Microarrayed allergen molecules: Diagnostic ...

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Jan 14, 2002 - Science and Culture, by grant Y078GEN of the Austrian Science Foundation, by Pharmacia. Diagnostics, Uppsala, Sweden, and by BIOMAY, ...
The FASEB Journal express article 10.1096/fj.01-0711fje. Published online January 14, 2002.

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment 1,*

2,*

1

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Reinhard Hiller, Sylvia Laffer, Christian Harwanegg, Martin Huber, Wolfgang M. 1 3 4 5 6 Schmidt, Anna Twardosz, Bianca Barletta, Wolf M. Becker, Kurt Blaser, Heimo 2 7 6 2 8 Breiteneder, Martin Chapman, Reto Crameri, Michael Duchêne, Fatima Ferreira, Helmut 9 2 10 5 Fiebig, Karin Hoffmann-Sommergruber, Te Piao King, Tamara Kleber-Janke, Viswanath P. 11 12 13 14 4 12 Kurup, Samuel B. Lehrer, Jonas Lidholm, Ulrich Müller, Carlo Pini, Gerald Reese, Otto 2 15 16 17 9 Scheiner, Annika Scheynius, Horng-Der Shen, Susanne Spitzauer, Roland Suck, Ines 17 18 4 19 Swoboda, Wayne Thomas, Raffaela Tinghino, Marianne Van Hage-Hamsten, Tuomas 20 2 1 2 Virtanen, Dietrich Kraft, Manfred W. Müller, and Rudolf Valenta 1

VBC-GENOMICS, A-1030 Vienna, Austria; 2Department of Pathophysiology, and 3Department of Internal Medicine IV, Division of Pulmology, University of Vienna, A-1090 Vienna, Austria; 4 Istituto Superiore di Sanitá, Laboratorio di Immunologia, 00161 Roma, Italy; 5Biochemical and Molecular Allergology, Research Center Borstel, D-23845 Borstel, Germany; 6Swiss Institute for Allergy and Asthma Research, CH-7270 Davos, Switzerland; 7Department of Internal Medicine, Asthma and Allergic Diseases Center, University of Virginia, Charlottesville, VA 22908, USA; 8 Department of Genetics and Biology, University of Salzburg, A-5020 Salzburg, Austria; 9 Allergopharma Joachim Ganzer KG, D-21465 Reinbek, Germany; 10The Rockefeller University, New York, NY 10021, USA; 11Allergy-Immunology Research, Medical College of Wisconsin, Milwaukee, WI 53295, USA; 12Section of Clinical Immunology and Allergy, Department of Medicine, Tulane University Medical Center, New Orleans, LA 70112, USA; 13 Pharmacia Diagnostics, Uppsala, Sweden; 14Department of Internal Medicine, Zieglerspital, CH-3007, Bern, Switzerland; 15Clinical Allergy Research, Karolinska Institutet, 17176 Stockholm, Sweden; 16Department of Medical Research and Education, Veterans General Hospital, Taipei, Taiwan 11217, Republic of China; 17Institute of Medical and Laboratory Diagnostics, University of Vienna, A-1090 Vienna, Austria; 18TVW Telethon Institute for Child Health Research, The University of West Australia, West Perth 6872, Australia; 19Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Institute and Hospital, 17176 Stockholm, Sweden; and 20Department of Clinical Microbiology, University of Kuopio, FIN-70211 Kuopio, Finland. *The first two authors contributed equally and are listed in alphabetical order. Corresponding authors: Rudolf Valenta, Department of Pathophysiology, Vienna General Hospital-AKH, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: [email protected]. Manfred Müller, VBC-GENOMICS, Rennweg 95B, A1030 Vienna, Austria. www.vbc-genomics.com

ABSTRACT Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases. Key words: type I allergy • IgE • microarray technology • recombinant allergen • diagnosis

