Allergen microarrays: a novel tool for high-resolution IgE profiling in adults with atopic dermatitis

ARTICLE

Auteur(s) : Hagen Ott¹, Regina Fölster-Holst², Hans F Merk¹, Jens Malte Baron¹

¹Department of Dermatology and Allergology, University Hospital Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany
²Department of Dermatology, Venerology and Allergology, University Hospital Schleswig-Holstein, Campus Kiel, Germany

accepté le 20 Août 2009

With a prevalence of up to 25% among children and 3% in adults, atopic dermatitis (AD) has become one of the most common dermatoses in industrialized countries [1]. Substantial scientific efforts have resulted in a better understanding of its complex pathogenesis, which involves genetically determined epidermal barrier dysfunction and characteristic immunological deviations. The latter include raised numbers of circulating regulatory T cells, eosinophilia, T helper type 2 (Th2)-skewed cytokine patterns and, in particular, elevated serum levels of total (tIgE) and allergen-specific IgE (sIgE) [2, 3]. Hence, a correct diagnosis of IgE-mediated sensitization is mandatory in patients affected by AD and if suspicion arises, several established in vivo and in vitro tools are readily available for sIgE determination [4, 5].

Furthermore, protein microarrays have recently been introduced into allergological research as promising tools for the assessment of sIgE in patients with atopic diseases [6-9]. Moreover, we have previously demonstrated in a proof of principle study that microarray-based detection of sIgE against nutritive proteins is clinically useful in children with food allergy [10]. Still, the clinical implications of component-resolved diagnosis (CRD) including microarrayed inhalant and food allergens have hitherto not been investigated in adult patients suffering from AD.

Thus, it was the principal objective of the present study to assess the clinical usefulness of an allergen microarray containing recombinant and highly purified natural allergen components for sIgE detection in adults with AD. Additionally, we aimed to correlate the assay performance of this allergen microarray to that of an established component-based fluorescence enzyme immunoassay (FEIA).

Materials and methods

Study design

Patients

In the second part of this study, we prospectively recruited a total of 20 consecutive adults who had been referred to our centre of dermatology and allergology for the evaluation of suspected allergic skin disease. In all included patients, the diagnosis of AD was established by a dermatologist performing a thorough clinical examination and applying a structured questionnaire adhering to the Hanifin and Rajka diagnostic criteria for atopic dermatitis [11]. Grade of illness was classified according to the severity scoring of AD (SCORAD) index devised by the European Task Force on AD [12]. Data processing was performed in compliance with ethical standards on human experimentation and with the Helsinki Declaration of 1975, as revised in 1983. The study was approved by the local ethical committee of the Medical Faculty of the RWTH Aachen.

Fluorescence enzyme immunoassays

Determinations of tIgE and sIgE levels were performed with a widely used, commercially available fluorescence enzyme immunoassay (FEIA) as proposed by the manufacturer (UniCAP™, Phadia, Uppsala, Sweden). For comparison with microarray data, we determined sIgE titres against a panel of 6 timothy grass pollen (Phleum pratense, Phl p) and birch pollen (Betula verrucosa, Bet v) allergen components as well as a house dust mite (Dermatophagoides farinae, d. farinae) and a cat dander allergen extract purchased from the same supplier (manufacturer’s code): Phl p 1 (g205), Phl p 5 (g215), Phl p 6 (g209), Phl p 7 (g210), Phl p 1 (g6), Bet v 1 (1215), d. farinae (d2), cat dander (e1). Total and specific IgE levels were quantified in protein units designated as kU/L with a lower detection limit of 0.35 kU/L.

Protein microarray test procedure

Microarray immunoassays were performed according to the manufacturer’s recommendations as recently published by Deinhofer and co-workers [15]. Briefly, each microarray reaction site was incubated with 20 μL of undiluted patient serum for 180 minutes in order to capture allergen-specific serum IgE antibodies by their corresponding allergen molecules. In a second step, the microarray slides were washed with a conventional TBS-T (Tris buffered saline/Tween) buffer solution two times for five minutes, rinsed with de-ionised water and dried under nitrogen flow. Hereafter, microarray-bound IgE was marked with a secondary, fluorescence-tagged antihuman IgE antibody for 60 minutes at room temperature. After a second washing procedure with TBS-T, the corresponding fluorescence signals were scanned at a 10 μm resolution using a conventional biochip reader (Scan Array Express™, Perkin Elmer Life Sciences, Boston, MA). Based on calibration sera of known specific IgE content, raw data analysis of the corresponding digitized microarray images was performed with the QuantArray™ 3.1 software (Perkin Elmer Life Sciences, Boston, MA) transforming the image information into numerical data, yielding four semi-quantitative classes of (negative, low, moderate, high) sIgE levels.

