HK1066276B - Allergen-microarray assay - Google Patents

Description

Allergen microarray analysis

Technical Field

The present invention relates to a method for detecting an allergen-binding immunoglobulin in a sample and a method for in vitro diagnosis of allergy (allergy) patients.

Background

Allergy is a reaction produced by the immune system of the body against a substance that is generally considered to be harmless. It is this reaction that results in a class of symptoms known as anaphylaxis. Thus, the development of allergy is not due to a failure of the immune system, but to an excessive activation of the immune system. The reaction of an allergic patient to an allergen can produce a wide range of symptoms. For example, some human symptoms may manifest as asthma, eczema, rashes, itchy eyes, sinusitis, nasal or runny nose, and hay fever, but more severe symptoms may also occur. Allergies caused by, for example, snake venom, nuts and shellfish can lead to a critical condition known as anaphylactic shock. This is due to the body producing so strong a response as to cause laryngeal edema, blood pressure drop and dyspnea. In some cases, such reactions are fatal.

In developed countries (i.e. europe, north america and japan), the incidence of allergy is increasing and it is estimated that 20-50% of the population is affected. It is now known that allergy is the result of an imbalance in the T cell component of the immune system. More specifically, the T helper cell type 2 (Th2) activity is increased relative to T helper cell type 1 (Th1) in allergy sufferers, which results in increased IgE production.

IgE (immunoglobulin E) is at least one of the substances that cause allergic reactions. Normally, IgE specific for one allergen is not detected in the blood, which is only produced after sensitization of the human body with one substance. An allergic reaction-causing substance (called an allergen) induces the production of a specific IgE, which reacts only with the substance. The reaction between IgE and allergen is like a lock and key, and IgE bound to allergen causes the release of chemicals (e.g. histamine) from cells, which can cause allergic symptoms. Humans may produce specific IgE to more than one substance and thus may develop an allergic reaction to more than one substance. The results of the IgE detection are presented as a gradient to show the amount of IgE specific to the substance detected in the blood. The higher the gradient, the more likely the patient will develop an allergic condition. In the last decades, the diagnosis of allergic diseases has used only a few unpurified extracts from major allergen sources. Obviously, the composition of these crude purified mixtures is complex and unstable. The result is that the diagnostic efficacy of these extracts varies frequently and leads to false negative results. The latter is mainly caused by instability of the allergens in the extract. Another disadvantage of using extracts for diagnosing allergy is the lack of standardization of the current products in terms of immunogenic compositions. The effectiveness units and calculation methods of various company products on the market are different. The result is a lack of comparability between diagnostic products of various companies.

The most important disease-related component, the major allergen, has been identified over the past decades, but it has not been quantified for use as a standardized basis for allergen extracts. Monoclonal antibodies against most major allergens have emerged.

The detection of allergies according to known methods is not a simple procedure and would be useful if a medical history were taken to identify which detection method is most appropriate. However, there are currently only a few medical examination methods that can identify allergies, and such examination methods are generally limited to use in clinical laboratories and specialized centers. Also, although the practitioner or allergy specialist can make a diagnosis of atopy or allergy relatively easily, further examination is often required to confirm.

Various types of allergy examination methods include:

1. skin pricking test

The suspected allergen is injected immediately beneath the skin surface and its response is observed by a qualified nurse or physician. If the reaction is positive, localized skin redness will appear, and the stronger the reaction, the larger the area of redness. The skin test has a high sensitivity, although this method does not allow the determination of all types of allergies.

2. Skin contact test

The skin contact test can be used to diagnose contact dermatitis. The substance to be tested is usually applied to the skin by means of a patch and left in place for 48 hours. A positive responder will develop a small eczema and again, the response is observed by a qualified nurse or physician.

3. Other allergy testing methods

Other types of allergy testing are numerous, but are not sufficiently accurate or approved by the medical community, and are often inaccurate and expensive.

Vega test

The Vege test is an electrical test (electrical test) belonging to the bioenergetic regulation technique (bioenergetic regulation technique). The instrument measures conductance through a Wheatstone circuit (Wheatstone circuit). During detection, the electrodes are connected to the acupuncture part or held by the patient, and then various solutions are put into a metal groove. A vial of glass containing the toxicant is then placed in the tank and the instrument calibrated, the vial causing a decrease in conductivity, and the other substance placed in the tank, if it produces a similar reading, is considered an allergic or "sensitive" reaction. This technique is widely used in health food stores, but its value has not been recognized, nor has it been confirmed by effective tests.

5. Leukotoxicity test

The leukotoxicity test involves mixing patient leukocytes with extracts of specific foods and then observing some altered forms of the cells through different routes. Because of its high false positive and false negative rates, the American Association for allergic diseases (American academy of Allergy) holds no evidence to show that it is helpful in the diagnosis of food and inhalation allergies.

6. Hair analysis

Another detection method is hair analysis. Hair analysis can find out whether toxic metals such as lead, mercury and cadmium are present in the body or whether selenium, zinc, manganese and magnesium are absent. There are many records in the judicial community about heavy metal poisoning, where hair analysis can be used to confirm the presence or absence of toxic metal exposure. However, hair analysis, a method similar to "interrogating a water source" (dowing), has never proven effective as a means of diagnosing allergies.

7. Applied kinematics (Applied kinesiology)

Placing the food sample under the tongue of the patient or in a glass container to be held by the patient, and then asking the patient to push the arm of the patient with the arm not holding the container, the allergic reaction is detected as the decrease of the muscle reactivity of the patient and the difficulty of lifting the arm. However, this detection technique was found to be ineffective by double-blind testing.

In addition, there are various in vitro tests for diagnosing allergy by detecting the presence of IgE or IgG antibodies in a blood sample from a patient:

8. conventional methods such as ELISA or western blotting detect the presence of antibodies (IgE) in the serum of patients by means of (recombinant) immunogens.

9. Radioimmunoassay (RAST)^®))：

Coupling specific immunogen to paper disc; if immunospecific IgE is present in the (patient) serum to be detected, it will bind to the disc; detection is then performed by radiolabeled anti-IgE. Various scoring systems compare the test results with the absolute binding of negative controls. In general, improved RASTs^®The method employs overnight incubation to achieve higher sensitivity.

10. RAST-based quantitative IgE inhibition experiments, in which allergen extracts are spotted onto nitrocellulose strips, a measured amount of a mixture of serum and specific recombinant allergens is preincubated, then the mixture is placed on the nitrocellulose strips for incubation, bound IgE is detected by anti-human IgE antibodies, and finally the percent inhibition of binding of the IgE to the natural extract after preadsorption by the recombinant allergens is calculated.