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he hallmark of type I allergy is the production of immunoglobulin E (IgE) antibodies against otherwise harmless antigens, termed allergens, which can originate from a multitude of allergen sources (e.g., mites, plant pollens, animals, insects, molds, and food) (1). IgE recognition of allergens per se would likely remain unnoticed because IgE represents the least abundant class of immunoglobulins (2, 3). However, receptor cross-linking on effector cells (i.e., mast cells, basophils) by IgE-allergen complexes triggers the release of biological mediators (e.g., histamine, leukotrienes) within a few minutes and thus causes the immediate symptoms of type I allergy (hay fever, allergic asthma, anaphylactic shock) (4). In addition, IgE-mediated presentation of allergens to T cells leads to T-cell activation and chronic allergic inflammation (e.g., chronic asthma, atopic dermatitis), particularly after repeated contact with allergens (5). The latter event also induces increases of allergen-specific serum IgE levels and patients’ sensitivity to allergens (6). Since 1880 when Charles Blackley performed provocation tests by administrating pollen extracts to his skin (7), diagnosis of type I allergy has been conducted by provocation testing with extracts prepared from the suspected allergen sources. Almost 90 years later, shortly after the discovery of IgE antibodies, the radioallergosorbent test (RAST) was introduced, which allowed the in vitro measurement of specific serum IgE antibodies by using radioisotope-labeled anti-IgE antibodies (2, 3, 8). However, until now allergen extracts consisting of variable mixtures of allergenic and nonallergenic components have been used for in vitro and in vivo diagnosis of allergy. With such diagnostic extracts, the allergen sources can be identified but not the molecules that cause disease. For this reason, it is impossible to discriminate with extract-based diagnosis whether a patient is cosensitized to a variety of unrelated allergen molecules in different allergen sources or is cross-sensitized to few, but cross-reactive, allergens. Extractbased diagnosis also cannot provide information regarding the individual reactivity profiles of allergic patients and thus fails to identify the precise target molecules for specific immunotherapy (9). Here, we present a novel principle for the diagnosis of type I allergy that allows the simultaneous determination of complex IgE reactivity profiles with purified allergen molecules that have been

printed as microarrays on preactivated glass slides. This approach is based on recent advances made in the field of molecular allergology and biochip technology. The application of molecular biology to the field of allergen characterization has led to the availability of a continuously growing number of recombinant allergen molecules that mimick the immunological properties of their natural counterparts (9, 10). Progress achieved in microchip technology allows precise delivery and immobilization of a great number of nanoliter aliquots of protein samples onto small surfaces, as well as analysis and quantification of interactions with potential ligands (1114). In this study, we combined the latter two technologies to develop a miniaturized allergy test that is based on microarrayed allergen molecules representative of important allergen sources, and we investigated microchip technology for possible use in the determination of allergic patients’ IgE reactivity profiles. MATERIALS AND METHODS Purified recombinant and natural allergens Table 1 presents characteristics of the purified allergen molecules used for the allergen microarray in this study. They were selected to represent the most common allergen sources. With the exception of Bos d 2, one Ves v 5 preparation and Pol a 5 (expression in yeast: Pichia pastoris), Api m 2 (expression in baculovirus-infected insect cells), and Mal d 2 (expression in TMV-infected tobacco), all recombinant allergens were expressed in Escherichia coli. Certain allergens were purified from natural allergen sources (e.g., albumins from cat, dog, cattle, mouse, rat, pig, sheep, chicken, rabbit, hamster, horse, pigeon, guinea pig; Phl p 4; Pen a 1; Pen n 13). References and GenBank accession numbers of the allergen molecules are displayed in Table 1. Characterization of allergic patients Serum samples were obtained from 20 allergic patients and, for control purposes, from nonatopic persons. The diagnosis of type I allergy was based on case history, skin prick testing, and IgE serology as described earlier (15, 16). To evaluate the performance of the microarray, we included patients with various manifestations of allergic disease (e.g., rhinoconjunctivitis, asthma, atopic dermatitis, and food allergy) who exhibited different sensitization spectra (oligosensitization, polysensitization) and who had different total serum IgE levels (28 to >2000 kU/L) (Table 2). Preparation of the microarray of purified allergens Preactivated glass slides were developed by VBC-GENOMICS (Vienna, Austria). The slides contained an amine-reactive coating optimized by VBC-GENOMICS for the covalent attachment of proteins and assay performance. Purified allergen components were diluted to a final spotting concentration of 0.3 mg/ml in 150 mM sodium phosphate buffer (pH 8.5) containing 0.1% sodium dodecyl sulfate. Allergen solutions were spotted robotically in an ordered array onto the preactivated glass slides by using an Affymetrix 417 Arrayer (Affymetrix, Santa Clara, CA). A 1

