TW200525150A - Microarray chip for detecting immunoglobulin - Google Patents

Abstract

The present invention provides a microarray chip, which is capable of detecting allergen and quantitatively detecting immunoglobulin. The microarray chip comprises a solid slide, a reacting layer, which is disposed on the solid slide, and at least one sample allergen or substances, which can combine with immunoglobulin, being disposed on the reacting layer. In addition, the present invention also provides a method for detecting allergen by means of this microarray chip. The method utilizes monoclonal antibody as secondary antibody to reduce non-specific combination and uses enzyme reaction as a signal-amplifying system to quantitatively detect immunoglobulin. The microarray chip and the detecting method of the invention can complete more tests in shorter time and can reduce cost while testing. Furthermore, the present invention can proceed with a quantatative and a semi-quantatative determination, so that has a greater market potential.

Description

200525150 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to the * wafers for detecting allergens in allergic patients, and in particular, it can be used for quantitative or semi-quantitative detection in a very small number of specimens. Concentrations of antibodies related to allergies such as IgE or IgG, and microarray biochips that can screen a large number of allergens. [Previous technology] Allergy (Allergy, Hypersensitivity) refers to the human immune system's excessive response to certain innocuous substances in the environment, and because of these abnormal immune reactions, the human body appears allergic symptoms, even severe symptoms May be life threatening. The most common type of allergic reaction in general is Type I hypersensitivity (Immediate Hypersensitivity Reaction). The mechanism of this allergy is that when a person is first exposed to an allergen, the body's immune system produces specific IgE antibodies to the allergen, and attaches to the body's obese cells (Mast cells) or basophils (Basophile) )surface. When in the environment with this allergen again, allergens bind to specific IgE antibodies to 'activate obese cells and basophils, and produce certain mediators that can cause reaction symptoms, such as Histamine, Leukotrienes and other substances. These media may cause the contraction or expansion of blood vessels and bronchial tubes, or attract some inflamed cells to move to the inflamed area ’and cause allergic symptoms such as sneezing, runny nose, skin rash, eye discomfort, and shortness of breath. In addition to the aforementioned allergic mechanisms, IgG and IgM antibodies are also antibodies that are related to the allergic mechanism. These 200525150 specific antibodies issued by certain antigens may cause the body to cause allergic symptoms through different immune responses. . In recent years, patients who have allergic symptoms seem to have a rising trend. With mouths 4 and 5, nearly one-third of them have allergies, and produce, for example, food allergies, allergic conjunctivitis, and skin allergies. , Rhinitis and asthma. Because allergies can cause a considerable degree of physical discomfort, affecting the quality of daily life, severe symptoms may cause death. Therefore, if patients can know the specific allergens that cause their own allergic reactions, on the one hand, they can avoid exposure to these allergens in their daily lives and reduce the chance of causing allergic symptoms. On the other hand, if these specific allergens can be accurately detected, they can be applied to hyposensitization therapy (Hyposensitization), which can reduce or even eliminate allergic symptoms in patients. Conventional allergen detection methods generally use skin testing or serum testing to determine the concentration of allergy-related antibodies in the blood. The skin acupuncture test uses a small amount of test allergen to be injected subcutaneously into the subject, and then measures the size of the swelling or inflammatory reaction zone on the skin. Although this method is highly accurate, it often causes discomfort to the subject. On the other hand, testing multiple allergens can take considerable