11. Cell antigen stimulation assay (CAST), in which basophils of a patient are stimulated with interleukin-3 (IL-3) and an allergen, followed by measurement of newly produced and released leukotriene Sulfide (SLT) by ELISA.

UniCAP: the recombinant allergen is immobilized on a solid support and the binding of antibodies present in the serum of a patient to the allergen is then detected.

However, the use of these methods for the detection of a large number (e.g. 100) of allergens requires the repetition of a large number of experimental steps. Furthermore, these detection methods do not show sufficient sensitivity and reliability and are very time consuming. Simultaneous performance of such tests requires large sample volumes (e.g. patient serum) as well as large amounts of purified, possibly expensive, recombinant allergen.

Us patent 4,444,879 relates to a method and apparatus for immunoassay for determination of total immunoglobulins and IgE. The extracted allergens are immobilized in the wells of a microtiter plate coated with a polymer, and then a sample to be tested is added to the wells for conventional immunoassay.

European patent 0,556,745a1 describes a method for detecting anti-wheat protein IgE antibodies in body fluids, wherein an immunogenic extract of wheat is immobilized on the surface of a solid support, such as a chromatographic and capillary material or a fiber, glass, nylon, cellulose or derivatives thereof, in the form of glass beads, microparticles, a membrane or the wells of a microtiter plate. Samples from patients are also tested using conventional immunoassay methods.

U.S. Pat. No. 3,720,760 relates to a method for analyzing immunoglobulins in body fluids. It also binds commercially available immunogen-containing extracts to water-insoluble polymers and then performs conventional immunoassays.

Us patent 4,849,337 relates to a method of identifying levels of immunogen specific IgE in the serum of a patient by binding IgE in the serum to an immunogen adsorbed to an insoluble support. Likewise, the immunogen may be an extract or immunogen directly from pollen, dust, animals, etc.

However, these conventional immunoassay methods are not very effective for detecting allergy in patients because the number of various allergies has exceeded hundreds and is still steadily increasing. In laboratories that test hundreds of patient samples per day, the analysis of patient samples to identify all possible and rare allergies is time-consuming and laborious and therefore impossible.

Disclosure of Invention

It is therefore an object of the present invention to provide a method for the detection of immunoglobulins which can be bound to allergens in a sample, which method overcomes the above-mentioned disadvantages, which method is short in operation time, which requires only a small amount of sample and allergen and which has a high degree of reliability and sensitivity, and above all which allows the detection of an almost unlimited number of allergens in one assay.

It is another object of the present invention to provide a method for in vitro diagnosis of allergy in a patient which overcomes the above-mentioned drawbacks.

The above method is characterized in that one or more allergens are immobilized on a microarray chip, after which the sample is incubated with the immobilized allergens so that immunoglobulins with allergen specificity can bind to the specific allergens and thereby the immunoglobulins to which the specific, immobilized allergens are bound are detected.

Microarray chips have found application in a range of well-established DNA manipulation techniques within the scientific field. Such DNA chips already exist in many different forms and will eventually change the way experimental design in existing common laboratory work. Since this novel in vitro DNA technology can perform complex detection and analysis in a "high-throughput format", the in vitro DNA diagnosis becomes more time-saving and labor-saving. In the field of "proteome" research, classical solid phase substrates, such as microtiter plates, filters, and microscope slides, are also being converted to high density microarray formats. This novel, versatile protein analysis technology ultimately enables high throughput screening for gene expression and molecular interactions (Walter et al, Curr Opin Microbiol 2000 Jun; 3 (3): 298-. Recently, the concept of protein microarrays has begun to be applied to immunoassays.

Biochips are believed to be capable of supporting High Throughput (HT) multiplex enzyme-linked immunosorbent assays (ELISAs). These biochips may consist of, for example, 96-well optical flat glass plates coated with a hydrophobic Teflon coating in the wells; however, biochips of other materials and formats are known for use. Experiments have shown that a specific multiplex detection of antigens arranged on a glass substrate is feasible. Other applications of this novel High Throughput Screening (HTS) approach include direct cellular protein expression assays, multiplexed detection of infectious agents, and diagnosis of tumors (Mendoza LG et al, Biotechniques 1999 Oct; 27 (4): 778-80).

However, in such a known microarray protein analysis method, an antibody is immobilized on a microarray chip. Surprisingly, not only antibodies but also allergens which are antigens corresponding to such specific antibodies, in particular IgE and IgG, can be bound to the microarray chip. It is particularly surprising that functional allergens can exhibit a particularly modified secondary structure when bound at high density to a solid support such as a microarray chip. Such modification of the structure of the allergen may affect the binding of the antibody to the allergen, thereby possibly leading to false negative results. Fragments of antibodies, antibodies or peptides are relatively small and have high stability, and thus antibodies have higher stability in solution relative to their cognate antigen. However, when immobilized on a solid support, both the antibody and only a small portion remain intact and active.

Haab et al (Genom Biology 2000, I (6): pre-print 0001, 1-0001, 22), which was accepted at 11/9/2000, relates to protein microarrays that detect and quantify proteins and antibodies in solution. The results of the analysis according to this document show that only 50% of the antigens in the array and 20% of the antibodies in the array have given specific and accurate measurements of the cognate allergens.

Furthermore, in the case of protein processing, in particular the immobilization and staining of a series of structurally different allergens, the difference in allergen is of course much higher than the difference in antibody. However, it has surprisingly been found that allergens immobilized on a microarray chip have high reliability and sensitivity in the detection of immunoglobulins in a sample.

This method allows the analysis of multiple immunoglobulins that bind to a specific allergen in one experimental step, and the analysis can be further refined (atomized): i.e. more than 100 different allergens are detected without the need to perform extensive experimental steps as required in conventional microtitre methods. Furthermore, the method has a high sensitivity and reliability. Meanwhile, the method can obtain the detection result in a short time, such as about 3 hours, and the detection time is shortened compared with the traditional RAST or ELISA detection.

Furthermore, microarray analysis is highly reproducible when repeated experiments are performed with chips containing samples from different patients, e.g. by including a number of spots of different allergens in each well of a microtiter plate-like coating and then adding a sample from one patient to each well. Another advantage of the method of the invention is that a large number of different probes occupy only a relatively small area and are therefore very dense. The small surface area of the microarray allows for high uniformity of binding conditions (temperature adjustment, salt concentration, etc.), while the large number of probes allows for large numbers of hybridizations to be performed in parallel. Because this high density microarray contains such a large number of probes, it is possible to provide a large number of controls including, for example, a mutation or mutation control for a specific allergen, a global hybridization condition control, a sample preparation condition control, and a mismatch control (mismatches control) for nonspecific binding or cross-hybridization.

