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

Overview

In this video, we describe an in vitro method to evaluate the allergic potential of an antigen to induce basophil degranulation. β-hexosaminidase is used as an index of chemical mediator release, and the enzyme activity of β-hexosaminidase is measured using a fluorogenic substrate.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board

1. Passive sensitization of huRBL cells

1. Centrifuge the pre-incubated Ag8/serum suspension for 5 min at 250 x g.
2. Transfer 50 µL of the centrifuged Ag8/serum suspension to each well containing huRBL cells without disturbing the Ag8 cell pellet.
   1. Include the no antigen control, which are sensitized, but unstimulated cells (do not add antigen), serving as an indication for the bottom signal plateau/background. Background and maximum lysis control wells do not need to be sensitized with serum. Add 50 µL of huRBL medium to control wells instead.
3. Cover the plate with the lid and incubate overnight at 37 °C and 5%-7% CO₂.

2. Antigen-stimulated degranulation and mediator release

1. Prepare the antigen dilution in 1x Tyrode's buffer (9.5 g/L Tyrode's salts, 0.1% bovine serum albumin (BSA), 0.5 g/L Sodium hydrogen carbonate (NaHCO₃) in dH₂O) in advance. A final amount of 100 µL per well is needed.
   NOTE: Not every allergen, either purified from natural sources or recombinantly produced, might be stable in 1x Tyrode's buffer. Therefore, perform stability tests in 1x Tyrode's buffer prior to the assay procedure. Alternatively, dilute 1x Tyrode's buffer in deuterium oxide (D₂O) to increase the signal-to-noise ratio of the assay.
2. Make 8 dilutions of the antigen of interest of a 1:10 dilution series in reaction tubes starting with either 10 or 1 µg/mL of protein.
   NOTE: Always test the dilution series beforehand. Alternatively, adapt the 1:10 dilution series (e.g., 1:5, 1:20, or 1:30) to cover the full release curve. In addition, the starting concentration can vary depending on the experimental setup.
3. To wash huRBL cells plated on the 96-well plate, remove the sera-containing cell medium first by carefully aspirating, inverting, and tapping the plate on absorbent paper.
4. Wash cells with 200 µL of 1x Tyrodes' buffer per well. Treat all wells similarly.
   NOTE: Add the washing solution slowly to the cells to not disturb them.
5. Leave it for approximately 30 s and repeat the washing step three times in total.
6. After adding the washing solution for the final time, leave the solution in the wells until ready to continue with adding the antigen dilution.
   NOTE: Avoid exposing cells to air for too long.
7. Transfer 100 µL of antigen solution to each well containing the pre-sensitized huRBL cells.
   NOTE: If analyzing several different parameters, transfer the individual samples of the dilution series into an additional non-binding 96-well plate (use the same layout as on the huRBL plate) and transfer them afterward with a multichannel pipette directly on the huRBL cell plate. This way, exposing the cells to air for too long can be avoided, which might result in poor assay performance (lower/no signal).
8. Cover control wells (maximum lysis and non-sensitized background cells) with 100 µL of 1x Tyrode's buffer. Do not stimulate these control wells with the antigen.
   1. Additionally, add 100 µL of 1x Tyrode's buffer to the sensitized no-antigen wells of the dilution series, which is needed to take the antigen-independent spontaneous release of sensitized cells into account during data analysis.
9. Incubate huRBL cells for 1 h at 37 °C and 5%-7% CO₂.

3. Fluorescence measurement of β-hexosaminidase activity

1. Treat the wells of the maximum lysis control with 10 µL of 10% Triton X-100 per well and mix properly in order to lyse the cells completely and obtain the 100% release of β-hexosaminidase.
2. Add 50 µL of substrate solution into a new non-binding 96-well plate. Substrate solution for one 96-well plate: 5 mL of 0.1 M citric assay buffer, pH 4.5; and 80 µL of 10 mM 4-methylumbelliferyl N-acetyl-β-D-glucosaminide.
3. Transfer 50 µL of cell supernatant of all wells to the new plate containing the substrate solution.
   NOTE: Pipette the supernatant carefully off the huRBL plate to not disrupt the huRBL cells.
4. Incubate the plate with substrate solution and cell supernatant for 1 h at 37 °C to allow conversion of the fluorogenic substrate.
   NOTE: Keep the huRBL plate for cell viability assay.
5. Add 100 µL of stopping solution (15 g/L glycine, 11.7 g/L NaCl dissolved in dH₂O, pH 10.7) per well.
6. Measure the fluorescence at 360 nm excitation and 465 nm emission using a plate reader.

Materials

Name	Company	Catalog Number	Comments
4-Methylumbelliferyl N-acetyl-β-D-glucosaminide	Sigma	M2133
96-well plate for huRBL cells (Nunc MicroWell 96-Well, Nunclon Deltatreated, flat-bottom microplate)	ThermoFisher Scientific	167008
96-well plate for substrate solution and cell supernatant (Greiner BioOne non-treated 96-well microplates)	Fisher Scientific	655101
Bovine serum albumin (BSA)	Sigma	10735078001
Citric acid	Applichem	131018
Glycine	Applichem	A3707
Sodium chloride (NaCl)	Applichem	A2942
Sodium hydrogen carbonate (NaHCO3)	Applichem	131638
Triton X-100	Sigma	X100
Tyrode’s salt	Sigma	T2145