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 the world and favorable clinical outcomes were achieved. A series of immunological changes, including antibody responses and cellular reactions, were observed during immunotherapy with different kinds of allergen. The specific IgG4antibody in serum is regarded as dominant component of inhibitory "blocking antibody" induced by immunotherapy. Despite, clinical improvement brought by immunotherapy could not be explained convincingly by the detection of expression level of any immunology product. It is desiderate to find a key biomarker to generally reflecte the inhibition function of serious antibody induced by immunotherapy.IgE-facilitated allergen presentation (FAP) is involved in many steps and aspects of pathology of allergy, and can be inhibited by immunotherapy. The IgE-facilitated allergen binding (FAB) assay, which can be detected by a flow cytometry-based method, represents an in vitro model of facilitated allergen presentation. The allergen-IgE complex, formed by the incubation of allergen and high-concentration specific IgE, can bind to the low-affinity IgE receptor expressed on EBV-B cells and be detected by flow cytometry. The formation of complexes and the following binding to B cells can be inhibited by the addition of immunotherapy serum. The inhibition, reflected as the decrease of percentage of B cells binding with allergen-IgE complexes, is correlated with the clinical improvement during allergen 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 the International Conference on Harmonisation (ICH). We established the intra-and inter-assay variability of IgE-FAB and have defined the detection limits of this assay. We have also demonstrated assay linearity and robustness.In conclusion, the IgE-FAB assay is reproducible, robust, sensitive and a specific method suitable as a tool for monitoring inhibitory antibody function from allergic rhinitis patients receiving allergen immunotherapy.