In addition, this assay requires only a minimum amount of material (allergen as well as sample) compared to conventional detection systems. This means that with the microarray method, more test kits can be made using the same amount of starting material, while a larger amount of immunoglobulin that can bind to the allergen can be detected using a smaller amount of sample. Also, binding can be performed very quickly in a small system.

A natural, intact "immunoglobulin" or antibody comprises a generally Y-shaped tetrameric molecule having an antigen binding site at the end of each "upper arm". The antigen binding site consists of the variable region of one heavy chain together with the variable region of one light chain. More precisely, the antigen-binding site of an antibody is essentially formed by 3 CDRs (complementarity determining regions) of a heavy chain variable region (v.sub.h) and 3 CDRs of a light chain variable region (v.sub.l).

In general, the term "antigen" refers to a substance capable of eliciting an immune response, and in general, the corresponding antibody can be detected using the substance by one of a variety of in vitro and in vivo immunological methods that are currently available for confirming antigen-antibody interactions.

Similarly, the term "allergen" refers to an antigen capable of inducing and binding to a specific antibody (e.g. IgE) which causes allergy in the general sense; the latter definition does not exclude the possibility that the allergen also induces reactive antibodies, which may include other types of immunoglobulins than IgE.

By "immobilized" of a protein or peptide herein is meant that the protein/peptide is bound to a solid support in a conventional manner, with or without a spacer between the solid support and the allergen. Immobilization of proteins/peptides to a support is well known in the art; within the scope of the present invention, any immobilization comprises interaction with e.g. membranes and synthetic surfaces, respectively, e.g. covalently, non-covalently, in particular by hydrophobic interaction.

A "microarray" is an arrangement of features (e.g., "spots") having a density of at least about 16/cm of non-contiguous features²Preferably at least about 64/cm²More preferably about 96/cm²Most preferably at least about 1000/cm². The features on the microarray have a typical dimension (typical dimension), for example a diameter in the range of about 10-2000 microns, preferably about 50-500 microns, more preferably about 150-250 microns, and are separated from other features on the microarray by the same distance. Thus, microarrays of the invention that can be used to perform routine large-scale screening can have at least 500, preferably at least 1000, different spots containing allergens, standards, and controls.

The "chip" of the invention may have virtually any shape, such as a well of a slide, a microtiter plate, or even a multitude of surfaces, although a flat array surface is preferred.

The detection step may be carried out by conventional methods known to those skilled in the art, such as physical, enzymatic, chemical reactions, and the like.

According to a preferred embodiment of the invention, IgE may be detected as immunoglobulins. IgE is known to be a substance that causes an allergic reaction, which is produced only by people allergic to one substance (i.e., an allergen). Thus, if a sample is to be tested for being from a patient allergic to a particular allergen, the IgE of the sample may be tested as immunoglobulin. The higher the amount of IgE in the sample, the more likely the patient is to be allergic to the specific allergen.

Preferably, IgG is detected as an immunoglobulin. IgG is known to be present in samples from patients with food allergy, and thus detection of IgG can be used as an indicator of food allergy.

One or more natural allergens may be immobilized on a microarray chip. These single allergens may for example be purified from allergen extracts, e.g. from specific foods or plants. One or more specific types of allergens may be analyzed simultaneously.

Methods for purification are generally known to those skilled in the art, such as chromatographic purification, mass separation and purification, purification by specific binding of an allergen to a solid phase (e.g., by an antibody), and the like, but application of highly purified allergens to mass production of allergy detection methods has not been proposed.

"purified" allergens are distinguished from extracts by the fact that they are individual allergens, for example natural, recombinant or synthetic allergens. These purified allergens immobilized on a solid support have many advantages over allergen extracts: since all extracts are mixtures of multiple proteins, the allergen concentration to be detected is much lower than in a purified single allergen preparation. Thus, purified single allergens can be immobilized on microarrays in very high concentrations. Since the molecules in the preparation containing the purified allergen are defined, it is only possible for the defined allergen to be immobilized on the microarray. Therefore, the microarray and the method for detecting antibodies with the aid of purified allergens allow standardized and precise quantitative control analyses.

Furthermore, due to the high concentration of purified allergen present in the composition, the allergen can be immobilized on the microarray at a higher density, thus allowing any detection method with this microarray to have a higher sensitivity compared to microarrays using extracts containing allergen. Another advantage of purified single allergens over allergen extracts is that the immobilized purified allergens are precisely defined. In contrast to the purified allergen, the allergen extract comprises e.g. two, three and more different microorganisms or subject allergens. For example, in the case of apples, the extract may comprise a mixture of apple allergens, whereas the composition of the invention comprises a purified apple allergen or, depending on the use, a defined mixture of two or more apples or other allergens.

Another advantage of purified allergens over non-purified allergens (e.g. mixtures) is that the extract becomes unstable over time, e.g. mouldy, due to the presence of other impurities in the extract. This would affect the detection of allergy, since in this case an allergy to mould may lead to false positive results.

Furthermore, the extract is unstable due to the presence of proteases in the extract, whereas the purified allergy principle does not.

Also, since there are currently therapeutic methods with the aid of purified allergens, a diagnosis determined by the same allergen component has great advantages over a diagnosis determined by a non-purified allergen. The former diagnosis is particularly advantageous when using purified allergens to track the effect of therapy and to judge the response of a patient to a specific therapy, and can thus provide the patient with a treatment called "tailor-made" (therapy).

For the above reasons, a method of detecting immunoglobulins in a sample that bind to one or more purified allergens by immobilizing the allergens on a microarray chip is of particular advantage.

Preferably, one or more recombinant allergens may be immobilized on a microarray chip. During the past years, large quantities of recombinant allergens have been produced. The production of recombinant allergens is also generally known in the art, but has little impact on conventional allergen detection methods. Using recombinant allergens, a large number of one or more specific allergens can be provided. In addition, specific modifications of recombinant allergens can be made to generate variant allergens for the detection of specific immunoglobulins. Moreover, the high use of recombinant proteins as allergens provides an alternative to extract-based detection methods in terms of stability of the detection and standardization of the diagnosis. Recombinant (or synthetic) allergens are also more amenable to standardization in terms of their mode of production. Furthermore, recombinant (or synthetic) allergens differ less between different production batches than allergens of natural origin. The present invention surprisingly shows that the use of these recombinant allergens in the conventional in vitro diagnostic assay of the present invention is equivalent to or superior to conventional extract-based assay systems. Different from the conventional series detection, the allergen detection method using the recombinant allergen brings brand-new quality assurance for standard allergen detection in the aspects of standardization, controllability, repeatability and the like.

