Establishment And Validation Of A Method For The Detection Of Inhibition By House Dust Mite Immunotherapy On The Ige-facilitated Allergen Binding

Allergen specific immunotherapy was used for the treatment of allergic rhinitis all over theworld and favorable clinical outcomes were achieved. A series of immunological changes,including antibody responses and cellular reactions, were observed during immunotherapywith different kinds of allergen. The specific IgG4antibody in serum is regarded asdominant component of inhibitory―blocking antibody‖induced by immunotherapy.Despite, clinical improvement brought by immunotherapy could not be explainedconvincingly by the detection of expression level of any immunology product. It isdesiderate to find a key biomarker to generally reflecte the inhibition function of seriousantibody induced by immunotherapy.IgE-facilitated allergen presentation (FAP) is involved in many steps and aspects ofpathology of allergy, and can be inhibited by immunotherapy. The IgE-facilitated allergenbinding (FAB) assay, which can be detected by a flow cytometry-based method, representsan in vitro model of facilitated allergen presentation. The allergen-IgE complex, formed bythe incubation of allergen and high-concentration specific IgE, can bind to the low-affinityIgE receptor expressed on EBV-B cells and be detected by flow cytometry. The formationof complexes and the following binding to B cells can be inhibited by the addition ofimmunotherapy serum. The inhibition, reflected as the decrease of percentage of B cellsbinding with allergen-IgE complexes, is correlated with the clinical improvement duringallergen immunotherapy.In this study, we describe the characterisation and analytical validation of the house dust mite-specific IgE-FAB assay according to methodology guidelines from theInternational Conference on Harmonisation (ICH). We established the intra-and inter-assayvariability of IgE-FAB and have defined the detection limits of this assay. We have alsodemonstrated assay linearity and robustness.In conclusion, the IgE-FAB assay is reproducible, robust, sensitive and a specificmethod suitable as a tool for monitoring inhibitory antibody function from allergic rhinitispatients receiving allergen immunotherapy.