The IgE-facilitated allergen binding (FAB) assay: validation of a novel flow-cytometric based method for the detection of inhibitory antibody responses.
The IgE-facilitated allergen binding (IgE-FAB) assay represents an in vitro model of facilitated allergen presentation. Allergen-IgE complexes are incubated with an EBV-transformed B cell line and complexes bound to CD23 on the surface of cells are detected by flow cytometry. The addition of serum from patients who have received allergen-specific immunotherapy has been shown previously to inhibit allergen-IgE complex binding to CD23 on B cells. In this study, we describe the characterisation and analytical validation of the grass pollen-specific IgE-FAB assay according to guidelines from the International Conference on Harmonisation. 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. Using the results from a randomised double-blind placebo-controlled trial of grass pollen immunotherapy (n=33), we have defined the clinical sensitivity and specificity of the IgE-FAB assay using ROC curve analysis. In conclusion, the IgE-FAB assay is reproducible, robust, sensitive and a specific method suitable as a tool for monitoring inhibitory antibody function from patients receiving allergen immunotherapy.
Shamji
Mohamed H
MH
Upper Respiratory Medicine, Allergy and Clinical Immunology, National Heart and Lung Institute, Faculty of Medicine, Imperial College, Dovehouse Street, London, SW3 6LY, UK.
Wilcock
Louisa K
LK
Wachholz
Petra A
PA
Dearman
Rebecca J
RJ
Kimber
Ian
I
Wurtzen
Peter A
PA
Larché
Mark
M
Durham
Stephen R
SR
Francis
James N
JN
D000485:Allergens / Q000276:immunology*; D000917:Antibody Formation / Q000276:immunology; D017951:Antigen Presentation / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D003888:Desensitization, Immunologic; D004311:Double-Blind Method; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D010919:Placebos; D006109:Poaceae / Q000276:immunology; D011058:Pollen / Q000276:immunology; D011485:Protein Binding; D012372:ROC Curve; D016032:Randomized Controlled Trials as Topic; D017455:Receptors, IgE / Q000276:immunology; D015203:Reproducibility of Results; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000517:prevention & control; D012680:Sensitivity and Specificity
D000485:Allergens; D010919:Placebos; D017455:Receptors, IgE; D007073:Immunoglobulin E