Characterization of Modified Allergen Extracts by in vitro β-Hexosaminidase Release from Rat Basophils

Background: To date, there is no well-established test available that can be used to measure functional properties of modified allergens (allergoids). Due to the cross-linking process, the IgE-binding capacity of the allergens, normally necessary for their characterization, is lost. The aim of this study was to test whether the rat basophilic leukaemia (RBL) cell assay (β-hexosaminidase release by rat basophils upon allergen stimulation) can be adopted to characterize allergoids and to evaluate the assay for testing allergoids and native allergens as well. Methods: Mice were immunized with native and modified Phleumpratense extracts in the presence of alum. Their sera were used to sensitize RBL-2H3 cells and measure basophil stimulation induced by different allergen extracts in the presence or absence of various additives. Results: Sera containing specific IgE against both extract formulations were obtained. Native as well as modified extracts induced dose-dependent β-hexosaminidase release from RBL cells. Both extracts were used to evaluate the characteristics of the assay, which showed high precision. Storage conditions were chosen to enhance extract degradation, which could be read directly from the altered stimulatory capacity of the extracts. Additives turned out to have diverse effects on the assay, whereas phenol had no measurable effect, alum had an inhibitory effect and glycerol elevated basophil activation. Conclusions: For the first time, a reliable, precise in vitro assay is available that is able to directly measure the properties of modified allergen extracts after their production process. The test is well evaluated and its advantages and limitations are discussed in this report.