time. Furthermore, there is still a risk of triggering a systemic allergic reaction in the subject. As for serum testing, immunoassays are generally used to determine the concentration of allergy-related antibodies in the blood, so that the degree of allergy to a specific allergen can be determined. Commonly used methods such as enzyme-linked immunosorbent assay 200525150 (Enzyme-linked immunosorbent assay, ELISA), radioimmunoassay (Ria), fluorescence immunoassay (FlU0resent immunoassay, FIA) or chemiluminescent analysis (Chemiluminescent assay, CLA) and other methods. With these methods, the total IgE concentration, specific IgE, or specific IgG concentration in the blood can be detected. Disadvantages of the above-mentioned detection method are that a large amount of serum is required, especially when a large number of different types of allergens are screened at the same time, more specimens are required. In addition, the yuancheng test takes a long time and the operation steps are complicated. Although the US patent application US 2003/0109067 proposes a method that can quantitatively measure allergens using micro-samples, the method of labeling allergens is very complicated, so it will be difficult to apply if a large number of allergens are detected at the same time. At the same time, the time it takes to analyze the data will increase significantly. Biochips are also used in the detection of allergens because they have the characteristics of a small amount of specimens and can be used to detect or screen a large number of samples at the same time. For example, WO 02/29415 and US 2003/0073249 both disclose wafers that can detect allergens. The wafer detection method disclosed in WO 02/29415 is a detection system of wafer-allergen-test immunoglobulin _ fluorescent antibody = grade antibody, and then read and test immunoglobulin Fluorescent signal on bound secondary antibody. As for us 20003 / 〇〇73249, it is the use of tablets-allergens-immunoglobulins to be tested-two labeled with biotin (Bi〇Un): antibodies-streptavidin labeled with fluorescent substances Detection system of streptavidin (str ^ tavidi, and then read the fluorescent signal on streptavidin. = Yes, both methods can only be used for qualitative interpretation, and cannot be determined exactly Measure the concentration of immunoglobulin. That is to say, only the subject can be measured, and an allergen produces an allergic reaction, and it is impossible to determine the degree of allergy by quantitative measurement. $ 200525150 For clinical detection or treatment Individuals have very different types of allergens or the degree of allergies. A clear understanding of the condition of allergic patients will help improve the accuracy of medication or determine whether it is suitable for desensitization therapy. Based on As mentioned above, there is an allergen detection method that can provide more and more accurate test data, including a larger number of allergen tests and the degree of allergy to allergens, which requires only a small number of specimens and can be completed in a short time. Its necessity [Summary of the Invention] One of the objectives of the present invention is to provide a microarray wafer. The basin can be used to detect allergens and quantitatively detect immunoglobulins. The microarray wafer can use a small number of specimens and test a large number of allergies at the same time. (1 () 25 ^ specimen can detect more than 150 antigens), and can be used to qualitatively or quantitatively detect allergy-related antibodies in serum. Another aspect of the present invention is to provide a method for manufacturing microarray wafers. The method of the invention can be used for the qualitative or quantitative detection of allergy-related antibodies in serum. Another object of the present invention is to provide a method for detecting allergens by using microarray wafers. A small number of specimens are required to detect allergens in the salamander. In addition, this method requires a short time, can complete the detection of 96-150 allergens in 1 hour, and can be detected qualitatively or definitely. The concentration of allergy-related antibodies in the specimen to determine: two:

