3. Technological Enhancements Leading to Microarrays
Over the years, significant technological developments have allowed the use of (a) non-isotopic poly- and monoclonal anti-human IgE Fc conjugates to detect bound IgE antibody; (b) the World Health Organization IgE reference preparation [6] to allow calibration of the allergen-specific assay which has enhanced inter-laboratory standardization; (c) new solid phase matrix materials with higher binding capacities for allergenic molecules; (d) engineering advances in robotics and electronics that resulted in current, computer-driven singleplex autoanalyzers; and most recently (e) the production of purified recombinant and native allergenic components [7,8]. These technological developments have resulted in our current state of the art singleplex standalone assays, autoanalyzers that are used throughout the world to measure total and allergen-specific IgE antibodies [7]. A positive IgE antibody response signifies that an individual has become sensitized, which is necessary, but is not sufficient to make the definitive diagnosis of allergic disease [9]. The IgE antibody’s concentration, strength of binding (affinity/avidity), specificity and the percentage of specific IgE to total IgE each plays an interrelated role in translating a humoral IgE antibody response into a clinical allergic symptom [7].
Microarray technology has been only recently applied to the field of diagnostic allergy serology. The first report of chip-based microarray technology being applied to the diagnosis of human allergic disease occurred in 2002. Hiller et al. immobilized 94 purified recombinant and natural allergen molecules at 0.3 mg/mL in 150 mM sodium phosphate buffer in triplicate onto a pre-activated amine reactive coated glass slide [10]. The slide was then used as a solid phase antigen to bind allergen-specific antibodies (principally IgG and IgE) from 200 microliters of human serum diluted 1:5 in an initial incubation. Following a buffer wash to remove unbound proteins, bound IgE was detected with a fluorophore-labeled anti-human IgE in a second incubation step. After a second buffer wash, bound fluorophore was detected in a fluorescent microarray reader and an individual’s IgE antibody specificity profile was obtained. This chemistry of the assay called the immunosorbent allergen chip or ISAC was patterned after the first singleplex radioallergosorbent test (RAST) for allergen-specific IgE antibody reported by Wide et al. [5]. The ISAC is currently the predominant IgE antibody microarray that is available to clinical immunology laboratories.
There are reports of the use of microarray technology in the detection of IgE antibody for a number of special patient care related services. Reported applications include the assessment of poly-sensitization (e.g. detection of IgE antibody to different allergen groups), clarification of the allergen triggers for patients with atopic dermatitis and anaphylaxis and the selection of appropriate allergens for use in immunotherapy [11,12,13,14,15]. However, it is rare to use microarray-based IgE antibody assay (e.g., ISAC) in routine patient testing for several reasons. First, microarrays cannot technically cover all clinically relevant allergen specificities so singleplex assays are additionally required when the patient’s history suggests an offending allergen not in the microarray. Second, the use of a fixed panel of allergens forces testing of IgE antibody to allergen specificities that may not be relevant to the patient based on their clinical history. Because IgE antibody measurements indicate sensitization and not allergic disease [3], this can create a problem that requires interpretation of low level IgE antibody to some allergen specificities that are clinically tolerated by the patient. Third, the panel-based microarray testing is not generally reimbursed by medical insurance and thus is often too expensive for the patient out of pocket for over 100 measurements. Finally, there are technical limitations associated with the microarray-based assays involving analytical sensitivity and specificity, IgG anti-allergen interference and quality control that are noted below which make the singleplex IgE antibody assays those of choice for routine diagnostic allergy testing.