The IgE-facilitated allergen binding (FAB) assay: Validation of a novel flow-cytometric based method for the detection of inhibitory antibody responses

Abstract

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. 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 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 specific method suitable as a tool for monitoring inhibitory antibody function from patients receiving allergen immunotherapy.

Sublingual grass pollen immunotherapy is associated with increases in sublingual Foxp3‐expressing cells and elevated allergen‐specific immunoglobulin G4, immunoglobulin A and serum inhibitory activity for immunoglobulin E‐facilitated allergen binding to B cells

“…Post-immunotherapy sera from SCITtreated patients have been shown to inhibit IgE-facilitated allergen binding to B cells and subsequent presentation to allergen-specific T cell clones [18][19][20]. The proliferative response of these T cell clones corresponds to the level of allergen-IgE binding to B cells, which can be determined by flow cytometry in a validated technique termed the IgE-facilitated allergen binding (IgE-FAB) assay [21]. The assay has been used to demonstrate a bona fide functional suppressive effect of induced allergen-specific antibodies in SCIT but has not as yet been extended to a study of SLIT.…”

“…The B cell IgE-facilitated allergen binding inhibition assay has been described and validated previously [21]. Briefly, stock serum indicator containing a high concentration of P. pratense-specific IgE (4100 IU/mL) was incubated with 1mm/ml allergen for 1 h at 37 1C to form allergen-IgE complexes.…”

Trimolecular Complex Formation of IgE, FcεRI, and a Recombinant Nonanaphylactic Single-Chain Antibody Fragment with High Affinity for IgE

“…10B, anti-IgE #2), isotype controls, or FACS buffer (PBS containing 0.1% BSA and 0.1% NaN 3 ) for 1 h at room temperature. Loading of IgE on CD23-expressing EBV-B cells was performed as described (35). The cells were washed with FACS buffer and incubated with monoclonal mouse anti-IgE biotin (SouthernBiotech) for 30 min at 4°C.…”

Birch Pollen-Related Food Allergy: An Excellent Disease Model to Understand the Relevance of Immunological Cross-Reactivity for Allergy

“…Again, the significant increase of Bet v 1-specific IgG4 antibody titers was not paralleled by a significant increase of Cor a 1-reactive IgG4 antibody levels. Nevertheless, after three months of treatment the sera contained significantly enhanced Cor a 1-reactive IgG levels and the sera obtained after 1 year of treatment showed IgE-blocking capacity in facilitated antigen-binding (FAB) assays (Shamji et al, 2006). Still, SIT with birch pollen extract did not result in clinical improvement of hazelnut allergy in these patients as analyzed by means of double-blind placebo-controlled food challenges at inclusion and after 1 year of treatment (van Hoffen et al, 2011).…”