Degree of allergy to allergens. X The secondary antibody system described herein refers to another antibody that can bind to the immunoglobulin to be tested 200525150. The microarray chip for detecting allergens according to the present invention includes a solid slide 'which is used to carry the allergen to be tested; a reaction layer, which is above the basin solid slide, which contains amine groups which can interact with proteins: = : And at least one allergen to be tested or a substance that can be bound to the immunoglobulin to be tested, which can be immobilized on the solid slide through the reaction layer. The microarray crystal is m- or quantitative Determination of allergy-related antibodies in the test object.

The solid slide used in the present invention may be glass, plastic or metal, and preferably glass. The allergen used in the present invention is not particularly limited, and can be used for allergen detection in allergen detection, or specific allergens for the sample to be tested. If the substance immobilized on the reaction layer is a substance compatible with the immunoglobulin to be tested, it can be used to detect the non-white concentration of a certain material in the test sample. This kind of substance that can be bound to the immunoglobulin to be tested only needs to be:

It is sufficient to include a portion that can bind to the immunoglobulin to be tested, preferably: ... the immunoglobulin to be specifically bound, such as an antibody against the immunoglobulin to be tested. In addition, the microarray wafer of the present invention may be provided with a dot matrix arrangement containing a plurality of over-primitive elements, and the dot density W -... may be 300-484 dots / cm2. An allergen / cm2 was measured at a weight of 2_3. It can be at least the same as that of the method for manufacturing a microarray wafer of the present invention. At least the steps include: firstly preparing a solution containing at least one allergen of a predetermined concentration or a substance that can be tested; 10 200525150 epidemic globulin-binding substance solution; In addition, a solid slide of a predetermined size is prepared; a reaction layer containing an amine group that can interact with the allergen is provided on the solid slide; a solution of the allergen or a substance capable of binding to the immunoglobulin to be tested Lattice the reaction layer according to a predetermined lattice density; cause the allergen or a substance capable of binding to the immunoglobulin to be tested to interact with the reaction layer and be immobilized on the solid slide; and make the reaction layer not contact with Allergens or functional groups that can react with the substance to be tested for immunoglobulin do not react. In the above manufacturing method, a dot matrix machine (Instnmentation for printing microai: rayS) can be used to dot the allergen solution on the reaction layer to accurately prepare the microarray wafer. Depending on the type of allergens, the appropriate temperature and humidity conditions can be used during the dot matrix. The preferred humidity range is 40-90% relative humidity. When the allergen reacts with the reaction layer, the allergen can be immobilized on the solid slide for a period of time under high humidity. After the aforementioned allergen immobilization step, a commonly known blocking buffer, such as PBST buffer (containing Tween 20 phosphate (PBS)) containing BSA (bovine serum albumin) can be used. Buffer) at room temperature to 42. Contact with the reaction layer for 15 minutes to 1 hour, so that the functional groups that have not reacted with the allergen are not reacted. When using the microarray wafer of the present invention for allergen detection, it includes at least the following steps: • A microarray wafer with an allergen immobilized on the surface is provided, and the test solution is brought into contact with the microarray wafer to perform the allergen and to be treated. Binding reaction of the test antibody in the test solution; after a predetermined time of reaction, remove the unreacted components in the test solution; make the test antibody and a secondary antibody that can bind to the test antibody 11 200525150 body In combination, this secondary antibody is linked to a site that can be combined with the signal generating unit 70; after a predetermined period of time, the secondary antibody that has not participated in the reaction is removed; and then the site that can be combined with the signal generating unit is made Combining with the howling generating unit; after a predetermined time of reaction, removing the Λ generating unit that is not involved in the reaction, and then causing the signal generating unit to generate a signal; and measuring the signal to determine the concentration of the test antibody. When the microarray wafer of the present invention is used to detect allergens, the secondary antibody can be a single antibody or multiple antibodies, preferably a single antibody, because a single antibody can be unitarily combined with a test antibody, so It can avoid the noise caused by non-uniform binding when using multiple antibodies. In addition, the portion of the secondary antibody that can be combined with the signal generating unit may be biotin (BiUn). The signal generating unit includes a part that can be linked to the secondary antibody (if the site that can be combined with the signal generating unit is biotin, then Streptavidin can be used as the link with biotin Part) and the part that generates the signal. The signal generating part is preferably an enzyme, such as Hydroperoxidase (HRP), Alkaline phosphorase (AP) or β-galactosidase (^ Galact〇sidase). Light substances (such as Alexa Flour 647, Alexa Flour * 546, Alexa Flour 532, Cy3 or Cy5, etc.) and can perform enzyme reactions with the aforementioned substrate Γ reaction 'can react the test antibody in the test solution with the allergen The signal is amplified, making quantitative measurement possible. Among them, the fluorescent substance is preferably labeled with Alexa fluorescent dye as the matrix, because its quantum yield is high. The schematic diagram in the first figure can more clearly show the