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

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells basophils, upon stimulation with serial dilutions putative allergens. Therefore, these represent an essential tool that mimics the vivo process, which occurs allergen exposure sensitized patients or skin prick tests. Additionally, are usually employed to investigate allergenic potential proteins reactivity patients' sera's reactivity. Herein, we describe a simple 2-day protocol using immortalized rat basophil leukemia cell line transfected humanized human high-affinity IgE plasma-membrane receptor (FcεRI). This variant mediator assay is robust, sensitive, reproducible cell-based system without need immobilize antigen solid matrices. The consists following steps: (1) complement inactivation sera, (2) harvesting, seeding, passive sensitization (3) cause release, (4) measuring β-hexosaminidase activity surrogate for released inflammatory mediators, histamine. represents useful assess capacity allergen-IgE cross-linking trigger can be implemented standardize extracts, compare minor major allergens extracts (pollen, cat dander, etc.), potency homologs, isoforms, fold-variants (e.g., hypoallergenicity), well effects ligands on activity. A more recent application includes use monitor treatment efficacy course immunotherapy.