Allergen microbead arrays: the future of allergy diagnostics?

Keywords::

• ABA
• allergy
• IgE
• microarray

The incidence of IgE-mediated allergic disorders has increased over the last decades in the western world [1,2]. Consequently, the availability of reliable tools for the diagnosis of allergic diseases is currently felt to be a very hot topic. At the moment, investigative work is widely performed by means of diagnostic reagents, directly derived from the biological sources responsible for allergic sensitization. These allergenic extracts are utilized both for cutaneous and in vitro IgE testing [3,4]. Unfortunately, a series of pitfalls actually impair the consistency of diagnostic tools based on extract: these reagents, in fact, provide no information on the specific culprit protein(s) responsible for IgE sensitization, it is impossible to distinguish panallergens’ IgE reactivity from a genuine antigen recognition, and contaminants or genuine but irrelevant components could prejudice a precise evaluation, both diluting or giving the possibility for a false IgE reactivity [5].

An outstanding increase in knowledge regarding the protein components that are recognized by IgE within the different allergenic biological sources has been achieved in the last few years, and an increasing number of new allergens have been identified, characterized and isolated or produced as recombinant molecules, to obtain increasing reliable diagnostic tools. It is currently possible to use natural or recombinant components for diagnostic purposes, both using a single reagent for each specimen (singleplex) or by means of a predefined panel of molecules to be tested in the same test, at the same time (multiplex) [6]. The latter approach allows a more precise depiction of a patient’s IgE sensitization profile, as demonstrated in a recently published survey [7]. The intrinsic novelty of multiplexing testing in allergy, in fact, is strictly dependent on the possibility to obtain hundreds of results at once from a single serum sample [8].

The major criticism raised to the afore-mentioned multiplexing approach is represented by the lack of flexibility in the currently available list of allergenic molecules spotted on microarrays [9]. This system is rigid, and the clinician is forced to evaluate the whole set of components selected by the seller, even in the case of monosensitization (~15% of allergic individuals) [7]. In the case of fixed microarrays, consequently, a patient-tailored approach, similar to what traditionally happens when the allergist uses the skin prick test, is not achievable. Furthermore, those systems do not allow the study of novel allergenic molecules. As a result, there is a clear need for more flexible multiplex immunoassay platforms that could allow both the choice of controls (internal assay controls) or the introduction of customized allergenic molecules.

The multiplex detection of a number of soluble factors, like cytokines, synthesized during spontaneous or experimentally induced immune responses, is currently achievable by some flow cytometric bead arrays [10–12]. Naked fluorescent microbeads can also be used in a customized approach [13]. Cytometric bead arrays such as Flex Set, unlike the other microbead-based systems [14–16], are designed for flow cytometry, an instrument widely used in almost all research or routine laboratories.

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). Up to 30 different microbeads can be measured at the same time without any further cost, just using the FACS facilities that are commonly available in almost all laboratories. Other multiplexed systems for specific IgE measurement are actually based on a comparable microbead system, but require a dedicated laser scanner (Luminex Corp. TX, USA) [16].

FACS analysis does not require extensive experience and expertise in the interpretation of positive stained beads, possibly reducing the intra- and inter-laboratory variability and thus improving the reproducibility. 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.

Therefore, the ABA system not only allows the precise recognition of culprit molecule(s) responsible for the IgE sensitization, as with other more rigid systems, but it also allows a patient-tailored approach. The ABA assay is, for example, useful for the IgE cluster reactivity evaluation of homologous molecules, as already published by Scala et al., where the mutually exclusive recognition of PR-10, profilin and tropomyosin molecules has been demonstrated [7]. The flexibility of ABA testing could represent a further advantage allowing the selection of the antigen mixture to analyze, after conjugation with color-coded polystyrene microbeads, when a limited panel of allergens for studying restricted aspects of IgE sensitization is required (i.e., latex reactivity, cow’s milk or hen’s egg allergy), or in the case of experimental evaluation of allergens not yet available on other microarrays.

Another great advantage is associated to the multiple fluorescence parameters evaluation that could allow the measurement in the same specimen of diverse antibody isotype allergen recognition. Alongside the flexibility in the number of allergenic components to be tested, the small amount of serum required (50 μl) is another important attribute, particularly when only limited amounts of biological fluids are available, such as in pediatric patient assessment.

In conclusion, allergen microbead arrays could represent the future in allergy diagnosis as it conjugates both the flexibility and the molecule-resolved evaluation necessary for a modern approach to allergy testing.

Financial & competing interests disclosure

E Scala, D Pomponi and M Giani are supported by grants from Ministero della Salute. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.