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

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.