Statistics

Results

Correlation of conventional enzyme immunoassay and allergen microarray data

The diagnostic accuracy of the allergen microarray, as defined by correlation with the conventional FEIA system, was calculated by matching the respective recombinant allergen components in performance analysis, if possible. However, in the case of 2 allergens (i.e. d. farinae, cat dander) crude allergen extracts had to be used in the FEIA analysis because the corresponding recombinant allergen components (Der f 1, Fel d 1) were not commercially available for UniCAP^™ testing at the time of this investigation. Using this approach, the correlation coefficients obtained showed slight variations with regard to different allergen components, as shown in table 2. Nevertheless, a high degree of correlation (r ≥ 0.8) could be demonstrated in the case of all recombinant allergen components, with a particularly high correlation coefficient in the case of timothy grass pollen components rPhl p 1 (r = 0.93) and rPhl p 7 (r = 0.99). Less correlation between the two methods was detected comparing the purified natural microarray allergens Der f 1 and Fel d 1 with their corresponding FEIA allergen extracts (r = 0.72 and r = 0.76 respectively).
Table 1 Fluorescence enzyme immunoassay (FEIA) data obtained from venous serum samples of 40 atopic adults. Specific IgE (sIgE) serum levels are subdivided into 7 semiquantitative classes (CAP). Positive results are indicated for allergen components and FEIA classes respectively

Allergen-specific IgE profiling in adult patients with AD

Microarray-based assessment of sIgE recognition patterns disclosed a mean of 21 ± 11 (range 1-46) sensitizations per patient composed of 11 ± 7 (range 1-25) sIgE responses to plant allergens and 10 ± 6 (range 1-21) sensitizations to non-plant allergens. Positive sIgE results in more than 50% of the patients investigated were elicited by 9 plant proteins (nCyn d 1 [75%], rPhl p 1 [65%], rCor a 1 [55%], rPru p 1 [55%], rAln g 1 [55%], rBet v 1 [55%], Ara h 8 [50%]) and 8 non-plant proteins (rAlt a 1 [90%], rFel d 1 [65%], nDer p 2 [65%], rDer f 2 [65%], nDer f 1 [60%], nDer p 1 [60%], rCan f 1 [55%], rAsp f 6 [50%]). In contrast, sIgE could not be detected at all in the case of 6 microarrayed plant allergens (nAct d 1, nAct d 5, rBer e 1, rTri a 19, rHev b 3, rHev b 5) and 3 non-plant allergens (nBos d 5, nGal d 5, rBla g 5) (figures 1A, B). The total number of sensitizations was shown to positively correlate with SCORAD scores (r_p = 0.46) (figures 2, 3) and serum tIgE levels (r_p = 0.45), respectively.

We also analyzed sIgE repertoires against the following groups of plant and non-plant molecules that have previously been described as potentially cross-allergenic [16-18]: pathogenesis-related proteins (PR-10), profilins, lipid transfer proteins (LTP), parvalbumins, cysteine proteases, tropomyosins, as well as the house dust mite minor allergens nDer f 2 and nDer p 2, both belonging to the group of Niemann Pick type C2 (NPC2) proteins (figures 1A, B). As a result, 13 patients (65%) displayed sIgE cross-reactivity against PR-10 proteins. Simultaneously, cross-reactions were elicited by NPC2 proteins in 13 patients (65%), of whom 12 individuals (60%) also mounted sIgE against ≥ 2 cysteine proteases. Profilins, tropomyosins, LTP and parvalbumins elicited in vitro cross-reactivity in a minority of patients (n ≤ 3), only. Group comparison of patients sensitized and non-sensitized to PR-10 proteins revealed a statistically significant difference with regard to disease severity, while this could not be observed for NPC2 (figure 4).

Discussion

Hence, in order to ensure feasibility and sufficient clinical performance of the allergen microarray employed, we compared the sIgE profiles obtained to those detected by an established FEIA. Viewing this first part of our investigation as a proof of principle study, we were able to further corroborate previous reports demonstrating high degrees of correlation between allergen microarray and FEIA data [19]. This was particularly evident in the case of sIgE antibodies against recombinant timothy grass pollen components (rPhl p 1, 5, 6, 7) and the recombinant birch pollen major allergen (rBet v 1). These findings are in accordance with a recent publication by Wöhrl and co-workers who also matched birch- and grass pollen-specific sIgE responses of both test systems and could not demonstrate any significant disparities [14]. These data also agree with another recent publication demonstrating equally high correlations of microarray, FEIA and ELISA results obtained with the same panel of recombinant pollen allergens [23]. Likewise, although not systematically evaluated in the current study, the inter-assay reproducibility of microarray testing has been reported to be comparable to that of FEIA analysis with coefficents of variation ranging from 17% to 26% depending on the grass and tree pollen major allergen components (Phl p 1, Phl p 5, Bet v 1) implemented [23].