Examples of recombinant allergens are described in the following publications: chapman et al, Allergy, 52: 374- (374) -. Other (recombinant) allergens such as, but not limited to, rBetv, rJunO, Cass, rPhlp5, rPhlp, rParj, Artyla, Mugwort profilen, Apig1.0201, Dauc1.2, rArah, Mald, rPena, recCarp, rDip 2.0101, rDip 2, rDip, rDipp 5, rDip, rTyrp, rLepd2.01, rLepd, rEurm2.0101, rFeld, rBo, rBollmin, rPimin, rPisca, rPimin, rPisca, rPimin, pii, Penn13, rPenc19a, rPenc19b, rAspf1, rAspf 11, rAspf 31, rAspf 41, rAspf 61, rAspf1, rAlta1, rMalf1, rHevb 36K, rBlag 1, rBl 1, rAgBl 1, phospholipase, Hyaluronidase, VesVes3672, rVes3672.

In another advantageous embodiment, one or more synthetic allergens are immobilized on a microarray chip. The advantages of the aforementioned recombinant allergens with respect to allergen extracts as well as with respect to purified natural allergens also exist here. Methods for synthesizing peptides or proteins are well known in the art; by synthesizing the allergen, a large amount of highly purified allergen can be provided at low cost, since such production methods are highly automated. In addition, the precise sequence of any allergen, modified or unmodified, may be provided, thereby increasing the sensitivity and reliability of the method to detection of immunoglobulins.

It is also possible to use only the allergenic determinants or allergenic domains of specific allergens in the detection method of the invention. This avoids the risks and disadvantages of handling large molecular weight proteins, since shorter peptide structures are easier to handle in the production of conventional biochips. This applies to all purified natural allergens, recombinant allergens and synthetic allergens. It further allows the detection of individual peptide epitopes, which will make diagnostic tests and treatments more optimal, especially in terms of specificity.

It is particularly advantageous to provide both the natural form of the allergen and the synthetic or recombinant form of the allergen in the same chip. But preferably mainly recombinant or synthetic allergens are used. The natural form of the allergen can provide at least control or additional quality information for the chip. A preferred chip of the invention therefore comprises one natural form and one synthetic or recombinant form of at least one allergen. This also improves quality control and standardization of different batches of allergen.

Preferably, one or more haptens are immobilized as allergens on a microarray chip. A "hapten" is a low molecular weight (typically less than 7000 Dalton) substance that generally does not itself cause significant antibody production upon administration to an animal, including a human. This is due to the hapten being too small to be recognized by the immune system of the body. However, when haptens are coupled to a larger carrier molecule, antigenicity of the hapten is obtained. In other words, the binding of the hapten to the carrier molecule (formation of an analyte-carrier molecule complex) enables the bound hapten to be recognized by the immune system of the animal. An immunoprecipitation reaction can occur between a hapten (conjugated to a carrier molecule) and the corresponding antibody. By using haptens immobilized to a microarray chip, higher densities can be achieved on the chip.

Of course, the different forms of allergens described above can be used in combination, for example using both recombinant and (purified) natural allergens. By combining these different forms, they can be compared and for example recombinant allergens can be compared, as well as various batches of natural allergens as controls, which differ in biological origin, preparation method, purity etc.

For an efficient detection method the allergen used preferably has optimal characteristics, such as high binding capacity. For the detection of allergen variants with low binding efficiency, however, it is preferred to use antigens with lower activity or different activity, such as in desensitization therapy (hyposensitizing transdermal).

In a preferred embodiment of the invention, the allergen is immobilized on a microarray chip at a spot having a diameter of 10-2000 microns, preferably 50-500 microns, more preferably 150-250 microns. Due to the small diameter of the spots, a large number of different kinds of allergens and various concentrations of specific allergens can be immobilized on separate spots of the microarray chip, so that a microarray chip capable of analyzing a large number of allergens or a large number of allergen concentrations in one automated step can be provided.

Preferably the allergen is immobilized on a solid support, which may preferably be a glass support, a synthetic support, a silicon wafer, and a membrane, respectively. These chips can be produced according to the following methods, for example: ge, H. (2000), Nucleic Acids Research, 28, e3 (i-vii); qian W, et al, (2000), Clinical Chemistry, 46 (1456-; MacBeath G, et al, (2000), Science, 289, (1760-. The respective characteristics and advantages of these materials are well known to those skilled in the art. Therefore, the material used for the microarray chip will be selected according to the allergen to be detected, the method for detecting the bound form of immunoglobulin, the buffer used, and the like. In addition, economic issues are also a factor in selecting materials.

Preferably the microarray chip is chemically modified, preferably by amino-reactive modification and carboxy-reactive modification, respectively. This allows for accurate determination of the manner in which the allergen binds to the microarray chip.

Preferably the allergen is covalently bound to the microarray chip. This provides an especially reliable method of providing stable binding of allergens to the microarray chip.

In another preferred embodiment of the invention, the immunoglobulin is detected in serum as a sample. In patients allergic to allergens, immunoglobulins are mainly present in the serum, and thus analysis of the serum is a reliable method for detecting immunoglobulins. Furthermore, the serum of a patient can be easily collected by a simple route, and the collection of the sample can be performed even in the patient's home.

Preferably, the serum is diluted 1: 1 to 1: 15, preferably 1: 5. It can be diluted with any suitable solution, such as 1 XTSST (10mM Tris-HCl, pH8.0, 150mM NaCl, 0.5% TWEEN 20).

The sample is preferably incubated with the allergen for 1 minute to 24 hours, preferably 1-2 hours. Of course, the sample may also be incubated with the allergen for several days. But this is not signal strength or specificity. In general, an incubation time of 1 hour is sufficient to obtain reliable and sensitive results.

Furthermore, the sample is incubated with the allergen preferably between 0-60 ℃, preferably 37 ℃. Lower incubation temperatures do not increase signal, but may increase non-specific binding and background. Incubation at 37 ℃ gives accurate and reliable results for detection of human allergens.

Preferably, the bound immunoglobulin is detected by at least one labeled anti-immunoglobulin antibody. The term "antibody" as used herein refers to intact molecules as well as fragments thereof, such as Fa, F (ab) sub.2, and Fv, which all bind epitope determinants. Antibodies can be prepared using intact polypeptides and fragments that contain the desired small peptide as an immunogen. The polypeptide or peptide used to immunize an animal can be obtained by translation of RNA or can be chemically synthesized, if desired, by attachment of a carrier protein. Commonly used carriers for chemical coupling to peptides include bovine serum albumin and thyroglobulin. The coupled peptide is then used to immunize an animal (e.g., a mouse, rat, or rabbit).

