BURSTTEST allows the quantitative determination of leukocyte oxidative burst in heparinized whole blood. It contains unlabeled opsonized bacteria (E.coli), phorbol 12-myristate 13-acetate (PMA) and the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as stimulants, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate and necessary reagents. It determines the percentage of phagocytic cells which produce reactive oxidants (conversion of DHR 123 to R 123) and their enzymatic activity (amount of R 123 per cell).

The evaluation of these bioactivities should be performed by flow cytometry.

APPLICATIONS

This test is intended to investigate the altered oxidative burst activity found in various disorders and to evaluate the effects of drugs.

Reduced or missing burst activity is observed in inborne defects like the chronic granulomatous disease (CGD). CGD is a heterogenous group of inherited disorders that usually manifests itself during the first two years of life (3, 4). The disease is characterized clinically by repeated and life-threatening infections caused by bacterial and fungal organisms. These infections typically consist of pneumonia, lymphadenitis, or abscesses that involve lymph nodes, lungs, and liver. The NADPH oxidase is the enzyme system responsible for producing superoxide anion, which is quickly converted to hydrogen peroxide and hydroxyl radicals. Abnormalities in the constituent peptides of the NADPH oxidase enzyme system lead to the dysfunctions characteristic of CGD. Neutrophils from CGD patients fail to produce a significant oxidative burst following stimulation. Different forms of CGD are described (classical X-linked CGD and autosomal recessive patterns). BURSTTEST is a rapid and sensitive method for the diagnosis of CGD and for the detection of X-linked carriers.
The oxidative burst of granulocytes is impaired in transplant patients and patients with AIDS (6).
The spontaneous and fMLP-induced neutrophil respiratory burst was shown to be increased in neonates with laboratory signs of infection (7).
Various immunomodulators (e.g., cytokines (GM-CSF, G-CSF, TNF) or drugs) seem to have effects on the oxidative burst. By using fMLP as a low stimulant one can investigate additive or priming effects (8) of test substances.

The test kit is compatible with blood of mice, rats, rabbits, dogs, cattle and other species.

TEST PRINCIPLES

Phagocytosis by polymorphonuclear neutrophils and monocytes constitutes an essential arm of host defense against bacterial or fungal infections. The phagocytic process can be separated into several major stages: chemotaxis (migration of phagocytes to inflammatory sites), attachment of particles to the cell surface of phagocytes, ingestion (phagocytosis) and intracellular killing by oxygen-dependent (oxidative burst) and oxygen-independent mechanisms (1, 2).

BURSTTEST allows the quantitative determination of leukocyte oxidative burst. The BURSTEST kit contains unlabelled opsonized E.coli bacteria as particulate stimulus, the protein kinase C ligand phorbol 12-myristate 13-acetate (PMA) as high stimulus and the chemotactic peptide N-formyl-MetLeuPhe (fMLP) as low physiological stimulus, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate (5) and necessary reagents. Heparinized whole blood is incubated with the various stimuli at 37°C, a sample without stimulus serves as negative background control. Upon stimulation, granulocytes and monocytes produce reactive oxygen metabolites (superoxide anion, hydrogen peroxide, hypochlorous acid) which destroy bacteria inside the phagosome. Formation of the reactive oxidants during the oxidative burst can be monitored by the addition and oxidation of DHR 123. The reaction is stopped by addition of LYSING SOLUTION, which removes erythrocytes and results in a partial fixation of leukocytes. After one washing step with WASHING SOLUTION, DNA STAINING SOLUTION is added to exclude aggregation artifacts of bacteria or cells. The percentage of cells having produced reactive oxygen radicals are then analyzed as well as their mean fluorescence intensity (enzymatic activity).

REAGENTS PROVIDED

BURSTTEST contains reagents for 100 tests:

• Opsonized Escherichia coli bacteria
• Chemotactic peptide f-formyl-MetLeuPhe
• Phorbol 12-myristate 13-acetate
• Fluorogenic substrate Dihydrorhodamine 123
• LYSING SOLUTION
• DNA STAINING SOLUTION
• WASHING SOLUTION

REFERENCES

(1)	Roitt, I.M., Brostoff, J. & D.K. Male. 1996. Immunology, 4th ed. Gower Medical Publishing Ltd., London.
(2)	Sawyer, D.W., Donowitz, G.R. & G.L. Mandell. 1989. Polymorphonuclear neutrophils: An effective antimicrobial force. Rev. Infect. Dis. 11: S1532-S1544.
(3)	Donadebian, H. D. 1989. Congenital and aquired neutrophil abnormalities. In: Klempner, M.S. et al. (eds) Phagocytes and Disease. Kluwer, Dordrecht Boston New York, pp 103-118.
(4)	Smith, R.M. & J.T. Curnutte. 1991. Molecular basis of chronic granulomatous disease. Blood 77: 673 -686.
(5)	Rothe G, Oser A & G. Valet. 1988. Dihydrorhodamin 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwissenschaften 75: 354 - 355.
(6)	Dobmeyer, T.S., Raffel, B. Dobmeyer, J.M., Findhammer, S., Klein, S.A., Kabelitz, D. Hoelzer, D., Helm, E.B. & Rossol.1995. Decreased function of monocytes and granulocytes during HIV-1 infection correlates with CD4 cell counts. Eur. J. Med. Res. 1: 9-15.
(7)	Gessler, P., Nebe, T. Birle, A., Haas, N. & W. Kachel. 1996. Neutrophil respiratory burst in term and preterm neonates without signs of infection and in those with increased levels of C-Reactive Protein. Pediatr. Res. 39: 843-848.
(8)	Elbim, C., Chollet-Martin, S., Bailly, S., Hakim, J. & M.A. Gougerot-Pocidalo. 1993. Priming of polymorphonuclear neutrophils by tumor necrosis factor in whole blood: Identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides. Blood 82: 663-640.

For further information please contact:

Dr. Werner Hirt
Head of Immunology, Cell Biology and Flow Cytometry
+49 6221 9105-50
e-Mail: w.hirt@orpegen.com