Microarray-Based Allergy Diagnosis: Quo Vadis?

Abstract

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.

Keywords: IgE; allergen; allergen chip; allergy; microarrayed allergens; molecular diagnosis; precision medicine.

Figure 1

Comparison of traditional allergy diagnosis and microarray-aided allergy diagnosis. In traditional allergy diagnosis (left part) an anamnesis of allergic symptoms is recorded which serves as the basis for targeted provocation testing, usually skin testing with a limited number of allergen extracts selected according to the anamnesis and eventually collection of a serum sample for measuring IgE specific for the suspected allergen sources. In the best case, the patient receives first treatment suggestions according to skin test results. Usually at least one, but often several additional visits are necessary to adjust the treatment to the IgE test results and/or to conduct further targeted testing to determine more precisely the patients sensitization profile and to further adjust treatment. Regarding microarray-aided allergy diagnosis (right part), it can be envisioned that the first visit can be conducted even in a virtual, telemedicine-like form because no provocation testing is required. The anamnesis and complete molecular IgE reactivity profile would be available to the specialist online who could then prescribe precise treatment taking clinical information and the complete sensitization profile into consideration.

Figure 2

Unmet needs in microarray-based allergy diagnosis.

Figure 3

Outlay of prototype allergen microarrays made by printing on glass slides and assembled silicon elements. Order of house dust mite, mite, cat, and PR10 allergens microarrayed in triplicates on glass slides (left) and precut and assembled silicon chips-derived elements.

Figure 4

Sensitivity of the reactivity of a human monoclonal Bet v 1-specific IgE antibody to Bet v 1 printed on glass versus silicon. Shown are the fluorescence light intensities (y-axis) corresponding to different concentrations (x-axis) of the monoclonal human Bet v 1-specific IgE antibody (IgEmoAb).

Figure 5

Comparison of IgE and IgG reactivity to HDM/mite, cat, and PR10 allergens which had been microarrayed on glass and silicon. A serum pool containing IgE and IgG antibodies against each of the tested allergens was tested for (A) IgE and (B) IgG reactivity to the individual allergens in different dilutions (x-axes). Fluorescence intensities corresponding to bound antibodies are shown on the y-axes.

Figure 6

IgE- and IgG-reactivity (y-axes: fluorescence intensities corresponding to bound antibodies) of allergic patients to (A) microarrayed HDM/mite allergens, (B) cat allergens and (C) PR10 allergens (x-axes) and of non-allergic subjects to all tested allergens (D). Glass: black dots; Silicon: red dots.

Figure 7

Different assembly of silicon-based microarray elements generates different microarray formats for different diagnostic needs. Currently available allergen chips represent a single format which cannot adapt to different needs as shown for ImmunoCAP ISAC. By contrast, silicon-based microarray elements can be assembled in different formats. For example, chips containing only one array for individualized rapid testing can be produced which due to the high sensitivity can be read in simple scanners or by using mobile phone-based cameras. Alternatively, chips containing up to eight microarrays can be assembled which allow manual testing of medium scale numbers of sera and subsequent analysis by inexpensive automated scanners. For large scale automated testing of large numbers of sera, silicon elements can be mounted in ELISA-type plates and subjected to automated testing in a closed ELISA-based instrument containing an incubation, washing and detection unit followed by a scanning unit.