In the context of the present invention, the term antibody refers to monoclonal or polyclonal antibodies, wherein monoclonal antibodies may be prepared by any technique that produces antibody molecules in continuous cell lines in culture. This includes, but is not limited to, hybridoma technology, human B-cell hybridoma technology, and IBV hybridoma technology. In addition, there are chimeric antibodies, single-chain antibodies and synthetic antibodies.

The antibodies can be labeled by any known method so that the bound antibodies can be characterized, and preferably quantified. Detectable labels suitable for use in the present invention include any composition detectable spectrophotometrically, photochemically, biochemically, immunochemically, electrically, optically, or chemically. Useful labels include biotin stained with a labeled avidin conjugate, magnetic beads, fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, Green fluorescent protein, etc.), radiolabels(e.g. using³H、¹²⁵I、³⁵S、¹⁴C or³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene latex, etc.) beads.

Methods for detecting such markers are known in the art. Thus, for example, a radioactive label can be detected by developed film or a scintillation counter, and a fluorescent label can be detected by a photodetector that detects the emitted light. Enzyme labels can typically be detected by providing a substrate to the enzyme and then detecting the reaction product of the enzyme with the substrate, while colorimetric labels can be detected by simply observing the colored label.

Preferably, the bound immunoglobulins are detected by at least one anti-immunoglobulin antibody which is fluorescently and radioactively labeled, respectively. These classical methods for the qualitative and quantitative analysis of bound antibodies are very specific and reliable.

Other preferred detection systems for microarrays are described, for example, below: schult K. et al, (1999), Anal Chem, 71 (5430-; Vo-Dinh T, et al, (1999), AnalChem, 71 (358-; brignac SJ Jr. et al, (1999), IEEE Eng Med Biol Mag, 18 (120-; otamiri M, et al, (1999), Int J Biol Macromol, 26 (263-268); wright, GL Jr. (2000), State Cancer and Static Diseases, 2 (264-); nelson RW, et al, (2000), Electrophoresis 21 (1155) -1163); RichRL et al, (2000), Curr Opin Biotechnol 11 (54-61); chen JJ. et al, (1998), Genomics, 51(313- & 324), the publication of which is incorporated herein by reference.

Preferably, one or more indoor allergens are immobilized as allergens. These may be, but are not limited to, Mites (Mites), tyr.put, lep.dest.or lep.mayrei, Felis, Bos, albumin, pen.cit., pen.not., asp.furigiatus, alt.alt., Malasseziafurfur, Latex (Latex), Plodia, Blatella.

In a more preferred embodiment of the invention, one or more outdoor allergens are immobilized as allergens. These may be, but are not limited to, Betula, Juniperus, Phleum, Parietaria judcea.

Further, one or more food allergens are immobilized as allergens. Such as selerie, karott, peanuts, apples, shrimp, fish.

Further, one or more poison allergens are immobilized as allergens. These may be, but are not limited to, bees or wasps.

Likewise, one or more auto-allergens are immobilized as allergens. These autoantigens may be, for example, liver cell membrane antigens, ssDNA antigens, skeletal muscle cell internal or surface antigens, and the like.

Another aspect of the present invention relates to a method for in vitro diagnosis of allergy in a patient, comprising taking a serum sample from the patient, analyzing the sample for immunoglobulins which bind to allergens according to the method of the invention as described above, in which method at least 10, preferably at least 50, more preferably at least 90 different allergens are immobilized on a microarray chip, and diagnosing allergy in a subject having a positive reaction between the sample and the immobilized allergens. The method for in vitro diagnosis of allergy as defined above has advantages compared to known methods for diagnosis of allergy. The method of the present invention is advantageous in that since all allergens to be tested are immobilized on one microarray chip, a large number of allergies can be simultaneously diagnosed using only one sample. Each step is carried out on the same chip, which also contains the spots of unfixed allergen, thus allowing a negative detection to be carried out simultaneously. The number of allergens to be tested is not limited, but two or more microarray chips may be used.

Another aspect of the present invention relates to a microarray chip having one or more allergens immobilized thereon. The above definitions and advantages apply equally to this aspect.

Preferably, the allergen is immobilized on a spot having a diameter of 100-.

Preferably the microarray chip is a glass carrier, a synthetic carrier, a silicon wafer, and a membrane, respectively.

Preferably, the microarray chip is chemically modified, preferably by an amino reaction and a carboxyl reaction, respectively.

Preferably, the allergen is covalently bound to the microarray chip.

Another aspect of the present invention relates to a kit comprising the above-described microarray chip of the present invention and a first reagent comprising at least one immunoglobulin detection reagent, preferably an anti-immunoglobulin antibody, preferably in a known concentration, and possibly a second reagent comprising as a positive sample at least one immunoglobulin capable of binding to an allergen. The first reagent comprises at least one immunoglobulin detection reagent, preferably an anti-immunoglobulin antibody useful for detecting bound immunoglobulin, wherein the anti-immunoglobulin antibody is preferably labeled as described above. Preferably the antibody of the first reagent is of known concentration, thereby providing reproducible results. Furthermore, the kit preferably comprises a second reagent as a positive sample comprising at least one immunoglobulin which binds to an allergen. Likewise, the immunoglobulin in the second reagent is preferably of known concentration, whereby the immunoglobulin in the sample can be quantified by comparing the results obtained for the patient sample with the results for a positive sample (second reagent).

Preferably, the kit is provided for use in the method of the invention as described above. To this end, the first and second reagents are designed to detect specific immunoglobulins which bind to a specific allergen or a specific allergy in a patient. The kit is also designed to detect multiple immunoglobulins that bind to multiple allergens in a patient or to diagnose multiple allergies.

The invention will now be described in more detail with reference to the following examples and the accompanying drawings, to which, of course, the invention is not restricted.

Drawings

FIG. 1 shows a schematic representation of an allergen microarray;

FIG. 2 shows a scanned image of serum from an allergic patient after allergen microarray analysis;

FIGS. 3a-3c show scatter plots from a primary triplet of analysis.

FIGS. 4a-4c show the% fluorescence intensity detected for the immobilized extracts and immobilized recombinant allergens.

Detailed Description

Example 1

Allergen spotting (Allergen spotting)

The allergens were aligned using a GMS 417 spotter from Genetic Microsystems. The protein was spotted onto a derivatized (derivanted) glass slide. Each allergen was spotted once on one dot for a total of 3 dots. The diameter of the dots (features) is about 200 microns.

