Allergy 2008: 63: 1521–1528

Clinical usefulness of microarray-based IgE detection in children with suspected food allergy

H. Ott, J. M. Baron, R. Heise, C. Ocklenburg, S. Stanzel, H.-F. Merk, B. Niggemann, K. Beyer

Abstract
Background: Component-resolved diagnostics using microarray technology has recently been introduced into clinical allergology, but its applicability in children with food allergy has hardly been investigated so far. The aim of this study was to evaluate the utility of microarray-based IgE detection in the diagnostic workup of food allergy and to compare this new diagnostic tool with established methods of allergen-specific IgE detection.

Methods: We investigated 130 infants and children with suspected allergy to cow´s milk (CM) or hen´s egg (HE). Serum IgE measurements, skin prick tests, allergen microarray assays and controlled oral food challenges with HE and CM were performed. Results: We analyzed 145 oral challenges that served as reference parameters for assay performance assessment. On this basis, the panel of microarrayed allergen components was shown to represent a comprehensive repertoire of clinically relevant CM and HE proteins. Additionally, the implemented CM and HE components respectively sufficed for equivalent test performance as compared to the corresponding fluorescence enzyme immunoassay extract and skin testing. However, component-resolved diagnostics for HE and CM allergy did not make oral food challenges superfluous. Clinical IgE decision points predicting positive oral food challenges could be calculated for both in vitro test methods.

Conclusions: Allergen microarrays provide a new tool to diagnose symptomatic CM and HE allergy. They show performance characteristics comparable to the current diagnostic tests and may be indicated in small children in whom only small blood volumes are obtainable. However, they are not capable of replacing double-blind, placebo-controlled food challenges in most cases.

Allergy. 2006 May;61(5):633-9.

The performance of a component-based allergen-microarray in clinical practice.

Wohrl S, Vigl K, Zehetmayer S, Hiller R, Jarisch R, Prinz M, Stingl G, Kopp T.

Abstract
Currently, the diagnosis of IgE-mediated allergy is based on allergen-specific history and diagnostic procedures using natural allergen extracts for in vivo and in vitro tests. OBJECTIVE: The aim of the study was to comparatively analyse a new component-based allergen-microarray and the 'quasi-standard' ImmunoCAP for their clinical relevance in patients with allergic rhinoconjunctivitis to five aeroallergens [house dust mite (HDM), cat dander, birch, grass and mugwort pollen] in a prospective, double-centre study. METHODS: We enrolled 120 subjects at the two study centres. Allergic patients were defined as having an allergen-specific history plus a concomitant positive skin-prick test (SPT) to natural allergen extracts and specific serum IgE was measured by both methods. Each allergen was analysed separately. RESULTS: The microarray performed equally well in receiver-operating characteristic curve (ROC) analyses when compared with the CAP in cat (23 allergic vs 97 non-allergic, ROC area under the curve microarray 0.950 vs CAP 0.894, P = 0.211), birch (31/89, 0.908 vs 0.878, P = 0.483) and grass pollen (47/73, 0.923 vs 0.915, P = 0.770). It was slightly less sensitive in HDM-allergic subjects (26 allergic vs 94 non-allergic, ROC area microarray 0.808 vs CAP 0.911, P = 0.053) and displayed a reduced sensitivity in the mugwort pollen-allergic patients (17/103, 0.723 vs 0.879, P = 0.032). CONCLUSIONS: Component-based testing and the whole-allergen CAP are equally relevant in the diagnosis of grass-, birch- and cat-allergic patients. Although slightly less sensitive, the microarray is sufficient for the diagnosis of HDM-allergic patients, but needs alternative and/or additional components for detecting mugwort allergy.

PMID: 16629796

Allergy Net 2006:61:1146–52.

Microarray-based IgE detection in capillary blood samples of patients with atopy.

Ott H, Schroeder CM, Stanzel S, Merk HF, Barom JM.

Abstract
Not available.

Clin Chem Lab Med. 2005;43(12):1321-6.

Protein microarrays for the diagnosis of allergic diseases: state-of-the-art and future development.

Harwanegg C, Hiller R.

Abstract
In the emerging field of Functional Proteomics, protein microarrays are considered to be one of the most promising tools for the simultaneous analysis of the a) abundance, b) function, and c) interaction of proteins on a system-wide scale. Resting on the technological grounds of widely used DNA biochips, the great power of microarray-based miniature solid-phase immunoassays lies in their potential to investigate in parallel large numbers of analyte pairs in a variety of biological samples. Consequently, this has fueled aspirations that protein microarrays may serve as tools for the high-throughput functional investigation of complete proteomes and, moreover, that they will develop into promising candidates for innovative in-vitro diagnostic (IVD) applications. To date, published examples of protein microarrays for IVD purposes have included tests for allergy, autoimmune and infectious diseases. Here, we discuss recent advancements in the development of protein microarrays for the profiling of IgE antibodies in the diagnosis of Type 1-related allergic diseases.

PMID: 16309367

Immunol Lett. 2005 Feb 15;97(1):81-9.

Affinity determinations of purified IgE and IgG antibodies against the major pollen allergens Phl p 5a and Bet v 1a: discrepancy between IgE and IgG binding strength.

Hantusch B, Scholl I, Harwanegg C, Krieger S, Becker WM, Spitzauer S, Boltz-Nitulescu G, Jensen-Jarolim E.