µg/ml dilution of labeled anti-IgE antibody was spotted as a guide dot to support automatic image analysis. Diagnosis of allergy based on microarray, dot blot, and skin test assays Allergen chips were washed in TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.5% Tween-20, pH 8.0) for 15 min, rinsed with double-distilled water (H2Odd), and air-dried. Slides were then incubated with 200 µl of patients’ serum diluted 1:5 in TBS-T by using Probe-Clip Incubation Press Seal Chambers (Sigma, St. Louis, MO) for 60-180 min at 37°C with shaking at 150 rpm. Slides were then rinsed with TBS-T and washed with TBS-T for another 15 min. Bound IgE antibodies were detected with a 1:1000 diluted mouse monoclonal anti-human IgE antibody (Pharmingen, San Diego, CA), which had been labeled by using the Alexa Fluor 546 proteinlabeling kit according to the manufacturer’s protocol to approximately 5.53 mol dye/mol protein. Slides were washed five times in TBS-T, rinsed with H2Odd, and air-dried. Allergen arrays were scanned by using an Affymetrix 418 scanner (Affymetrix), with the laser power and PMT gain set to 100%. Data were evaluated with the GenePix Software Version 3.0 (Axon Instruments, Union City, CA) and Microsoft Excel. The mean signal of each triplicate determination was considered positive when it was at least five times higher than the signal obtained for spotted human serum albumin. The latter was usually not higher than signals obtained for buffer dots. In parallel to the microarray, IgE reactivity to nitrocellulose-dotted allergen molecules was analyzed for each serum sample. Aliquots of 2 µl containing 100 ng of each allergen were dotted to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and probed with 1:5 serum dilutions from the allergic patients and nonatopic persons as described earlier. Bound IgE antibodies were detected with 125I-labeled anti-human IgE antibodies (RAST, Pharmacia, Uppsala, Sweden) (17). Skin prick tests were performed with 20-µl aliquots of purified allergens diluted in H2Odd to final concentrations of 20 µg/ml, with test areas on the backs of selected atopic persons, as described previously (18). No skin tests were performed with purified natural albumins. For control purposes, prick tests were performed with diluent (H2Odd) alone and histamine (1 mg/ml) (Allergopharma, Reinbek, Germany). RESULTS Selection, purification, and characterization of allergen molecules We purified 78 recombinant and 16 natural allergen molecules representative of most of the important allergen sources (Table 1). Allergen molecules that according to IgE competition studies resemble the epitope complexity of tree (birch), grass (timothy grass), and weed pollens were prepared as described before (15, 16). We also produced recombinant mite, animal, mold, insect, food, and latex allergens and one autoallergen according to the protocols described in the supplementary references for Table 1 (19-26). Purified allergen components that remained soluble in aqueous solutions up to a concentration of approximately 1 mg/ml were obtained for most allergens.