The functional combination of allergens is as follows:

(A) outdoors (i. trees [1 ═ rBetv1, 2 ═ rBetv2, 3 ═ rBetv4, 4 ═ rJunO2, 5 ═ Cass1], ii. grasses [6 ═ rpllp 2, 7 ═ rpllp 4,8 ═ rpllp 5a, 9 ═ rpllp 1, 10 ═ rpllp 6, 11 ═ rpllp 7, 12 ═ rpllp 11, 13 ═ rpllp 12], iii. seeds [14 ═ rpinj 2, 15 ═ Artv1a, 16 ═ mogfilt profile ]),

(C) food allergens (x. vegetables [17 ═ Apig1, 18 ═ Apig1.0201, 19 ═ dauc1.2, 20 ═ rArah2, 21 ═ rArah5], xi. fruits [22 ═ Mald1, 23 ═ Mald2], xii. shrimps [24 ═ rPena1], xiii. fish [25 ═ recCarp ]),

(B) indoor (iv.mites [26 ═ rDerp1, 27 ═ rDerp2.0101, 28 ═ rDerp2, 29 ═ rDerp2b, 30 ═ rDerp5, 31 ═ rDerp5a, 32 ═ rDerp7, 33 ═ rDerp8, 34 ═ rDerp10, 35 ═ rTyrp2], v. animals [36 ═ rLepd2.01, 37 ═ rLepd13, 38 ═ eurrm 2.0101, 39 ═ rFeld1, 40 ═ rFeldla, 41 ═ rBosd2, 42 ═ representative allergens from dolicha, 43 ═ representative allergens from dog, 44 ═ BSA, 45 ═ allergen from rat, 56 ═ allergen from rabbit, 19 ═ allergen representative from rabbit, 52 ═ allergen from rabbit, 52 ═ allergen representative allergen from rabbit, 52 ═ allergen from pig ═ monkey, 52 ═ allergen representative allergen from pig ═ allergen representative allergen from rabbit, 52, rabbit-representative allergen from pig ═ monkey, rabbit-monkey-horse-representative allergen-horse-allergen-representative-allergen-horse-allergen-54, pig-allergen-representative-allergen, 58-Penn 13, 59-rAspf 1, 60-rAspf 3, 61-rAspf 4, 62-rAspf 6, 63-rAspfla, 64-rAspf 3a, 65-rAspf 4a, 66-rAspf 6a, 67-rAspf 2, 68-rAspf 8, 69-rastal, 70-rasta 2, vii-yeast [ 71-rMalf 7, 72-rMalf 1, 73-rMalf 5, 74-rMalf 6, 75-rMalf 8, 76-rMalf 8 ], viii-lactic acid [ 3677-henb ], blr 8, blr 3678, blr 8, herx 3679, hevrefb 72, hevrefb 3679, hevretf 3679, hevrefb 72, hevrelf 363678, hevre 3680, hevrefb 72, hevretfi 3678, hevrefm 3680, hevrefm 363678, hevrefm 3678, hevrefm 8478, hevrefm 3678, hevrefm 72, hevrefm 3678, hevrefm 3680, hevrefm 3678,

(D) poisons (xiv. bees [88 ═ Ag5, 89 ═ phospholipase a, 90 ═ hyaluronidase, 91 ═ rveasv 5, 92 ═ rveasg 5, XV. wasps [93 ═ Ag5]),

(E) auto-allergens (94 ═ HSA, 95 ═ a-NAC)

In addition, buffer dots are interspersed between allergen subgroups as background controls, indicated with "X" and "ab" as labeled antibody. (FIG. 1: 1864 × 1182 pixels, 1.86 × 1.18cm, 1000 pixels/cm, pixel depth/color 8/256, 1. pixels correspond to 10 microns).

A 100 microliter assay amount of allergen is dispensed into the wells of a 96-well microtiter plate at a concentration of about 200 ng/microliter in the spotting buffer (300mM sodium phosphate pH 8.5). The optimal concentration for immobilizing the allergen was calculated from titration experiments using allergen in several different buffers and different slides. When the concentration reached or exceeded 100 ng/l, the analyzed protein showed saturation and became a persistent intense white signal when scanned with a GMS scanner. At this concentration, the binding of the allergen is independent of the stored composition (of the storage) and the spotting buffer.

Example 2

SOPHIA (solid phase immunoadsorption test)

After incubation overnight, slides purchased from CEL a ssociates (aldehyde slides) or home-made were washed in Falcon tubes with shaking at ambient temperature using 1 × TBST (10mM Tris pH 8.0/150mM NaCl/0.5% Tween 20). The slides were then transferred to a1 × TBST solution containing 0.01% BSA and blocked at ambient temperature for 2 hours. The slides were then washed in 1 × TBST for 15 minutes and rinsed slightly with distilled water.

Allergen microarrays were incubated with diluted sera (diluted 1: 5 in 1 × TBST) at 37 deg.C for (at least) 60 minutes with shaking. Serum has been tested at various dilutions ranging from 1: 1 to 1: 15. The best results are usually obtained with a dilution of 1: 5. 30 microliters of diluted serum was added to a slide in a pressure-tight cassette (PressSeal Chamber) purchased from SIGMA Technologies. The use of the pressurized capsule is according to the protocol provided by the manufacturer. Incubation times with serum ranged from 60 minutes to overnight, and various incubation times within this range were analyzed. However, prolonged incubation with serum did not increase signal intensity or specificity. After incubation with serum, the slides were washed with 1 × TBST (15 min, ambient temperature) and shaken.

5 sera from different patients who had been tested by conventional diagnostic methods (skin prick test, RAST, ELISA) and 1 serum from a patient with non-hereditary allergy were selected for the baseline test of allergen microarrays. Each serum was analyzed at least 2 times with different batches of allergen microarrays.

Fluorescently labeled alpha IgE antibodies in 0.01% BSA/1 XTBST solution labeled with AlexaFluor fluorochrome according to the manufacturer's protocol were added to the allergen microarray and incubated for 60 min at ambient temperature. Depending on the time and efficiency of labeling, the working dilution concentration of antibody ranges between 1: 1000 and 1: 5000. After immunoassay, slides were washed with 1 × TBST for 15 minutes at ambient temperature and shaken, then rinsed slightly with distilled water.

After the immunoadsorption analysis, the slides were evaluated using a GMS two-color scanner. There is usually only a slight difference in signal intensity between different assays and different slides. But the type of slide used will cause a difference in background values. FIG. 2 shows an example of a scanned image of serum from an allergic patient after analysis with an allergen microarray. The patient had a strong response to Phleum, Mite, Felis and bees (see FIG. 1). No background signal due to buffer dots or nonspecific reaction of the autoallerogens was found. After adequate evaluation of the serum from the allergy sufferers, the results were compared with preliminary data obtained from testing the same serum using the RAST method. The results obtained by the method of the invention have good consistency with the existing data.

Example 3:

reproducible assay of serum from patient C

Serum from patient C was analyzed 3 times using separately prepared allergen microarrays. The experimental procedure was essentially as described above.

