Humanized Mediator Enzyme Release Assay: An Immunochemical Method to Screen Allergic Potential of Test Antigen by Monitoring Basophil Degranulation Response In Vitro
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When exposed to an allergen – non-pathogenic antigen – antigen-presenting cells process and present the allergen to type-2 helper T-lymphocytes. These T-lymphocytes trigger B-lymphocytes to differentiate into plasma cells and produce immunoglobulin E, IgE, antibodies that bind to high-affinity IgE-specific Fc-receptors on the surface of circulating basophils – granule-containing secretory cells.
Upon subsequent exposure to the allergen, the IgE-Fc complexes recognize and crosslink with the allergen, activating basophils to release granules comprising mediators like cytokines and lysosomal enzymes including β-hexosaminidase, evoking a potent inflammatory response.
To screen a test antigen's allergic potential in vitro, begin with a suspension of transduced rat cancerous basophils expressing human IgE-specific Fc-receptors. Treat with IgE-containing human serum from an allergic donor. The IgE antibodies bind to and sensitize the basophil Fc-receptors.
Next, add buffer and an adequate volume of test antigen to the sensitized basophils. The antigen, depending on its affinity for IgE bound to Fc-receptors, crosslinks with the antibodies and initiates basophil degranulation, releasing mediators including β-hexosaminidase into the solution.
Collect the solution into a fresh multi-well plate containing a fluorogenic substrate of β-hexosaminidase and incubate. β-hexosaminidase hydrolyzes the substrate, generating a fluorescent product. Post-incubation, add high pH buffer to inhibit β-hexosaminidase activity and stop the reaction.
Using a microplate reader, measure the formed product's fluorescence, indicative of released β-hexosaminidase activity, and in turn, the antigen's allergic potential to induce basophil degranulation.