Abstract
Allergen-specific IgE and IgG antibodies coexist in allergic individuals, but only IgE has anaphylactogenic capacity. This study aimed to determine the association, dissociation and equilibrium constants for the interaction of allergen-specific IgE and IgG with the major grass and birch pollen allergens Phl p 5a and Bet v 1a. We isolated specific IgE and IgG antibodies from pollen allergic patients' sera by a two-step affinity chromatography protocol and controlled the high purity in a recombinant allergen chip microarray. Surface plasmon resonance measurements of polyclonal IgE and IgG species revealed that their affinities diverge widely, being in the range of 10(-10) and 10(-11) M for IgE, but only 10(-6)-10(-7) M for IgG. Moreover, murine monoclonal IgG1 antibodies against the allergens showed affinities of 10(-7)-10(-8) M. Thus, we conclude from our data that even stringently affinity matured IgG cannot score the superior affinity of IgE antibodies to allergens.

PMID: 15626479

Expert Rev Mol Diagn. 2004 Jul;4(4):539-48.

Protein microarrays in diagnosing IgE-mediated diseases: spotting allergy at the molecular level.

Harwanegg C, Hiller R.

Abstract
Over the last few decades, the prevalence of allergic diseases has increased dramatically in developed nations. The resulting worldwide burden on healthcare systems has provoked a whole series of research initiatives among allergy experts and commercial companies that aim to develop novel tests to improve the diagnostic risk assessment and early preventive treatment of disease. The advent of protein microarray technology has fueled aspirations of multianalyte immunological applications that permit the simultaneous analysis of huge numbers of disease-related parameters that will hopefully become amenable in the near future. Allergen microarrays have been developed for the monitoring of patient-specific antibody profiles to a previously unknown variety of allergens in a single analytical step. This review describes significant discoveries and developments in allergy research against a background of the increasing prevalence of disease and hence the emerging challenges for national healthcare systems. The development of novel protein microarray-based allergy diagnostic tests is portrayed in concert with the recent advances and benefits of this technology, along with the challenges that must be met by manufacturers in order to succeed with innovative allergen microarrays in a highly competitive market. Copyright Future Drugs Ltd.

PMID: 15225101

Methods. 2004 Mar;32(3):249-54.

Microarrayed allergens for IgE profiling.

Deinhofer K, Sevcik H, Balic N, Harwanegg C, Hiller R, Rumpold H, Mueller MW, Spitzauer S.

Abstract
Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.

PMID: 14962759

Clinical Laboratory International November Issue 2004

Recombinant allergen based approaches for the diagnosis of IgE-mediated type I allergies.

Harwanegg C, Hiller R.

Abstract
In the last decades single allergen molecules have been identified for many important sources of allergic diseases and the advent of recombinant DNA technology has since led to the characterisation of many of these allergens at the molecular level. Recombinant allergens can be arranged in diagnostic panels and used to resolve the IgE reactivites of an individual patient at the level of single disease-eliciting components, so called component-resolved diagnosis (CRD). This article reviews the advantages and shortcomings of CRD of allergy with purified or recombinant allergen molecules and discusses how this approach could change the way allergy is diagnosed and treated in the future.

Clin Exp Allergy. 2003 Oct;33(10):1443-9.

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E.

Jahn-Schmid B, Harwanegg C, Hiller R, Bohle B, Ebner C, Scheiner O, Mueller MW.

Abstract
The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen-specific IgE. OBJECTIVE: To test the performance of this allergen microarray in a serological analytical study. METHODS: Standard allergens contained in grass pollen (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) and tree pollen (Bet v 1 and Bet v 2) were used as a model system. The detection of allergen-specific serum IgE using microarrays was compared with standard test systems: CAP/RAST and an in-house ELISA. In order to test the analytical sensitivity of the assays, geometric dilutions of a serum pool containing high levels of pollen-specific IgE from allergic individuals were tested in each system. To assess the analytical specificity, the sera of 51 patients with presumptive allergic symptoms were collected before diagnosis. Thereafter, the results for grass/tree-pollen-specific IgE were compared. RESULTS: The microarray has a good dynamic range similar to the CAP/RAST system. Microarray and ELISA showed comparable analytical sensitivity exceeding the CAP/RAST system. With respect to the analytical specificity, no significant cross-reactivity of the allergens was observed. For two of the allergens tested, weak positive signals were detected in the microarray test system, whereas they were not detectable by CAP/RAST. CONCLUSION: A good correlation of presently used methods to detect serum IgE and the novel microarray test system was observed. As a next step, a careful validation of this method for a multitude of allergens and a thorough clinical evaluation has to be provided. Microarray testing of allergen-specific IgE can be presumed to be the method of choice for a prospective component-resolved diagnosis of Type I allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future.

PMID: 14519153

Clin Exp Allergy. 2003 Jan;33(1):7-13.

Microarrayed recombinant allergens for diagnosis of allergy.

Harwanegg C, Laffer S, Hiller R, Mueller MW, Kraft D, Spitzauer S, Valenta R.

Abstract
We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi-allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease-eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi-allergen tests consisting of microarrayed recombinant allergen molecules. Microarrayed recombinant allergens can be used to determine and monitor the profile of disease-eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray-based allergy testing will likely improve diagnosis, prevention and treatment of allergy.

PMID: 12534543

FASEB J. 2002 Mar;16(3):414-6. Epub 2002 Jan 14

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.

Hiller R, Laffer S, Harwanegg C, Huber M, Schmidt WM, Twardosz A, Barletta B, Becker WM, Blaser K, Breiteneder H, Chapman M, Crameri R, Duchene M, Ferreira F, Fiebig H, Hoffmann-Sommergruber K, King TP, Kleber-Janke T, Kurup VP, Lehrer SB, Lidholm J, Muller U, Pini C, Reese G, Scheiner O, Scheynius A, Shen HD, Spitzauer S, Suck R, Swoboda I, Thomas W, Tinghino R, Van Hage-Hamsten M, Virtanen T, Kraft D, Muller MW, Valenta R.

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.CTTEXT

PMID: 11790727

Tecan Application Note

Comparison of Manual vs. Automated Protein Microarray Processing

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