Allergen Micro-Bead Array for IgE Detection: A Feasibility Study Using Allergenic Molecules Tested on a Flexible Multiplex Flow Cytometric Immunoassay

Abstract

BackgroundAllergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing.Objective and MethodsTo provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Molecule-based micro-bead Array system (ABA) was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems.ResultsSuccessful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE to nAct d 1, nAct d 11, rAln g 1, nAmb a 1, nArt v 3, rBet v 1, rCor a 1, nCup a 1, nDer p 1, nDer s 1, rHev b 5, nOle e 1, rPar j 2, nPen m 1, rPhl p 1, and nPru p 3. Results obtained by ABA IgE testing were highly correlated to ISAC testing (r = 0.87, p<0.0001). No unspecific binding was recorded because of high total IgE values.ConclusionThe ABA assay represents a useful and flexible method for multiplex IgE detection using allergenic molecules. As also shown by our initial experiments with monoclonals and polyclonals, ABA is suitable for detecting other human and non-human immunoglobulins.

Allergen microbead arrays: the future of allergy diagnostics?

“…Furthermore, flow cytometric assessment may be more sensitive when compared with other ELISA-based systems for the recognition of extremely low levels of IgE reactivity. The cytometric ABA testing, in fact, seems not to be affected by problems of background noise, commonly observed in other immunoassays [17]. We found a very good linear relationship, in terms of IgE molecule recognition, between the flexible ABA system and static microarrays like the ImmunoCAP-ISAC.…”

“…Taking advantage of previously published reports [10,11] and subsequent applications [13], the feasibility of this approach for specific IgE detection using allergenic components coupled upon the naked fluorescent microbeads undergoing FACS analysis has also been recently demonstrated [17]. This 'flexible' new multiplex system customized for specific IgE testing was named allergen bead array (ABA).…”

An Immunodiagnostic Assay for Quantitation of Specific IgE to the Major Pollen Allergen Component, Pas n 1, of the Subtropical Bahia Grass

“…Whilst we have utilized the ImmunoCAP assay format in this study, the indirect coupling method mediated by the biotin-streptavidin interaction means that the measurement of Pas n 1 IgE could be transferrable to a number of alternative assay formats, including the IMMULITE system [23], solid-phase multi-allergen arrays (e.g. ISAC [24]), multi-allergen bead arrays [25], or point-of-care devices [26,27]. …”

Recombinant Allergens in Structural Biology, Diagnosis, and Immunotherapy

“…The technological advancements for in vitro allergy diagnosis do not stop there [44]. Small-scale suspension arrays for a small number of purified recombinant and natural allergens have been developed on the Luminex xMAP® platform [45] and on the Becton Dickinson cytometric bead array system [46]. Indoor Biotechnology produces an IgE quantitative binding array for the simultaneous detection and quantitative determination of total and allergen-specific human IgE against 11 allergens.…”