BASOTEST allows the quantitative determination of human basophil degranulation in heparinized whole blood. It contains the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as stimulant, a two-colour antibody reagent for detection of basophilic granulocytes and their activation and necessary reagents. It determines the percentage of basophilic granulocytes which have degranulated after incubation with allergen or fMLP.

APPLICATIONS

BASOTEST is intended to investigate allergen induced activation of basophilic granulocytes.

Basophilic granulocytes are the least common circulating leucocyte in blood and account for only 0.5-1% of the total white blood cell population. Immediate-type hypersensitivity is the hallmark of any allergic reaction. Basophils have high affinity Fc receptors for IgE antibodies on their surface. Allergic reactions are predominantly due to the immunoglobulin E (IgE) class of antibodies and develop when contact with a specific antigen triggers the formation of IgE antibodies which bind to the Fc receptors of basophilic leukocytes. Renewed contact with the same antigen then leads to bridging: adjacent antibodies on the cell surface are interconnected by antigen molecules through their antigen-binding portions. This bridging of IgE molecules on the basophil cell surface by antigen or allergen activates the cell to secrete its mediators. A number of chemical mediators are released from basophils. The secretory granules contain a preformed mediators, such as histamin, heparin, neutral protease, and a number of acid hydrolases and chemotactic factors. Secondary mediators (leukotrienes) are also generated as a result of cell activation. Allergic rhinitis, bronchial asthma and urticariia are typical allergic diseases.

The flow cytometric method correlates very well with histamine release. BASOTEST therefore allows the diagnosis of immediate-type hypersensitivity.

PRINCIPLES

BASOTEST allows the quantitative determination of human basophil activation and degranulation.

The test kit contains the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as positive control, a two-colour antibody reagent for detection of basophilic granulocytes and determination of basophil activation and necessary reagents. Heparinized whole blood is incubated with allergen at various concentrations for 30 min at 37°C. The chemotactic peptide N-Formyl-Met-Leu-Phe (fMLP) is used as a positive control at a concentration of 1 µM, 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. After incubation at 37°C the cell suspension is washed once with WASHING SOLUTION. The cells are then stained 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), which is expressed on activated basophils. After staining of basophils with this antibody reagent the cells are washed with WASHING SOLUTION, erythrocytes are removed by addition of LYSING SOLUTION. After one additional washing step, the percentage of activated basophilic granulocytes is determined by flow cytometry.

REAGENTS PROVIDED

• STAINING REAGENT (anti-IgE-PE and anti-gp53-FITC)
• fMLP
• LYSING SOLUTION
• WASHING SOLUTION