Microarrayed allergen molecules allow the determination of IgE reactivity profiles of allergic patients The allergen components were microarrayed as triplicates onto preactivated glass slides in groups representing individual allergen sources (Fig. 1a, Table 1). For certain allergens, several recombinant protein preparations were immobilized (e.g., Der p 2: mite; Fel d 1: cat) (Fig. 1a, Table 1). Microarrayed allergen components were then exposed to serum samples from allergic patients who were sensitized against a broad variety of different allergen sources and who exhibited total serum IgE levels ranging between 28 and higher than 2000 kU/L (Table 2). Only a small amount of serum (40 µl) from each patient was needed to test IgE reactivity to almost 100 allergen triplicates in a single test, whereas most currently used diagnostic tests require 50 µl of serum for testing of IgE reactivity to one single component or extract. To examine the performance of the allergen microarray, several control experiments were performed. First, individual allergen components were printed as triplicates to assay for homogeneity of the spotting process. Second, sera were assayed three times on individually printed allergen arrays to determine the reproducibility of the microarray-based allergy test system. The mean Pearson correlation coefficient calculated from these experiments was >0.95. Figure 1b and c exemplifies the overall performance of the allergen array. The polysensitized patient A showed IgE reactivity to microarrayed allergen components consistent with the clinical sensitization profile (Fig. 1b, Table 2), whereas the nonatopic person’s serum (Fig. 1c) did not react with any of the immobilized allergen components. Both microarrays shown in Fig. 1b and c contained comparable amounts of immobilized labeled anti-human IgE antibodies. As exemplified by patient A, we found that the IgE reactivity profiles determined with the allergen microarrays were in good agreement with those determined by skin prick testing or with allergen components that had been spotted onto nitrocellulose under conditions of allergen excess (0.2 µg/spot) (Fig. 1b, d, e). Microarray-based allergy diagnosis reflects the clinical sensitization pattern of patients To evaluate the allergen microarray, we tested serum from patients who were allergic to various numbers of different allergen sources and who showed low serum IgE levels (500 kU/L) (patients A, B, D, H, J, L, N) (Table 2). We found that clinical sensitivity to the different allergen sources was well reflected by the IgE reactivity profile to the microarrayed allergen components from the respective sources, e.g., patient A with sensitivity to birch, grass pollen, animal dander, mites, and molds reacted with the following allergens: recombinant birch (Bet v 1), grass pollen (Phl p 1, Phl p 2, Phl p 5, Phl p 6), mite (Der p 2, Lep d 13, Eur m 2.0101), insect (Plo i 1), animal (cat: Fel d 1, albumins from various animals), and yeast (Mal f 5) (Table 2, Fig. 1a, b). Likewise, we found that an atopic patient with allergic bronchopulmonary aspergillosis reacted with several of the recombinant Aspergillus allergens (patient N, Table 2). However, in agreement with previously reported results on skin prick and RAST testing (27), we observed no strict association between the magnitude of the

wheal reactions (i.e., mean wheal diameters) in skin prick testing and the fluorescence intensity as measured in the microarray-based IgE detection system (data not shown). DISCUSSION In this study, we present a novel principle for the diagnosis of type I allergy with purified allergens printed as microarrays on preactivated glass slides (Fig. 2). Allergen molecules representing important allergen sources were produced as recombinant proteins or, in certain cases, purified as natural proteins (Fig. 2, parts 1 and 2). The purified allergen molecules were then microarrayed on preactivated glass slides to obtain allergen chips that allow the determination of patients’ IgE reactivity profiles with minute amounts of serum in single tests (Fig. 2, parts 3-5). A major difference between the allergen microarray described and current allergy tests is that the microarray contains defined allergen molecules as reactants, whereas currently used tests are based on allergen extracts consisting of mixtures of allergenic as well as nonallergenic molecules. Thus, the latter do not allow identification of the disease-eliciting molecules. Because IgE antibodies are present in serum in only trace amounts, detection of IgE antibodies specific for microarrayed allergen components represented a major challenge for the application of protein chip technology to the field of allergy diagnosis. Nevertheless, we found that a single fluorescence-labeled monoclonal anti-human IgE antibody allowed the detection of IgE reactivities to immobilized allergens in serum samples containing high or low total IgE levels as well as allergen-specific IgE levels (Fig. 1b, Table 2). We also obtained comparable results over a range of several serum dilutions (undiluted, 1:2, 1:5, 1:10) that are commonly applied in various established IgE detection systems (RAST, ELISA, immunoblot) (data not shown). Current allergen test systems utilize very high amounts of allergen extracts that are tested separately to obtain maximal sensitivity. In contrast, the allergen microarray is based on the simultaneous exposure of rather low amounts of purified allergens to serum IgE, but the obtained results were comparable to those of RAST-based tests. It may even be argued that the principle of simultaneous multiallergen testing more closely mimicks the natural exposure to a variety of tiny amounts of allergen molecules that occurs in allergic patients. Nevertheless, it will be possible to increase the sensitivity of the allergen microarray by using a mixture of different monoclonal anti-human IgE antibodies for the detection of bound IgE antibodies or by other amplifying techniques (e.g., rolling circle DNA amplification) (28). Compared with currently used extract-based serological diagnosis, the recombinant allergenbased microarray offers many fundamental advantages. It allows the detection of specific IgE reactivities to defined allergen components and thus the precise identification of the diseaseeliciting allergens. The IgE reactivity profile to a great number of different allergen molecules can be analyzed in a single and fast determination. Furthermore, only small amounts of serum are required, making the allergen microarray particularly useful for the determination of complex IgE reactivity profiles when it is difficult to obtain large amounts of serum from patients (e.g., for the monitoring of the onset and development of type I allergy in early childhood).

