Shamji, M. H. et al. The IgE-facilitated allergen binding (FAB) assay: validation of a novel flow-cytometric based assay for the detection of inhibitory antibody responses. J. Immunol. Methods 317, 71-79

ALK-Abelló, København, Capital Region, Denmark
Journal of Immunological Methods (Impact Factor: 1.82). 01/2007; 317(1-2):71-9. DOI: 10.1016/j.jim.2006.09.004
Source: PubMed


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.

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Available from: Mohamed H Shamji, Dec 31, 2013
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    • "In addition to these clinically well-established serological tests, several methods are under investigation that may help assess and predict the success of SIT [93] or distinguish clinically beneficial from adverse immune responses to SIT [94]. Assays like the basophil activation test, for example, could serve as surrogates for in vivo tests [95], while the IgE-FAB assay allows the determination the blocking effect of SIT on IgE-facilitated allergen presentation [96]. "
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    ABSTRACT: Opinion statement Allergic diseases are among the most common health issues worldwide. Specific immunotherapy has remained the only disease-modifying treatment, but it is not effective in all patients and may cause side effects. Over the last 25 years, allergen molecules from most prevalent allergen sources have been isolated and produced as recombinant proteins. Not only are these molecules useful in improved allergy diagnosis, but they also have the potential to revolutionize the treatment of allergic disease by means of immunotherapy. Panels of unmodified recombinant allergens have already been shown to effectively replace natural allergen extracts in therapy. Through genetic engineering, several molecules have been designed with modified immunological properties. Hypoallergens have been produced that have reduced IgE binding capacity but retained T cell reactivity and T cell peptides which stimulate allergen-specific T cells, and these have already been investigated in clinical trials. New vaccines have been recently created with both reduced IgE and T cell reactivity but retained ability to induce protective allergen-specific IgG antibodies. The latter approach works by fusing per se non-IgE reactive peptides derived from IgE binding sites of the allergens to a virus protein, which acts as a carrier and provides the T-cell help necessary for immune stimulation and protective antibody production. In this review, we will highlight the different novel approaches for immunotherapy and will report on prior and ongoing clinical studies.
    Full-text · Article · Mar 2014
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    • "Secondly, we shall investigate if low dose intradermal desensitisation is associated with a systemic immunological effect. We will therefore measure serum allergen-specific IgE and IgG responses and test the biological inhibitory activity of IgG antibodies with an in-house validated assay of IgE-allergen complex binding to B cells [26]. We shall also examine the effect of low dose intradermal allergen treatment on peripheral blood basophil activation in response to grass pollen stimulation ex vivo. "
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    ABSTRACT: Subcutaneous immunotherapy with high dose grass pollen (typically microgram quantities) was first described over 100 years ago. This treatment suppresses allergen-induced cutaneous late responses, with lesser effects on early responses. We previously reported that repeated 2-weekly intradermal injections of grass pollen - containing approximately 7 ng of major allergen Phl p 5 -- led to a progressive suppression of the allergen-induced cutaneous response, and that by the sixth injection, this was inhibited by over 90%. The purpose of this trial is to investigate the clinical efficacy of intradermal desensitisation with low doses (i.e. nanogram quantities) of grass pollen allergen for seasonal allergic rhinitis.Methods/designThe Pollen Low dose Intradermal therapy Evaluation (PollenLITE) is a single centre double-blind randomised parallel group controlled trial of the efficacy and safety of intradermal grass pollen injections plus standard treatment, versus histamine injections plus standard treatment, in adults with moderate-severe grass pollen-induced allergic rhinitis ('summer hay fever'). A minimum of ninety adults with a history of moderate-severe persistent allergic rhinitis during the UK grass pollen season will be randomised into two equal groups to receive 7 or 8 intradermal injections of grass pollen extract (containing approximately 7 ng of major allergen Phl p 5) or histamine, before the grass pollen season. In the summer, participants will score their symptoms, medication requirements, visual analogue scores, and complete EuroQOL (EQ-5D-5 L) and mini Rhinoconjunctivitis Quality of Life Questionnaires. Global assessments will also be recorded at the end of the pollen season. Blood samples will be collected from all participants for mechanistic immune assays. Skin punch biopsies will also be collected in 40 participants selected at random from intradermal injection sites after the grass pollen season for mechanistic assays. Finally, to investigate if the desensitising effect of intradermal immunotherapy on cutaneous responses is long-lasting, all participants will be randomised to receive a follow up intradermal injection after 3, 6 or 12 months with measurement of early and late response sizes. Randomisation began in February 2013 and the final participant will complete the trial protocol in August 2014.Trial registrationISRCTN 78413121EudraCT number 2012-002193-31.
    Full-text · Article · Aug 2013
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    • "In some cases, histamine release assays can be used to detect the presence of an antigen-neutralizing blocking antibody in a subject's serum, although this approach has been largely been replaced by radioimmunoassays (RIAs), radioenzymatic assays (REAs), and most recently by the facilitated allergen binding (FAB) assay, which also detects blocking antibody activity (Shamji et al., 2006). These types of assays are useful in the setting of allergen immunotherapy, which is often associated with a rise in antigen-specific IgG that can compete with IgE for binding to allergen. "
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    ABSTRACT: In vitro basophil responses have long been used in mechanistic studies to help assess the human allergic diathesis, particularly during therapeutic intervention. Recent evidence points to the use of dendritic cells (DCs) in also being valuable in evaluating therapies aimed at lessening disease through immunomodulation. This review article therefore takes a look at some of the recent advances in old and new assays employing both basophils and DCs, with the added perception that the responses mediated by two cell types are insightful towards understanding immune cell mechanisms underlying allergic disease.
    Preview · Article · Jan 2012 · Journal of immunological methods
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