above-mentioned measuring method 12 200525150 ^. : First obtain the micro P column wafer (1) with the allergen (2) immobilized on the surface as shown in the first figure; then, as in the first figure (b), contact the liquid to be tested with the surface of the microarray wafer (1), The IgE antibody (3) contained in the specimen is bound to the specific substrate (2) on the surface of the wafer; as shown in Fig. ⑷, it can be combined with ㈣ antibody⑺ grade antibody (41) and IgE antibody (3). ), And the secondary antibody ⑼ has a site (42) that can be combined with the signal generating unit; then, as shown in the second figure Γ ', the second part (42) that allows the signal molecule to be linked to the site (42) ) Combination, the signal molecule produced by this method is marked with enzyme ⑼; as shown in Figure ，, it will react with enzyme and marked with glory substance = (6) and react with enzyme (52) to generate glory signal; Light blunts and calculates the concentration of IgE antibody (3). Qiao Shi! ^ Said 'In order to be able to determine the monoclonal antibody and allergen-producing monoclonal antibodies in the test solution: this test is possible. Xi Zhong's signal amplification system makes the quantitative detection in the following specific embodiments and the attached drawings ::: Solution: Yes ": The embodiments and drawings described are not intended to be within the scope of this artist. Without departing from the spirit and advantages of the present invention, the scope of protection of the present invention is determined by the scope of the appended patent application. [Embodiment] An allergen-containing egg protein solution can be prepared in advance using commercially available: J 2 allergens with allergens, or 13 200525150 allergen powder can be obtained, and the extraction solution can be obtained through the extraction step. Depending on the allergen, the concentration of the protein solution with 3 allergens was adjusted to 50 pg / mM 00 mg / ml. This solution can be added with 50% glycerol to facilitate subsequent immobilization steps and storage. In addition, a glass slide is used as a solid slide, and an amino-terminal silane containing an amine group is coated on the glass solid slide as a reaction layer that reacts with an allergen. The dot matrix machine was used to dot the above allergen-containing solution on a glass solid slide coated with an amine silane compound. The diameter of each dot was about 150 μηι, and the dot pitch was about 300 μπ1, which could be set according to actual needs. The number of points can be at least 300-484 points / cm2. The temperature of the dot matrix is room temperature, and the humidity is 40-90% relative to the degree. After the completion of 放置, put a section under high fishing degree = the immobilization of the allergen, and then add the pbst buffer containing 3% BSA to react with the residual amine group of the reaction layer for 15 minutes to 丄 hours, and the invention can be obtained Microarray wafer. Example 2 Zhao · The microarray crystal of the present invention is used to prepare a microarray wafer according to the method of Example 1. The serum sample (the dilution factor can be 2-10 times) and the microarray are prepared. The wafer is contacted and the IgE antibody in the specimen is bound to the allergen at a temperature of room temperature to 42 ° C. After 15 minutes to 丄 hours of use, use a washing buffer (such as buffers such as pBs, PBST, and TBST (Tris phosphate)) to remove the unbonded portion. Next, an anti-IgE unibody linked with biotin (tin) was added to bind to the IgE antibody. After 15 minutes to i hours of action, the unbound anti-IgE monoclonal antibody was removed using a washing buffer. After that, 14 200525150 key avidin (Streptavidin) linked with hydroperoxidase (HRp) was added to bind to biotin. After 15 minutes to i hours of action, the unreacted HRP_streptavidin was removed with a washing buffer. Tyramide bonded with Aiexa 647 was added as a matrix of HRP, and a color reaction was performed. After the color reaction is completed, the excess substrate is washed away, the microarray wafer is blown dry, and the fluorescence signal is read with a laser conjugate focus scanner (eg, GenePix4000B). Depending on the allergens and specimens tested, the time to complete the test can be completed in about 1.5-4 hours. The calibration curve of the IgE antibody is determined, and the analysis result of each point is determined by the calibration curve. In Example 3, the total IgE of the blood line of the present invention was divided and divided into the total IgE of Tang Xuehao. The microarray wafer was prepared according to the method of Example i, but a plurality of anti-Eb antibodies were latticed on the reaction layer of a solid slide. Next, IgE solutions of different concentrations were measured with this microarray wafer. During the measurement, except that the sample's partial injury was replaced with the human IgE antibody standard solution, the rest of the steps were the same as in Example 2. The concentration of the human IgE antibody standard solution used for the test was 5, 1, 100, 500, and 2500 IU / Furthermore, serum samples W23 and rhenium at concentrations of 1522 m / mi and 12.8 IU / ml, respectively, were tested by a commercially available testing machine (UniCAP-100 of Pharmacia Diagnostics, Sweden) as positive and negative serum samples. : As shown in the second figure, the R value (regression coefficient) of the detection line obtained by using the microarray wafer of the present invention is 0 983301, so it can be used as a calibration curve. In addition, the fluorescence intensity of W23 and Rhenium measured on a microarray chip was compared with the calibration line of 15 200525150. It was found that these two points did not deviate from the calibration line, and the fluorescence signal ratio between them and UniCAP The obtained ratio of -100 is approximate. Example 4 Invented microdisplay wafers and commercially available machines were tested against the semi-volume grade of Dexgiotophagoides farinae, and the semi-quantitative grades referred to here are compared with commercially available machines (Swedish Pharmacia Diagnostics UniCAp_1〇〇)