It will be certainly necessary to add additional molecules or epitopes to the panel of purified allergen components established in our study to cover the full spectrum of all important allergen sources and to accommodate the sensitization profiles of different populations of patients. The latter will not represent a major problem because the number of available recombinant allergen molecules is rapidly increasing. Furthermore, the glass slides used for the production of allergen arrays can in principle accommodate several thousands of individual components and therefore could include a large number of additional allergen molecules and, perhaps, even allergen epitopes. One of the advantages of allergen microarrays used for diagnostic purposes is, therefore, that their allergen or epitope repertoire can be continuously expanded and improved depending on the availability of newly isolated components. Microarrays will allow the monitoring of the IgE reactivity profiles during the development and course of the disease as well as during treatment, and they will also allow correlation of eventual changes of the reactivity profiles with clinical parameters (e.g., progression of the allergic rhinoconjunctivitis to asthma, or development of clinical sensitivity to additional allergen sources). By using specific antibody probes to immunoglobulin classes or subclasses other than IgE, it will be possible to study in detail the allergen and epitope recognition by antibodies that may compete with IgE and to evaluate their possible protective role. On the basis of the precise IgE reactivity profile determined by allergen microarrays, it will be possible to improve the selection of currently available forms of therapy as well as to develop new allergen-specific therapeutic strategies. In this context, it is noteworthy that for several important allergens, derivatives have been produced by recombinant DNA and synthetic peptide technology that exhibited a greatly reduced allergenic activity and that may be used to improve the safety and efficacy of allergenspecific immunotherapy (29, 30). It is therefore likely that microarrayed allergen components will fundamentally change the current practice of allergy diagnosis, prevention, and therapy. In addition, the principle of component-resolved diagnosis based on microarrayed purified antigens may find application to autoimmune and infectious diseases. ACKNOWLEDGMENTS This study was supported by the ICP program of the Austrian Federal Ministry for Education, Science and Culture, by grant Y078GEN of the Austrian Science Foundation, by Pharmacia Diagnostics, Uppsala, Sweden, and by BIOMAY, Vienna, Austria. REFERENCES 1.

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SUPPLEMENTARY MATERIAL: REFERENCES FOR TABLE 1

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Hoffman, D. R. (1993) Allergens in Hymenoptera venom XXV: the amino acid sequences of antigen 5 molecules and the structural basis of antigenic cross reactivity. J. Allergy Clin. Immunol. 92, 707-716 Hoffmann-Sommergruber, K., O´Riordain, G., Ahorn, H., Ebner, C., Laimer da Camara Machado, M., Pühringer, H., Scheiner, O., and Breiteneder, H. (1999) Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1. Clin. Exp. Allergy 29, 840-847 Hoffmann-Sommergruber, K., Ferris, R., Pec, M., Radauer, C., O´Riordain, G., Laimer da Camara Machado, M., Scheiner, O., and Breiteneder, H. (2000) Characterization of Api g 1.0201, a new member of the Api g 1 family of celery allergens. Int. Arch. Allergy Immunol. 122, 115-123 Kleber-Janke, T., Crameri, R., Appenzeller, U., Schlaak, M., and Becker, W. M. (1999) Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. Int. Arch. Allergy Immunol. 119, 265-274 Kos, T., Hoffmann-Sommergruber, K., Ferreira, F., Hirschwehr, R., Ahorn, H., Horak, F., Jager, S., Sperr, W. R., Kraft, D., and Scheiner, O. (1993) Purification, characterization and N-terminal amino acid sequence of a new allergen from European chestnut pollen, Cas s 1. Biochem. Biophys. Res. Commun. 196, 1086-1092 Laffer, S., Valenta, R., Vrtala, S., Susani, M., van Ree, R., Kraft, D., Scheiner, O., and Duchene, M. (1994) Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergens from eight different grass species. J. Allergy Clin. Immunol. 94, 689-698 Leitermann, K., and Ohman, J. L. (1984) Cat allergen 1: biochemical, antigenic, and allergenic properties. J. Allergy Clin. Immunol. 74, 147-153 Lin, K-L., Hsieh, K-H., Thomas, W. R., Chiang, B-L., and Chua, K-Y. (1994) Characterization of Der p V allergen, cDNA analysis, and IgE-mediated reactivity to the recombinant protein. J. Allergy Clin. Immunol. 94, 989-996 Lindborg, M., Magnusson, C. G. M., Zargari, A., Schmidt, M., Scheynius, A., Crameri, R., and Whitley, P. (1999) Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface display technology. J. Invest. Dermatol. 113, 156-161 Monsalve, R. I., Lu, G., and King, T. P. (1999) Expressions of recombinant venom allergen, antigen 5 of yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast. Protein Express. Purif. 16, 410-416 Morgenstern, J. P., Griffith, I. J., Brauer, A. W., Rogers, B. L., Bond, J. F., Chapman, M. D., and Kuo, M.-C. (1991) Amino acid sequence of Fel d I, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning. Proc. Natl. Acad. Sci. USA 88, 9690-9694

