Instructions BASOTEST Version 05/12 page 1 of 8

1 Instructions BASOTEST Version 05/12 . page 1 of 8. Reagent kit for the quantitative determination of the degranulation of basophilic granulocytes in heparinized human whole blood Reagent kit containing antibodies, allergens and reagents for 100 tests Glycotope Biotechnology GmbH. Czernyring 22. 69115 Heidelberg Germany Tel. +49 (0)62 21 91 05-0. Fax +49 (0)62 21 91 05-10. E-Mail: Key to symbols used * See chapter MATERIALS AND REAGENTS for a full explanation of symbols used in reagent component naming. Instructions BASOTEST Version 05/12 . page 2 of 8. genetic, developmental, and environmental fac- tors (6, 7, 8).

2 SUMMARY and EXPLANATION The flow cytometric method correlates well with histamine release assays. BASOTEST there- This reagent kit allows the quantitative determina- fore allows the diagnosis of immediate-type hy- tion of basophil degranulation in heparinized hu- persensitivity (type I reactions), especially in re- man whole blood. It contains the chemotactic sponse to natural allergens. Furthermore, the peptide N-formyl-Met-Leu-Phe (fMLP) as positive success of immunotherapies ( , hyposensitisa- control, important allergens, a two-colour antibody tion) may be monitored. However, this test is not reagent for assessing the activation of human suited to detect any delayed type hypersensitivity basophilic granulocytes and necessary reagents.

3 ( , type IV reactions). It determines the percentage of basophilic granu- locytes which have degranulated after incubation PRINCIPLES of the PROCEDURE. with allergen or fMLP. The evaluation of basophil degranulation should BASOTEST allows the quantitative determina- be performed by flow cytometry. The detailed tion of human basophil degranulation (1-3). Instructions result from specific experience and The test kit contains the chemotactic peptide N- precise validation assays. Critical steps are in formyl-Met-Leu-Phe (fMLP) as positive control (8), bold letters. important allergens, a two-colour antibody rea- gent for detection of human basophilic granulo- APPLICATIONS cytes and determination of basophil activation and necessary reagents.

4 Heparinized whole blood is incubated first with reagent B (stimulation BASOTEST is intended for investigation of al- buffer) for 10 min at 37 C, then with allergen at lergen induced activation of basophilic granulo- various concentrations for 20 min at 37 C. Rea- cytes. gent E contains the chemotactic peptide N- Basophilic granulocytes are the least common Formyl-Met-Leu-Phe (fMLP) which is used as a circulating leukocyte in blood and account for only positive control, addition of reagent A (wash solu- - 1% of the total white blood cell population tion) serves as negative background control. Acti- (1-3).

5 Immediate-type hypersensitivity (1-3) is vation of basophilic granulocytes induces fusion characterized by allergic reactions immediately of cytoplasmic granules with the plasma mem- following contact with innocuous foreign sub- brane and the sucessive release of inflammatory stances (allergens or antigens). Allergic reactions mediators. The degranulation process is stopped are predominantly due to the immunoglobulin E by incubating the whole blood samples on ice. (IgE) class of antibodies (4) and develop when The cells are then labelled reagent F which is a contact with an antigen triggers the formation of two-colour antibody reagent consisting of two specific IgE antibodies by B cells with T cell help.

6 Different murine monoclonal antibodies conjugat- The antigen-specific IgE antibodies bind to the ed with various fluorochromes. The monoclonal very high-affinity Fc receptors I in the membrane antibody anti-IgE-PE is conjugated with the fluo- of tissue mast cells and basophilic leukocytes (5). rescent dye Phycoerythrin, reacts with human IgE. Renewed contact with the same antigen then and therefore detects basophilic granulocytes. leads to bridging: adjacent antibodies on the cell The monoclonal antibody anti-CD63-FITC is con- surface of sensitized cells are crosslinked by anti- jugated with the fluorochrome fluorescein and gen molecules.

7 This bridging of IgE molecules on recognizes a glycoprotein (gp53) expressed on the basophil cell surface by antigen or allergen activated basophils. After staining of basophils activates the cell to secrete a number of pre- with this antibody reagent, erythrocytes are re- formed chemical mediators stored in secretory moved by addition of reagent G (lysing solution). granules, such as histamine, heparin, neutral After one washing step with reagent A (wash protease, and a number of acid hydrolases and solution), the percentage of activated basophilic chemotactic factors. Secondary mediators (leuko- granulocytes is determined by flow cytometry.)

8 Trienes 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 Instructions BASOTEST Version 05/12 . page 3 of 8. 5. Ultrapure, apyrogenic water or water for in- MATERIAL and REAGENTS jection for reconstitution of wash solution (re- agent A). The reagent kit contains: 6. Double distilled water for dilution of 10 x lys- ing solution (reagent G). REAG A. 7. Various other allergens or allergen mixtures.

9 1 bottle of Instamed-Salts to be reconstituted in 1 L of ultrapure, apyrogenic water, provides 1 L Required apparatus: ready-to-use 1 x wash solution. 1. Variable volume micropipettes 20 - 200 l, 100 - 1000 l and disposable tips. REAG B 2. Dispenser pipette and dispenser tips. 1 vial containing lyophilized stimulation buffer to 3. Bottle-top dispensers for wash solution and be reconstituted by addition of 2 ml of ultrapure, 1 x lysing solution. apyrogenic water. Store in aliquots at -20 C after 4. Waterbath. reconstitution. 5. Digital thermometer. REAG C 6. Vortex mixer. 1 vial (200 l) containing the allergenic extract 7.

10 Refrigerated centrifuge with swinging buckets Mite mix, concentrated stock solution. and 12 x 75 mm tube carriers. Dilute 10 l in 1 ml of WASHING SOLUTION for 8. Flow cytometer with 488 nm excitation wave- use. length (argon-ion laser). REAG D. 1 vial (200 l) containing the allergenic extract 7 WARNING. grass mix, concentrated stock solution. Dilute 10 l in 1 ml of WASHING SOLUTION for use. 1. Blood samples must always be regarded as potentially infectious. Wear disposable gloves REAG E and protective clothing while handling blood 1 vial (200 l) containing the chemotactic peptide samples. fMLP (200 x stock solution, mM).