The results obtained by the microarray wafers are semi-quantitatively divided into grades. A microarray wafer was prepared according to the method of Example 1, but the dust snail allergen extract was dot-arrayed on a solid slide. According to the method of Example 2, 32 microspheres containing this dust mite allergen were used to detect 32 serum. In addition, the same serum samples were detected using a commercially available machine (UnlCAP_lG0 of the Swedish pharmacia Diagnoses company) and the same allergen. Regression analysis was performed on the measured values of the two aforementioned measurement methods.

The results are shown in the third figure. The 32 serum samples were measured. The r value (regression coefficient) of the two serum samples was 0.90447, indicating that there is a very high correlation between the two detection methods. The two test results are comparable

It is proposed that the method provided by the present invention for Lüshi and L 7 branches can be used as a semi-quantitative grade analysis. Realize "5 J Don't use the main film and commercially available machines for house dust $ (e ^^^^^^ Elgronvssiniis, over the original implementation of semi-fixed feces and worms according to the method of Example 1 to prepare a microarray allergen extract The wafers are listed on the solid slide, but they are house dust. According to the method of Example 16 200525150 of Example 2, this microarray wafer containing house dust mite allergens was used to detect 32 serum. In addition, a commercially available machine (Swedish Pharmacia) UniCAMOO (Diagnostics) and the same allergen detected the same serum specimens. Then the regression analysis was performed on the measured values of the two previous measurement methods. The results are shown in the fourth figure: 'The 32 gold specimens, The r value (regression coefficient) of the two is 0.93799, which indicates that there is a relatively high correlation between the two detection methods. For the determination of this allergen, the two test results can be compared to 'the method provided by the present invention' It can be used as a semi-quantitative level analysis. Example 6 and the use of this hair film and commercially available machines to measure the main hair volume level of 赦

A microarray wafer was prepared according to the method of Example 1, except that the tropical five-claw snail allergen extract was dot-arrayed on the solid slide 1. Thirty-two sera were detected according to Example 2 using such a microarray wafer with three tropical claw allergens. In addition, the same serum samples were detected using commercially available equipment and the same allergens. Regression analysis was performed on the threshold values of the two aforementioned measurement methods. The results are shown in the fifth figure. The 32 serum samples were measured.