Moser, M., Crameri, R., Menz, G., Schneider, T., Dudler, T., Virchow, C., Gmachl, M., Blaser, K., and Suter, M. (1992) Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test reactivity. J. Immunol. 149, 454-460 Natter, S., Seiberler, S., Hufnagl, P., Binder, B. R., Hirschl, A. M., Ring, J., Abeck, D., Schmidt, T., Valent, P., and Valenta, R. (1998) Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients. FASEB J. 12, 1559-1569 Niederberger, V., Hayek, B., Vrtala, S., Laffer, S., Twardosz, A., Vangelista, L., Sperr, W. R., Valent, P., Rumpold, H., Kraft, D., Ehrenberger, K., Valenta, R., and Spitzauer, S. (1999) Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phlp7. FASEB J. 13, 843-856 Olsson, S., van Hage-Hamsten, M., Whitley, P., Johansson, E., Hoffman, D. R., Gafvelin, G., and Schmidt, M. (1998) Expression of two isoforms of Lep d 2, the major allergen of Lepidoglyphus destructor, in both prokaryotic and eukaryotic systems. Clin. Exp. Allergy 28, 984-991 O' Neill, G. M., Donovan, G. R., and Baldo, B. A. (1994) Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase. Biochim. Biophys. Acta 1219, 521-528 Pandjaitan, B., Swoboda, I., Brandejsky-Pichler, F., Rumpold, H., Valenta, R., and Spitzauer, S., (2000) Escherichia coli expression and purification of recombinant dog albumin, a cross-reactive animal allergen. J. Allergy Clin. Immunol. 105, 279-285 Rasool, O., Zargari, A., Almqvist, J., Eshaghi, H., Whitley, P., and Scheynius, A. (2000) Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8, Mal f 9. Eur. J. Biochem. 267, 4355-4361 Rautiainen, J., Auriola, S., Rouvinen, J., Kauppinen, J., Zeiler, T., Novikov, D., Virtanen, T., and Mäntyjärvi, R. A. (1998) Molecular and crystal properties of Bos d 2, an allergenic protein of the lipocalin family. Biochem. Biophys. Res. Commun. 247, 746-750 Reese, G., Jeoung, B. J., Daul, C. B., and Lehrer, S. B. (1997) Characterization of recombinant shrimp allergen Pen a 1 (tropomyosin). Int. Arch. Allergy Immunol. 113, 240-242 Schmidt, M., Olsson, S., van der Ploeg, I., and van Hage-Hamsten, M. (1995) cDNA analysis of the mite allergen Lep d 1 identifies two different isoallergens and variants. FEBS Lett. 370, 1114 Schmidt, M., Zargari, A., Holt, P., Lindbom, L., Hellmann, U., Whitley, P., van der Ploeg, I., Harfast, B., and Scheynius, A. (1997) The complete cDNA sequence and expression of the first major allergenic protein of Malassezia furfur, Mal f 1. Eur. J. Biochem. 15, 181-185

