Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.
BACKGROUND
The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens.


METHODS
Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously.


RESULTS
Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement.


CONCLUSIONS
The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.
Kahlert
H
H
Allergopharma Joachim Ganzer KG, R&D Department, Reinbek, Germany. helga.kahlert@allergopharma.de
Cromwell
O
O
Fiebig
H
H
D000328:Adult; D000485:Allergens / Q000276:immunology*; D000080384:Allergoids; D000911:Antibodies, Monoclonal; D001491:Basophils / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010727:Phosphoric Diester Hydrolases / Q000032:analysis*; D010936:Plant Extracts / Q000276:immunology; D006109:Poaceae / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D011755:Pyrophosphatases / Q000032:analysis*; D011995:Recombination, Genetic
D000485:Allergens; D000080384:Allergoids; D000911:Antibodies, Monoclonal; C427944:ENPP3 protein, human; D010936:Plant Extracts; D007073:Immunoglobulin E; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases