Humanized Mediator Enzyme Release Assay: An Immunochemical Method to Screen Allergic Potential of Test Antigen by Monitoring Basophil Degranulation Response In Vitro

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.

Centrifuge the pre-incubated Ag8/serum suspension, and transfer 50 microliters of the centrifuged Ag8/serum suspension to each well containing humanized RBL cells without disturbing the Ag8 cell pellet.

Use sensitized, unstimulated, cells without antigen, as no-antigen control for indication of the bottom signal plateau or background, and do not sensitize the background and maximum lysis control wells. Cover the plate with the lid, and incubate overnight at 37 degrees Celsius and 5% to 7% carbon dioxide.

Aspirate the sera-containing cell medium. Invert and tap the plate on the absorbent paper to empty the plate for washing humanized RBL cells. Wash the cells three times by adding 200 microliters of Tyrode's buffer per well, and incubate for approximately 30 seconds per wash, for the first two washes. After adding Tyrode's buffer for the third time, aspirate the buffer and leave the solution in the wells, until ready to add the antigen dilution.

Transfer 100 microliters of antigen solution to each well containing the pre-sensitized, humanized RBL cells, but do not stimulate the maximum lysis and non-sensitized background cells with antigen.

Add 100 microliters of Tyrode's buffer in the maximum lysis control wells, non-sensitized background control wells, and sensitized no-antigen wells. Incubate the cells for one hour at 37 degrees Celsius and 5% to 7% carbon dioxide.

Treat the maximum lysis control wells with 10 microliters of 10% Triton X-100 per well, and mix properly to lyse the cells completely for 100% release of beta-hexosaminidase. Add 50 microliters of substrate solution into a new non-binding 96-well plate.

Transfer 50 microliters of supernatant from the wells of humanized RBL cells-containing plate into the new plate containing the substrate solution, and incubate the plate for one hour at 37 degrees Celsius to allow conversion of the fluorogenic substrate.

Add 100 microliters of stopping solution per well, and measure the fluorescence as described in the text manuscript.