Biologic allergen assay for in vivo test allergens with an in vitro model of the murine type I reaction

Background: The determination of the biologic activity of allergenic extracts in human beings is limited for ethical and practical reasons. The establishment of a simplified in vitro model, which mimics a main event of the type I reaction, should provide true benefit for manufacturers, researchers, and clinicians. Objective: This study was designed to develop and evaluate a mediator release assay based on rat basophil leukemia cells for the purpose of investigating allergenic extracts. Methods: Rat basophil leukemia cells were passively sensitized with murine IgE raised against allergens and stimulated by serial dilutions of allergenic extracts in a dose-related manner. The allergen-specific degranulation was monitored by measuring the release of beta-hexosaminidase. Results: The investigation of standardized commercial allergen products for in vivo diagnostics (birch pollen, cat dander, and bee venom) allowed a quantitative description of differences in biologic activity in accordance with the declared activity units. The Fel d 1 content was determined in 17 cat dander extracts and correlated well with the results of a two-site binding ELISA (r = 0.93, log/log). Extremely low allergen amounts in the range of 10 to 100 pg/ml could be easily detected. Moreover, the cross-reactivity pattern of patients allergic to birch pollen could be reproduced with extracts of hazel, alder, apple, and celery. Conclusion: The assay is suitable for supplementing quality control of allergenic extracts and for the determination of biologic activity of final allergen products. As a research tool, it allows the study of IgE cross-linking properties of modified and recombinant allergens at an early stage before they are tested in human beings.