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

Background Allergies 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 Methods To 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. Results Successful 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. Conclusion The 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.