Value coefficient) is 0.9925, indicating that there is a relatively high correlation between the two detection methods. For the determination of this allergen, the results of the two tests can be compared with the methods provided by this local Mingming, and can be used as semi-quantitative Hierarchical analysis. "In addition to the actual yoke example, commercially available equipment UniCAP, MAST master) ', Ben & Ming's microarray chip to determine the allergen, the f' monthly testing costs, whether the total IgE concentration can be measured Table 1 lists whether the quantitative analysis and the time required for testing can be compared as shown in Table 1. It can be found that compared with a commercially available machine, the 17 200525150 microarray chip provided by the present invention can save a lot of money in a shorter test. The Λ-S test enables the determination and simultaneous detection of the surnamed fruit I and: advance determination: and semi-quantitative measurement of each fruit — highly correlated with commercially available machines. In practical use, it is extremely Market Potential. The testing of allergens and microarray wafers provided by the present invention is simple and simple. On the other hand, the method for detecting allergens provided by the present invention can be used. A very small number of serum samples can screen a large number of allergens in a short period of time, and can provide quantitative or semi-quantitative results, so it can provide more accurate test results for individual allergies.

18 200525150 Table 1. UniCAP, MAST, and microarray chips of the present invention.

Comparative item The serum volume required for the UniCAP MAST of the microarray wafer of the present invention is 25μ1 / 96-150 tests 150μ1 / 1 tests 1300μ1 / 36 tests The test cost is about NT $ 110/96 tests about NT $ 180/1 tests; 96 Kinds of test estimates: about NT $ 17280, about NT $ 1500/36 tests; 96 kinds of tests, estimates: about NT $ 4500, whether total IgE concentration can be measured, whether quantitative analysis is possible (quantitative and semi-quantitative), yes (quantitative) And semi-quantitative) The time required for semi-quantitative detection is 1.5-4 hours / 96-150 kinds of tests 8 hours / 88 kinds of tests> 16 hours / 36 kinds of test formulas 19 200525150 [Schematic description] The first picture is using this Schematic diagram of the invention allergen microarray wafer detection of allergens. The second figure shows the use of a microarray wafer of the present invention to establish a calibration curve and analyze the total IgE concentration in serum. The first figure is a semi-quantitative special-level measurement of a dust mite (Dermotophagoides fadnae) allergen using a microarray wafer of the present invention and a commercially available machine, respectively. The fourth figure is a semi-quantitative level measurement of the house dust mite (Dermota Phageides pteronayssinus) using the microarray wafers and commercially available machines of the present invention. The twenty-fifth figure is a semi-quantitative level measurement of the thermal I-allergen (Blomia tropiCais stolige) using a microarray wafer of the present invention and a commercially available machine, respectively. [Explanation of component number] 2 Allergen 41 Secondary antibodies

1 Microarray chip 3 IgE antibody 42 A site that can be combined with a signal generating unit 51 A site that can be connected to site (42) 52 An enzyme 6 A substrate labeled with a fluorescent substance 20

Claims (1)

