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

Abstract

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.

Chemical Modification of Birch Allergen Extract Leads to a Reduction in Allergenicity as well as Immunogenicity

“…These reports all conclude that allergoids show reduced allergenicity in IgE binding and IgE inhibition assays, different basophil activation tests, or skin tests [10,13,14,15]. This was confirmed in the present study (fig.…”

“…Studies of allergoids have demonstrated reduced allergenicity by various in vitro methods as well as skin prick testing [10,13,14,15], and treatment with allergoids has shown efficacy in reducing symptom and medication scores in some studies [7, 8]. However, the clinical effect of allergoids may be compromised because they seem to lack the ability to modify allergen-specific T cell responses [12, 17].…”

“…In vitro studies have shown that allergoids exhibit reduced IgE binding and lead to abrogated activation of human basophils and basophil cell lines [10, 13, 14], and this has been confirmed in vivo by human skin prick tests [10, 15]. On the other hand, in vitro T cell responses to allergoids have been reported to be markedly reduced compared to responses to intact allergens [16,17,18], even though other reports seem to suggest that this mainly depended on the type of APC used [18, 19].…”

Close-up of the Immunogenic α1,3-Galactose Epitope as Defined by a Monoclonal Chimeric Immunoglobulin E and Human Serum Using Saturation Transfer Difference (STD) NMR

“…In Vitro Mediator Release Assay with Humanized Rat Basophilic Leukemia Cells (RBL-SX38)-In vitro degranulation was analyzed as described previously (27). Soluble ␣-Gal proteins were biotinylated using TFPA-PEG3-biotin (tetrafluorophenylazide-(triethyleneglycol)-biotin (Pierce) according to the recommendations of the manufacturer and incubated with streptavidin-coated Roti-MagBeads (Roth).…”

Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors

“…After washing twice with Tyrode buffer (pH 7.7), the cells were incubated with 120 l/well of Tyrode buffer containing 10 g/ml DNP-BSA (Molecular Probes, Eugene, OR) for 30 min. The released ␤-hexosaminidase was measured (22).…”