties

Excitation (nm)571
Emission (nm)584



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View all 27 references: Citation Explorer
Detection of myeloperoxidase activity in primary leukemic cells by an enhanced chemiluminescent assay for differentiation between acute lymphoblastic and non-lymphoblastic leukemia
Authors: Tan S, Wang G, Peng M, Zhang X, Shen G, Jiang J, Chen F.
Journal: Clin Chim Acta (2009): 216
Determination of myeloperoxidase in EDTA plasma: comparison of an enzyme-linked immunosorbent assay with a chemiluminescent automated immunoassay
Authors: Zelzer S, Khoschsorur G, Stettin M, Weihrauch G, Truschnig-Wilders M.
Journal: Clin Chim Acta (2009): 62
Myeloperoxidase assay in plasma and peritoneal fluid of horses with gastrointestinal disease
Authors: Grulke S, Franck T, Gangl M, Peters F, Salciccia A, Deby-Dupont G, Serteyn D.
Journal: Can J Vet Res (2008): 37
Measurement of equine myeloperoxidase (MPO) activity in synovial fluid by a modified MPO assay and evaluation of joint diseases - an initial case study
Authors: Fietz S, Bondzio A, Moschos A, Hertsch B, Einspanier R.
Journal: Res Vet Sci (2008): 347
A novel assay system for myeloperoxidase activity in whole saliva
Authors: Sakamoto W, Fujii Y, Kanehira T, Asano K, Izumi H.
Journal: Clin Biochem (2008): 584
Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood
Authors: Franck T, Grulke S, Deby-Dupont G, Deby C, Duvivier H, Peters F, Serteyn D.
Journal: J Vet Diagn Invest (2005): 412
A sensitive and selective assay for chloramine production by myeloperoxidase
Authors: Dypbukt JM, Bishop C, Brooks WM, Thong B, Eriksson H, Kettle AJ.
Journal: Free Radic Biol Med (2005): 1468
Cytofluorimetric assay for evaluation of CD16 receptor expression and myeloperoxidase (MPO) activity of neutrophils in patients with psoriasis vulgaris treated with PUVA
Authors: Kapuscinska R, Wysocka J, Niczyporuk W, Ratomski K.
Journal: Wiad Lek (2004): 599
The relationship between subclassification of anti-myeloperoxidase IgG by enzyme-linked immuno-sorbent assay analysis and vasculitis activity
Authors: Yu F, Zhao MH, Zhang YK, Wang HY.
Journal: Zhonghua Nei Ke Za Zhi (2003): 27
A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol
Authors: Haqqani AS, S and hu JK, Birnboim HC.
Journal: Anal Biochem (1999): 126