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

Abstract

Source: Wenger, M. et al. Humanized Mediator Release Assay as a Read-Out for Allergen Potency. J. Vis. Exp. (2021)

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 bot....

Materials

List of materials used in this article

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