Fluoro EPO

Product Description

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.

Assay Principle

Eosinophil peroxidase (EPO) is the most abundant enzyme found in eosinophils. It is the major cytotoxic agent released by activated eosinophils and uses hydrogen peroxide to generate reactive oxidants from halides and pseudo halide thiocyanate [1,2]. Eosinophils peroxidase has been shown to have antimycobacterial activity [3], however it is also implicated in tissue damage that occurs in asthma and other diseases [4,5]. Currently, the function of eosinophil involvement in the immune response is being redefined. Once considered a cell involved in host protection of parasitic infection, eosinophils multiple functions as leukocytes involved in the initiation and propagation of diverse inflammatory responses are being investigated. Eosinophils are further involved as modulators of innate and adaptive immunity [6].

H2O2 + Detection reagent (non-fluorescent)+ EPO ———> fluorescent analog

Excitation: 530-571nm; Emission: 590-600nm

Figure 1. Eosinophil peroxidase was titrated in 1X reaction buffer and 50uL of each point was added in triplicate to 96 black wells. 50uL of reaction cocktail was added and the reaction was incubated in the dark at room temperature for 30 minutes.

Fluorescence was read at 530nm excitation and emission detected at 590nm.

Applications

• Detection of EPO activity in isolated eosinophils.
• Functional studies of eosinophil degranulation.
• In-vitro eosinophil chemotaxis studies.

References

• Mayeno, A. N., Curran, A. J., Roberts, R. L. and Foote, C. S. (1989) Eosinophils preferentially use bromide to generate halogenating agents. J. Biol. Chem. 264, 5660±5668 .
• Slungaard, A. and Mahoney, J. R. (1991) Thiocyanate is the major substrate for eosinophil peroxidase in physiologic ¯uids. J. Biol. Chem. 266, 4903±4910 (Pruitt, K. M. and Tenovuo, J. V., eds.), pp. 31±53, Marcel Dekker, New York
• Human Eosinophil Peroxidase Induces Surface Alteration, killing, and lysis of Mycobacterium tuberculosis", Infection and Immunity, Feb. 2003, p. 605-613 by The American Society for Microbiology.
• Bousquet, J., Chanez, P., Lacoste, J. Y., Barneon, G., Ghavanian, N., Enander, I., Venge, P., Ahlstedt, S., Simony-Lafontaine, J., Godard, P. and Michel, F. (1990) Eosinophilic in¯ammation in asthma. New Engl. J. Med. 323, 1033±1039
• Parrillo, J. E., Borer, J. S., Henry, W. L., Wolff, S. M. and Fauci, A. S. (1979) The cardiovascular manifestations of the hypereosinophilic syndrome. Prospective study of 26 patients, with review of the literature. Am. J. Med. 67, 572-582
• Marc E. Rothenberg and ­Simon P. Hogan.­. THE EOSINOPHIL Annual Review of Immunology, Vol. 24: 147-174 (Volume publication date April 2006.)
• 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).
• 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).
• 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).
• 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).
• Samantha C. Richer and W.C.L. Ford. A critical investigation of NADPH oxidase activity in human spermatozoa. Mol Hum Reprod 7, 237 (2001).
• William G. Gutheil, Miglena E. Stefanova and Robert A. Nicholas. Fluorescent Coupled Enzyme Assays for Image-Alanine: Application to Penicillin-Binding Protein and Vancomycin Activity Assays. Anal Biochem 287, 196 (2000).
• 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).
• 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).
• # D. W. R. Hall, Bridget W. Logan and G. H. Parsons. Further studies on the inhibition of monoamine oxidase by M & B 9302 (clorgyline)—I .Substrate specificity in various mammalian species. Biochemical Pharma

Kit contents and Long Term storage