After analysis, slides were scanned using the same hardware setup. Data analysis was performed using the GenePix software package. The data from 3 replicates were calculated and the average of the signal intensity of 3 experiments for each allergen was compared.

Only the corresponding values of the signal which are at least 1.5 times higher than the mean value of the signal at the buffer point are used for the final analysis. Table 1 shows the mean, standard deviation and percent difference of the respective signals.

TABLE 1

Allergens	Detection 1	Detection 2	Detection 3	Mean value of	Standard deviation of	% average
							rBetv1rPhlp2rPhlp4rPhlp5arPhlp6rPhlp12rParj2Artv1 aMugwort-profilenmald 1HAS sheep albumin horse rAspf1(b) rMalf5rAK	207311717636134566005624462917552699779019890986898589602122161345920680	2132515529335115306851359700376787931766915295873282959061101291101814202	25060142272932855594376251228994441125892608176107081022210559107811203519978	22372.0015644.0032991.0055087.3348409.338527.678224.678728.678276.6711120.339769.339458.339740.6711042.0012170.6718286.67	1916.111206.672802.761485.777882.142675.50863.731828.70701.743033.74809.71835.92619.37871.771001.142902.48	8.567.718.502.7016.2831.3710.5020.958.4827.288.298.846.367.908.2315.87

The mean standard deviation calculated from the values obtained from the analysis of 3 independent experiments was 12.36%. This demonstrates that the chip-based immunological assay has good reproducibility for detecting IgE in patient serum. This can also be seen in the scatter plots obtained in 3 of the triple analysis, see figures 3a-3c, where figure 3a shows the scatter plot of test 1 versus test 3, figure 3b is the scatter plot of test 2 versus test 3, and figure 3c is the scatter plot of test 1 versus test 2; a represents an allergen and FI represents fluorescence intensity.

Example 4:

comparison of different microarrays

To test the best slide for use in the present invention, the slides prepared separately were evaluated according to the following criteria:

-a method of production: the prepared slides are evaluated according to parameters such as hazardous chemical content and production time.

-production costs: the cost of home-made and purchased slides were compared.

-binding capacity: different slides were evaluated for their ability to bind to fluorescently labeled proteins.

Repeatability: serial experiments were performed with slides subjected to different pretreatments, which were evaluated for overall analytical performance.

-overall background: before and after allergy analysis, all slides were evaluated for their systematic surface background, which is likely to reduce the signal/noise ratio.

-a detection limit: all slides were evaluated for the minimum protein concentration detectable at each spot in the allergy screening assay.

-serum tolerance: in general, patient serum is a complex mixture of proteins, and batches of patient serum often negatively interfere with microarray-based immunoassays.

-closing: all slides were evaluated for the necessity of a blocking step prior to allergy analysis.

-storing: after the allergen chips were prepared, all slides were evaluated for long-term storage.

Table 2 shows the results of the above evaluation study:

TABLE 2

Reactive chemical surface/slide type	Production of	Cost of	Binding capacity	Specificity of	Repeatability of	General background	Detection limit	Allergen screening	Blocking/coupling	Storage of
											ProteoBind	+	++	++	++	++	++	++	++	Whether or not	-
CEL(1)	/	++	++	++	-	-	+	++	Is that	++
											3D-Link(2)	/	--	++	++	+	+	+	-	Is that	-
Superaldehyde(1)	/	--	+	+	++	-	+	+	Is that	++
											Aminosifane(3)	+	++	+	+	--	-	+	+	Whether or not	++
Glyoxal	-	+	++	++	--	-	+	++	Is that	++
											EGS(3)	-	+/-	++	++	--	-	+	+	Whether or not	-
Sulfo KMUS(3)	-	+/-	++	+	/	-	/	/	Whether or not	/
											Glutaraldehyde(3)	-	+/-	/	/	--	-	/	/	/	++
Photocrosslinking agent (3)	-	+/-	+	+	/	-	/	/	/	/
											PEI/EGS(3)	-	--	++	+	+	+	/	/	/	/
FAST slide (4)	/	--	+	--	-	--	--	-	Whether or not	++
											CAST slide (4)	/	--	+	+	+	--	--	--	Is that	++
Unmodified glass	+	++	-	/	--	++	--	/	/	/

Table 2 shows the results of the evaluation study described herein. The following symbols and their meanings are (++) Excellent, (+) good, (-) poor and (- -) poor, respectively. (/) represents that the slide was not analyzed for this term. (1) A slide having a functional surface containing aldehyde groups. (2) The exact chemistry of the slide with the amino reactive surface is unknown. (3) The homemade slide glass has functional groups shown in Table 2. (4) A slide with a membrane required for protein immobilization. The surface derivative used therefor has the working name ProteBind, which has the best performance in microarray-based allergy diagnosis and comprises (1- [3 "- [ trimethoxysilyl ] propyl ] -1' (4" -isothiocyanatophenyl) thiourea), prepared according to the method of Chen et al (Nucleic Acids Research, 199, vol.27, No.2), which is incorporated by reference.

According to the above evaluation studies, ProteBind was selected as an excellent surface derivative for producing allergen microarrays.

Example 5:

comparison of detection of immunoglobulins in samples Using immobilized recombinant allergens with immobilized extracts

To determine the sensitivity of the purified recombinant allergens immobilized on the microarray and the allergen extracts immobilized on the microarray and to diagnostically relevant information, specific allergens of the same origin are compared with the allergen extracts.

After the allergen composition is immobilized on the microarray, different samples containing the specific serum are added to the microarray, and then fluorescence corresponding to the amount of allergen-bound antibody is detected. The results are shown in tables 3, 4 and 5 and FIGS. 4a, 4b and 4c, where "FL" represents% fluorescence. These results clearly indicate that more sensitive detection and quantification results can be obtained using recombinant allergens than using allergen extracts. The fluorescence intensity of the single recombinant allergen is significantly higher than that of the extract. Furthermore, unlike detection using recombinant allergens, the use of an extract only detects the source of the extract and cannot determine which specific antigen caused allergy.

Thus, the method of the invention using a single purified allergen, in particular a recombinant allergen, is superior to the method using an extract comprising an allergen.

