Clinical Diagnostic for the Determination of Allergen Induced Basophil Degranulation
 | BASOTEST© allows the quantitative determination of basophil degranulation in heparinized human whole blood. It contains the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as positive control, important allergens, a two-colour antibody reagent for assessing the activation of human basophilic granulocytes and necessary reagents. It determines the percentage of basophilic granulocytes which have degranulated after incubation with allergen or fMLP. |
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APPLICATIONS
BASOTEST© is intended for investigation of allergen induced activation of basophilic granulocytes.
Basophilic granulocytes are the least common circulating leukocyte in blood and account for only 0.5 - 1% of the total white blood cell population (1-3). Immediate-type hypersensitivity (1-3) is characterized by allergic reactions immediately following contact with innocuous foreign substances (allergens or antigens). Allergic reactions are predominantly due to the immunoglobulin E (IgE) class of antibodies (4) and develop when contact with an antigen triggers the formation of specific IgE antibodies by B cells with T cell help. The antigen-specific IgE antibodies bind to the very high-affinity Fc* receptors I in the membrane of tissue mast cells and basophilic leukocytes (5). Renewed contact with the same antigen then leads to bridging: adjacent antibodies on the cell surface of sensitized cells are crosslinked by antigen molecules. This bridging of IgE molecules on the basophil cell surface by antigen or allergen activates the cell to secrete a number of preformed chemical mediators stored in secretory granules, such as histamine, heparin, neutral protease, and a number of acid hydrolases and chemotactic factors. Secondary mediators (leukotrienes and cytokines) are also generated as a result of cell activation. Allergic rhinitis or hay fever, asthma bronchiale, systemic anaphylaxis and urticaria are typical allergic diseases.
The immune response to environmental allergens is believed to depend on multiple factors including genetic, developmental, and environmental factors (6, 7, 8).
The flow cytometric method correlates well with histamine release assays. BASOTEST© therefore allows the diagnosis of immediate-type hypersensitivity (type I reactions), especially in response to natural allergens. Furthermore, the success of immunotherapies (e.g., hyposensitisation) may be monitored. However, this test is not suited to detect any delayed type hypersensitivity (e.g., type IV reactions).
PRINCIPLES
BASOTEST© allows the quantitative determination of human basophil degranulation (1-3).
The kit contains the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as positive control (8), important allergens, a two-colour antibody reagent for detection of human basophilic granulocytes and determination of basophil activation and necessary reagents. Heparinized whole blood is incubated first with STIMULATION BUFFER for 10 min at 37°C, then with allergen at various concentrations for 20 min at 37°C. The chemotactic peptide N-Formyl-Met-Leu-Phe (fMLP) is used as a positive control, addition of WASHING SOLUTION serves as negative background control. Activation of basophilic granulocytes induces fusion of cytoplasmic granules with the plasma membrane and the sucessive release of inflammatory mediators. The degranulation process is stopped by incubating the whole blood samples on ice. The cells are then labelled with the two-colour antibody reagent consisting of two different murine monoclonal antibodies conjugated with various fluorochromes. The monoclonal antibody anti-IgE-PE is conjugated with the fluorescent dye Phycoerythrin, reacts with human IgE and therefore detects basophilic granulocytes. The monoclonal antibody anti-gp53-FITC is conjugated with the fluorochrome fluorescein and recognizes a glycoprotein (gp53) expressed on activated basophils. After staining of basophils with this antibody reagent, erythrocytes are removed by addition of LYSING SOLUTION. After one washing step with WASHING SOLUTION, the percentage of activated basophilic granulocytes is determined by flow cytometry.
REAGENTS PROVIDED
- STAINING REAGENT (anti-IgE-PE and anti-gp53-FITC)
- STIMULATION BUFFER
- Allergenic extract 7 GRASS MIX
- Allergenic extract MITE MIX
- fMLP
- LYSING SOLUTION
- WASHING SOLUTION
REFERENCES
(1) Roitt, I.M., Male, D.K. & J. Brostoff. 1996. Immunology, 4th ed. Mosby, London.
(2) Klein, J. 1990. Immunology. Blackwell Scientific Publications Limited, Oxford.
(3) Janeway, Jr., C.A & P. Travers. 1996. Immunobiology: The Immune System in Health and Disease. Second ed. Garland Publishing Inc. (New York, London) and Current Biology, Ltd. (London, San Francisco, Philadelphia).
(4) Ishizaka, K., Ishizaka, T. & M.M. Hornbrook. 1966. Physiochemical properties of reaginic antibody. IV. Presence of a unique immunoglobulin as a carrier of reaginic activity. J. Immunol. 97: 75-85.
(5) Kinet, J.P. 1990. The high affinity receptor for immunoglobulin E. Curr. Op. Immunol. 2: 499-505.
(6) Suoniemi, I., Björkstén, F. & T. Haahthela. 1987. Dependence of immediate hypersensitivity in the adolescent period on factors encountered in infancy. Allergy 36: 263-268.
(7) Huang, S.K. & D.G. Marsh. 1993. Genetics of allergy. Ann. Allergy 70: 347-358.
(8) Hopkin, J.M. 1995. Genetics of atopy. Pediatr. Allergy Immunol. 6: 139-144.
(9) Siraganian, R.P. & W.A. Hook. 1977. Mechanism of histamine release by formyl methionine-containing peptides. J. Immunol. 119: 2078-2083.