Shen, H-D., Chua, K-Y., Lin, K-L., Hsieh, K-H., and Thomas, W. R. (1993) Molecular cloning of a house dust mite allergen with common antibody binding specificities with multiple components in mite extracts. Clin. Exp. Allergy 23, 934-940 Shen, H-D., Au, L-C., Lin, W-L., Liaw, S-F., Tsai, J-J., and Han, S-H. (1997) Molecular cloning and expression of a Penicillium citrinum allergen with sequence homology and antigenic crossreactivity to a hsp 70 human heat shock protein. Clin. Exp. Allergy 27, 682-690 Shen, H-D., Lin, W-L., Tam, M. F., Wang, S-R., Tzean, S-S., Huang, M-H., and Han, S-H. (1999) Characterization of allergens from Penicillium oxalicum and P. notatum by immunoblotting and N-terminal amino acid sequence analysis. Clin. Exp. Allergy 29, 642-651 Shen, H-D., Wang, C-W., Chou, H., Lin, W-L., Tam, M. F., Huang, M-H., Kuo, M-L., Wang, SR., and Han, S-H. (2000) Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3). J. Allergy Clin. Immunol. 105, 827-833 Simon-Nobbe, B., Probst, G., Kajava, A. V., Oberkofler, H., Susani, M., Crameri, R., Ferreira, F., Ebner, C., and Breitenbach, M. (2000) IgE-binding epitopes of enolases, a class of highly conserved fungal allergens. J. Allergy Clin. Immunol. 106, 887-895 Smith, W., Mills, K. L., Hazell, L. A., Hart, B. J., and Thomas, W. R. (1999) Molecular analysis of the group 1 and 2 allergens from the house dust mite, Euroglyphus maynei. Int. Arch. Allergy Immunol. 118, 15-22 Soldatova, L. N., Crameri, R., Gmachl, M., Kemeny, D. M., Schmidt, M., Weber, M., and Müller, U. R. (1998) Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli. J. Allergy Clin. Immunol. 101, 691-698 Suck, R., Weber, B., Kahlert, H., Hagen, S., Cromwell, O., and Fiebig, H. (2000) Purification and immunobiochemical characterization of folding variants of the recombinant major wasp allergen Ves v 5 (antigen 5). Int. Arch. Allergy Immunol. 121, 284-291 Tinghino, R., Barletta, B., Palumbo, S., Afferni, C., Iacovacci, P., Mari, A., Di Felice, G., and Pini, C. (1998) Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: a novel calcium-binding allergen. J. Allergy Clin. Immunol. 101, 772-777 Twardosz, A., Hayek, B., Seiberler, S., Vangelista, L., Elfman, L., Grönlund, H., Kraft, D., and Valenta, R. (1997) Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4. Biochem. Biophys. Res. Commun. 239, 197-204 Valenta, R., Duchene, M., Pettenburger, K., Sillaber, C., Valent, P., Bettelheim, P., Breitenbach, M., Rumpold, H., Kraft, D., and Scheiner, O. (1991) Identification of profilin as a novel pollen allergen; IgE autoreactivity in sensitized individuals. Science 253, 557-560

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Table 1 (part 1)

Table 1 (part 2)

Table 2

Fig. 1

Figure 1. Allergen microarray. A) Scheme of environmental allergens (1-93), an autoallergen (94), and human serum albumin (negative control: 95), which were microarrayed as triplicates and numbered as in Table 1. Allergens reactive with serum IgE of a representative patient (patient A in Table 2) (B) were boxed in gray and denoted with black numbers. Triplicates of buffer dots (x) and labeled anti-IgE (yellow dot) were spotted as negative and positive controls, respectively. B, C) Scan images obtained with serum of an allergic (B) and a nonallergic (C) individual. (Continued on following page)

Fig. 1 (continued)

Figure 1. (continued) D) IgE reactivity of patient A to nitrocellulose-dotted allergens. E) Skin prick test with purified allergens (application order as in d) for patient A. H2Odd and histamine were applied to fields H5 and H6, respectively.

Fig. 2

Figure 2. Schematic overview: Diagnosis of type I allergy with microarrayed allergen components. 1, 2) Purified allergen molecules were obtained from important allergen sources by recombinant DNA technology as recombinant allergens and/or as purified natural allergens. 3) Preparation of microarray of purified allergen molecules to obtain allergen chips. 4) Allergen chips were incubated with serum IgE, and bound IgE antibodies were traced with fluorescencelabeled anti-human IgE antibodies. 5) The patients’ IgE reactivity profile can be obtained and quantified by scanning the fluorescence intensity of the signals.