200525150 Scope of patent application: 1 · A slave array chip, which is used to detect allergens and to quantitatively measure immunoglobulins, which includes: a solid slide, which is used to carry the allergen to be tested; The reaction layer is disposed on the solid slide and contains an amine functional group; and at least one test allergen or a substance capable of binding to the test immunoglobulin is fixed by the reaction layer. The microarray chip can be used for qualitatively or quantitatively determining allergy-related antibodies in the test object. 2. The microarray wafer according to item i of the patent application scope, wherein the solid slide is glass, plastic or metal. 3. The microarray wafer according to item i in the scope of the patent application, wherein the binding reaction between the substance capable of binding to the immunoglobulin to be tested and the immunoglobulin to be tested is a specific binding reaction. 4. The microarray wafer according to the first item of the scope of the patent application, wherein the substance capable of binding to the immunoglobulin to be tested is an antibody against the immunoglobulin to be tested. 5. A method of manufacturing a microarray wafer, wherein the microarray wafer is used to detect allergens and to quantitatively detect immunoglobulins, the method includes at least the following steps: U) preparing at least one allergen solution containing a predetermined concentration Or a substance capable of binding to the immunoglobulin to be tested; (b) preparing a solid slide of a predetermined size; 21 200525150 ⑷ placed on the solid slide and provided with an amine group, which can interact with the allergen or with the test A reaction layer for the reaction of the immunoglobulin-bound substance; (d) a solution of the allergen 4 substance capable of binding to the tritiated immunoglobulin on the reaction layer according to a predetermined lattice density; A substance capable of binding to the immunoglobulin to be tested reacts with the reaction layer and is immobilized on the solid slide; and does not react the functional groups on the reaction layer that have not undergone the reaction of the europium. 6. The method for manufacturing a microarray wafer according to the scope of application patent (5), wherein the lattice density is at least 3GG-484 dots / cm2. 7. The method for manufacturing a microarray wafer according to item 5 of the scope of patent application, wherein step (d) is performed by a dot matrix machine. 8. The method for manufacturing a microarray wafer according to item 5 of the scope of patent application, wherein the relative humidity at the step (d) of the matrix is 40.9%. 9. The method for manufacturing a microarray wafer according to item 5 of the scope of the patent application, wherein the step (f) uses a blocking buffer solution (51, also called ^ to contact the reaction layer, so that the The functional group performing the reaction of step (e) is not reacted. Melon-A method for detecting allergens or quantitatively detecting immunoglobulin by using a microarray chip, the method includes at least the following steps: (a) providing a surface fixation A microarray wafer having an allergen or a substance capable of binding to the immunoglobulin to be tested; 〇 >>) contacting the test solution with the microarray wafer, and performing a substance binding to the immunoglobulin to be tested with The test solution in the test solution 22 200525150 should be used; After removing the unreacted immunoglobulin-associated binding reaction (C) reaction in the test solution for a predetermined time, divide; (d) Make the test immunoglobulin clean. One ☆ scholar can be combined with the secondary antibody bound to the immunoglobulin to be tested. Protein ^ ^ 4-Grade antibody is linked to a site that binds to a discriminating signal generating unit; /, (e) Reaction is scheduled After time, remove% grass Two anti-reaction of the thigh (f) may be such that the signal generating unit with last name 'means the combination;, - ... and bit signal generating ⑷Reaction-after a predetermined time, remove the non-reactive elements; (h) make the signal generation unit generate a signal; and (1) measure the signal to determine the concentration of the antibody to be tested. 11. If the application of the patent No. 10 of the brake field throw #, said the method of using the microarray chip to detect allergy 3 or quantitative detection of immunoglobulin ^, + ^ f method, wherein the secondary antibody is the body Strain antibodies. 12. For example, a method for detecting allergens or quantitatively detecting immunoglobulins using a microarray chip as described in the patent application No. 1G, wherein the site that can be combined with the signal generating early element is biotin. 13. The method of detecting allergens or determining immunoglobulins using a microarray chip as described in item 12 of U.S., wherein this step is combined with streptavidin and the biotin. 14. A method for detecting allergens or quantitatively detecting immunoglobulins using a microarray chip as described in the scope of the patent application (1G), wherein the signal Yansheng unit contains an enzyme. 23 200525150 15. The method for detecting allergens or immunoglobulins using microarrays, or tablets, as described in item 14 of the scope of patent application, 1 by ^, where the enzyme is selected from the group consisting of hydro-rolling enzymes (Hydroperoxidase HRP, κρ), alkaline phosphatase (AP), or β-galactosidase (β-Galactosidase). 16 · A method for detecting allergens using a material array, a tooth whistle, and a 锨 array chip as in item 10 of the scope of patent application, wherein (g) the method of generating a signal is by an enzyme reaction. 17. The method for detecting allergens or quantitatively measuring immunoglobulins by using a microarray wafer according to the scope of application patent No.16, wherein the substrate used for the enzyme reaction is a substrate labeled with a fluorescent dye. 18 • The method for detecting allergens or quantifying m. Pylori using microarray wafers according to the scope of patent application (17), wherein the fluorescent dye is one of Alexa fluorescent dye, Cy3 or Cy5. 19. A method for detecting an allergen using a microarray chip as claimed in item 17 of the patent scope, wherein the Alexa fluorescent dye is one of Alexa F10ur 647, Alexa Flour 546, or Alexa Flour 532. twenty four