TABLE 3

Chart 1	Serum 1-Sp	Serum 4-Sp	Serum 19-Sp	Serum 30-Sp
					Betv1a	52028	62529	62932	18847
Betv1a Mut	6590	237	905	139
					Betv1d	1576	14352	252	89
Betv2	2196	1408	273	1253
					BP-extract	21013	39438	11390	3174

TABLE 4

Chart 2	Serum 5-Sp	Serum 7-Sp	Serum 9-Sp	Serum 17-Sp	Serum 18-Sp	Serum 40-Sp
							PhlpI	45611	12148	21588	5838	23497	23424
PhlpII	209	52043	814	2562	10984	14753
							PhlpV	64005	63481	62953	40770	63530	62029
Phl-extract	63987	40318	14504	10183	44405	19798

TABLE 5

Chart 3	Serum 11-Sp	Serum 16-Sp	Serum 39-Sp	Serum 40-Sp
					Hevb3	416	387	108	235
Hevb5-Mal	350	519	30601	63897
					Hevb8	11146	7977	134	853
Hevb9	460	631	105	188
					Hevb10	360	429	73	152
Latex B-extract	264	202	80	3426
					Latex C-extract	769	866	5343	30970

Claims (51)

1. A method for detecting an immunoglobulin binding to an allergen in a sample, characterized in that one or more purified allergens are immobilized on a microarray chip and the sample is incubated with the immobilized allergens, whereby immunoglobulins specific for the allergens bind to the specific allergens and the immunoglobulins bound to the immobilized specific allergens are detected.

2. The method according to claim 1, characterized in that the immunoglobulins detected are IgE.

3. The method of claim 1, characterized in that the detected immunoglobulin is IgG.

4. The method according to any one of claims 1 to 3, characterized in that one or more recombinant allergens are immobilized on the microarray chip.

5. The method according to any one of claims 1 to 3, characterized in that one or more synthetic allergens are immobilized on the microarray chip.

6. The method according to any one of claims 1 to 3, characterized in that one or more haptens are immobilized as allergens on the microarray chip.

7. The method according to any one of claims 1 to 3, characterized in that the allergens are immobilized on spots of 10 to 2000 μm diameter on the microarray chip.

8. The method according to any one of claims 1 to 3, characterized in that the allergens are immobilized on spots on the microarray chip having a diameter of 50 to 500 microns.

9. The method according to any one of claims 1 to 3, characterized in that the allergens are immobilized on spots on the microarray chip having a diameter of 150 and 250 μm.

10. The method according to any one of claims 1 to 3, characterized in that the allergens are immobilized on a solid support used as the microarray chip.

11. The method according to any one of claims 1 to 3, characterized in that the allergens are immobilized on glass and synthetic carriers, respectively, which are used as the microarray chip.

12. The method according to any one of claims 1 to 3, characterized in that the allergens are immobilized on a silicon wafer used as the microarray chip.

13. The method according to any one of claims 1 to 3, characterized in that the allergens are immobilized on a membrane used as the microarray chip.

14. The method according to any of claims 1 to 3, characterized in that the microarray chip is chemically modified.

15. The method of claim 14, characterized in that said chemical modification is an amino reactive modification and a carboxyl reactive modification, respectively.

16. The method according to any one of claims 1 to 3, characterized in that the allergens are covalently bound to the microarray chip.

17. The method according to any one of claims 1 to 3, characterized in that the immunoglobulins are detected in a serum sample.

18. The method according to claim 17, characterized in that the serum is diluted 1: 1 to 1: 15.

19. The method according to claim 17, characterized in that the serum is diluted 1: 5.

20. The method according to any one of claims 1 to 3, characterized in that the sample is incubated with the allergen for 1 minute to 24 hours.

21. The method according to claim 20, characterized in that the sample is incubated with the allergen for 1-2 hours.

22. The method according to any one of claims 1 to 3, characterized in that the sample is incubated with the allergen at 0-60 ℃.

23. The method according to claim 22, characterized in that the sample is incubated with the allergen at 37 ℃.

24. The method according to any one of claims 1 to 3, characterized in that the bound immunoglobulins are detected with at least one labeled, specific anti-immunoglobulin antibody.

25. The method according to claim 24, characterized in that the bound immunoglobulins are detected with at least one fluorescently labeled, specific anti-immunoglobulin antibody.

26. The method according to claim 24, characterized in that the bound immunoglobulins are detected with at least one radiolabeled, specific anti-immunoglobulin antibody.

27. The method according to any one of claims 1 to 3, characterized in that one or more indoor allergens are immobilized as allergens.

28. The method according to any one of claims 1 to 3, characterized in that one or more outdoor allergens are immobilized as allergens.

29. The method according to any one of claims 1 to 3, characterized in that one or more food allergens are immobilized as allergens.

30. The method according to any one of claims 1 to 3, characterized in that one or more venom allergens are immobilized as allergens.

31. The method according to any one of claims 1 to 3, characterized in that one or more auto-allergens are immobilized as allergens.

32. A method for in vitro diagnosis of allergy in a patient, characterized in that a serum sample is taken from the patient, after which the sample is analyzed for allergen-binding immunoglobulins according to the method of any one of claims 1-31, wherein a microarray chip is used, on which at least 10 different allergens are immobilized, after which a positive reaction between the sample and the immobilized allergens is diagnosed as allergy.

33. The method of claim 32, wherein at least 50 different allergens are immobilized on the microarray chip.

34. The method of claim 32, wherein at least 90 different allergens are immobilized on the microarray chip.

35. A microarray chip on which one or more purified allergens are immobilized.

36. The microarray chip according to claim 35, characterized in that the allergens are immobilized on spots having a diameter of 100-500 μm.

37. The microarray chip according to claim 35, characterized in that the allergens are immobilized on spots having a diameter of 200-300 μm.

38. The microarray chip according to any of claims 35 to 37, characterized in that it is a glass carrier.

39. The microarray chip according to any of claims 35 to 37, characterized in that it is a synthetic support.

40. The microarray chip according to any of claims 35 to 37, characterized in that it is a silicon wafer.

41. The microarray chip according to any of claims 35 to 37, characterized in that it is a membrane.

42. The microarray chip according to any of claims 35 to 37, characterized in that it has been chemically modified.

43. The microarray chip according to claim 42, wherein the chemical modification is an amino-reactive modification and a carboxyl-reactive modification, respectively.

44. The microarray chip according to any of claims 35 to 37, characterized in that the allergen is covalently bound thereto.

45. A kit characterized by comprising the microarray chip of any one of claims 35-44 and a first reagent comprising at least one immunoglobulin detection reagent.

46. The kit of claim 45, wherein the immunoglobulin detection reagent is an anti-immunoglobulin antibody.

47. The kit of claim 45, wherein the concentration of the immunoglobulin detection reagent is known.

48. The kit of claim 46, wherein the concentration of the anti-immunoglobulin antibody is known.

49. The kit according to any one of claims 45 to 48, characterized in that it further comprises a second reagent comprising, as a positive sample, at least one immunoglobulin binding to an allergen.

50. The kit of any one of claims 45 to 48 for performing the method of any one of claims 1 to 34.

51. The kit of claim 49 for performing the method of any one of claims 1-27.