Humanized Mediator Release Assay as a Read-Out for Allergen Potency

The assay represents an important research tool to simulate the degranulations of basophils induced by the cross-linking of patients'IgE and the allergens. Thus the assay can be used to mimic type 1, allergic reactions. The assay is robust, reproducible, and tailorable.

Moreover, it is highly sensitive as it can be performed with small amounts of recombinant or purified allergen or even with complex allergen extracts. The method is used to diagnose allergies as it analyzes the reactivity of patients'IgE to allergens. Also this method is suitable to analyze cross-reactivity and to monitor AIT treatment efficacy.

Begin by harvesting Ag8 cells from the cell culture flask and transfer the cells into a centrifugation tube. Pellet down the cells by centrifugation for five minutes, at 250 times g at room temperature. Aspirate the supernatant, and resuspend the cell pellet to a final concentration of approximately one times 10 to the sixth cells, per milliliter in humanized, RBL cells medium.

Dilute human sera one to 10 in Ag8 cell suspension for the final serum dilution of one to 20 in the assay, and incubate for one hour at 37 degrees Celsius and five to 7%carbon dioxide. When the humanized RBL cells reach 50 to 90%confluency, aspirate the medium from a T-75 cell culture flask carefully without touching the adhered humanized RBL cells. Wash the cells twice by adding 10 milliliters of DPBS to the opposite side of the flask and not directly onto the cells.

Aspirate DPBS, add five milliliters of pre-warmed one times trypsin-EDTA for cell detachment and incubate for five minutes at 37 degrees Celsius. Gently tap the flask to detach the cells. Transfer the cell suspension into a 15 millimeter centrifugation tube and fill the tube with humanized RBL cell medium or DPBS to dilute the trypsin-EDTA.

Centrifuge the cells at 250 times g for five minutes at room temperature. Aspirate the supernatant, and resuspend the pellet in five milliliters of humanized RBL cell medium for cell counting. After counting the cells, dilute the cells in humanized RBL cell medium to obtain a final concentration of two times 10 to the sixth cells per milliliter.

Add 50 microliters of humanized RBL cell suspension, per well, equivalent to one times 10 to the fifth cells per well in a sterile 96-well plate. 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 five 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 five 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. The bell shaped curve obtained in the beta-hexosaminidase activity assay, indicating the monovalent occupation of antigen epitopes of immunoglobulin E due to the excess of allergen, which inhibits the allergen immunoglobulin E cross-linking at high antigen concentrations.

The antigen concentration necessary for the half maximum mediator release was calculated using the linear regression analysis. The cell viability assay was performed to exclude the cytotoxic effects derived from either the sensitizing serum or the antigen used for stimulation. Five different human serums were used for the beta-hexosaminidase activity assay, and four out of five sera derived from the birch pollen allergy patients responded to Bet v 1 stimulation, demonstrating that Bet v 1 is a potent allergen responsible for immunoglobulin E mediated allergic symptoms.

The cross-reactivity of immunoglobulin E to homologous allergens was assessed. The response to Bet v 1 was found in both patients, but patient two also responded to Cor a 1. The Bet v 1 homologous food allergen, indicating higher Cor a 1 cross-reactive immunoglobulin E levels in patient two.

The hypoallergenic nature of mutant variants of the allergens was assessed and compared to their wild type counterpart. The release curve of the Bet v 1 fold variant shifted towards a higher antigen concentration compared to the wild type allergen, resulting in a significantly higher concentration of antigen to provoke the half maximal release, which makes the mutant less allergic and therefore a candidate for allergen specific immunotherapy. Do not forget to leave the washing solutions on the cells until applying the antigen dilution to avoid the cells from drying out, otherwise, this would result in poor performance of the assay.

The assay can be used for many different applications, including the standardization of allergenic products, based on their biological activity, such as skin prick test solutions or extracts used for allergen specific immunotherapy.