For the first time, the current study applied microarray technology for high-resolution sIgE detection in adult patients suffering from AD. We were able to simultaneously analyze in vitro reactivity to nearly 100 proteins of more than 40 allergen sources in minimal-volume serum samples. Consequently, pronounced inter-individual variations could be observed with regard to both the total number of specific sensitizations and the corresponding sIgE recognition patterns. Microarray-based CRD permitted the identification of a panel of 16 allergen components eliciting positive sIgE responses in the majority of patients. Of note, these proteins belonged to plant and non-plant allergens of well-known significance in AD pathogenesis. In particular, grass and tree pollen proteins as well as mould, animal dander and house dust mite components induced positive sIgE results. This is consistent with FEIA-based investigations describing similar sensitization rates, and it clearly implies that the allergenic proteins investigated in our study represent a comprehensive repertoire of clinically relevant allergen components [24-27].

In addition, the instated microarray indicated a possible clinical role of cross-allergens in the context of AD. While sensitization to some of these partially homologous proteins has been shown to be associated with a more severe course of food allergy, their impact on AD is still poorly understood [17, 28, 29]. In the current study, AD severity was elevated in patients who were sIgE-positive to PR-10 proteins, a family of highly cross-allergenic molecules contained in a wide range of flowering plants [18]. In like manner, the total number of sensitizations encountered in our patients was positively, albeit moderately associated with AD severity. Still, these data should be interpreted with caution, because pre-test probability could have been influenced by a selection bias in favor of individuals with serious AD who represent the majority of patients presenting to our tertiary care centre. Additionally, the facts that no control patients were analyzed and that there was a comparatively small number of patients included both hamper the transferability of the current results to other, non-selected patient populations in whom PR-10 sensitization or multiple sIgE responses might not be linked to the clinical grade of illness. Furthermore, the microarray investigated represents a diagnostic platform that can be loaded with virtually all allergen components of relevance in the context of AD. This would potentially empower future epidemiological investigations, especially cohort studies in search of marker allergens, other than hen’s egg extract [30], for a longer disease duration or the later development of bronchial asthma, for example. Moreover, allergen microarrays represent a sensitive tool to differentiate the intrinsic variant of AD, characterised by a lack of IgE-mediated sensitization, from the extrinsic AD type, which is associated with the presence of allergen-specific IgE antibodies. A more reliable distinction of these two phenotypes would clearly enhance AD patient care, because both disease types are known to differ with regard to further diagnostic steps and effective allergological treatment [31, 32].

Additionally, if a strong suspicion of immediate-type food allergy arises which cannot be confirmed by extract-based skin prick testing or FEIA analysis, patient safety could be maximized by extensive scanning for markers of food-induced anaphylaxis, such as sIgE against wheat omega-5-gliadin (Tri a 19), peanut major allergens (Ara h 1, Ara h 2, Ara h 3) or LTP from different allergen sources (nPru p 3, rCor a 8) [33-35]. This is of major importance, particularly in the context of AD where up to one third of affected children and up to 10% of adults additionally display IgE-mediated food hypersensitivity [36, 37]. Unfortunately, this could not be systematically explored in the present study due to the low prevalence of food hypersensitivity. Yet, out of 10 patients mounting sIgE against peanut proteins, microarray analysis identified sIgE to peanut major allergens (Ara h 1, Ara h 2) only in the patient with symptomatic peanut anaphylaxis, whereas a cross-allergenic PR-10 plant protein (Ara h 8) was found to be the culprit antigen in the remaining, asymptomatic individuals.

In conclusion, allergen microarrays provide a novel tool to diagnose IgE-mediated sensitization in adults suffering from AD. They show performance characteristics comparable to the current CRD-based sIgE detection method and allow for the simultaneous detection of sIgE antibodies against multiple allergen components in minimal volumes of patient serum that can easily be obtained by capillary blood sampling. Thus, they might serve as sensitive and minimally-invasive sIgE screening tools improving patient care, especially in pediatric patients suffering from AD. However, further large-scale studies in unselected patient populations and in distinct high-risk groups such as AD patients with food-induced disease exacerbations are warranted before the allergen microarray can be introduced into daily clinical practice.

Acknowledgements

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