Humanized Mediator Enzyme Release Assay: An Immunochemical Method to Screen Allergic Potential of Test Antigen by Monitoring Basophil Degranulation Response In Vitro
Published: April 30, 2023
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 Centrifuge the pre-incubated Ag8/serum suspension for 5 min at 250 x g. Transfer 50 µL of the centrifuged Ag8/serum suspension to each well containing huRBL cells without disturbing the Ag8 cell pelle…
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 |
Tags
Citazione di questo articolo
Humanized Mediator Enzyme Release Assay: An Immunochemical Method to Screen Allergic Potential of Test Antigen by Monitoring Basophil Degranulation Response In Vitro. J. Vis. Exp. (Pending Publication), e21217, doi: (2023).