KR102070794B1 - Method to diagnose allergy by determination of Immunoglobulin E using an immunoassay based on based on enzyme-mimicking nanozymes - Google Patents

Abstract

The present invention relates to an allergy diagnostic enzyme-mimicking nanoparticles, lateral flow immunochromatic chip using allergy diagnostic enzyme-mimetic nanoparticles, and a manufacturing method, and an allergy diagnostic Immunoplate method and kit using allergy diagnostic enzyme-mimetic nanoparticles. In order to achieve the above object, an aspect of the present invention for diagnosing allergy, characterized in that the first anti-IgE antibody is labeled on the surface by a physical bond, monodisperse, and comprises an inorganic metal catalyst for oxidizing the chromogenic substrate Enzyme mimic nanoparticles. According to the present invention, the stability of the diagnostic kit is increased by using an inorganic metal catalyst-based diagnostic immunoassay that improves enzyme instability and variability caused by temperature changes and pH changes, which are problematic in conventional enzyme-based diagnostic immunoassays. Allergy diagnostic side flow immunochromic chips and allergy diagnostic Immunoplate kits can be provided to reduce the time required for testing, reduce the cost of manufacturing, transport, storage and inspection.

Description

Method to diagnose allergy by determination of Immunoglobulin E using an immunoassay based on based on enzyme-mimicking nanozymes}

The present invention relates to an allergy diagnostic enzyme-mimicking nanoparticles, lateral flow immunochromatic chip using allergy diagnostic enzyme-mimetic nanoparticles, and a manufacturing method, and an allergy diagnostic Immunoplate method and kit using allergy diagnostic enzyme-mimetic nanoparticles.

Allergy is a word derived from the Greek word "allos", which means "transformed," which means hypersensitivity reactions. In Korea, allergy and English pronunciation allergy are used interchangeably, but the standard language is allergy. In this specification, “allergy” is a word that includes both allergy and allergy.

Allergy refers to the appearance of several symptoms of immune hypersensitivity to environmental factors with little or no effect on most people. Types of allergy include hay fever, food allergies, atopic dermatitis, allergic asthma, and anaphylaxis. Symptoms of allergies include redness of the eyes, itching, runny nose, shortness of breath and swelling.

In general, allergic factors such as constitutional factors and acquired environmental factors appear to interact. In particular, environmental factors are called allergens. In general, allergens such as pollen, certain foods, insect stings, and medicines are present. When allergen enters our body, it acts as an antigen, resulting in an antigen-antibody reaction as an immune response, resulting in allergic symptoms.

An allergen-induced immune response involves an antibody called Igunoglobulin E antibodies (IgE). IgE receptors are known as Fc receptors, FcεRI and FcεRII (CD23). FcεRI is expressed in mast cells, basophils, and antigen-presenting dendritic cells, and IgE is present in FcεRI binding to mast cells. When allergens and IgE bind to antigen-antibody reactions, cross-linking occurs between the bound IgE and FcεRI receptors gathered around it, and degranulation occurs in mast cells, resulting in the release of immune mediators. do. In the case of basophil cells, immune mediators such as IL-4 (interleukin-4) are secreted. FcεRII is always expressed in B cells, and expression is induced in macrophages, eosinophilic cells, platelets, and some T cells by IL-4 secreted from basophils. . FcεRI has a high affinity for IgE, while FcεRII has a low affinity for IgE. It is hypothesized that the concentration of IgE is regulated by the involvement of FcεRII with low affinity for IgE at the stage of differentiation of B cells into plasma cells that secrete IgE.

Allergy tests include total IgE test in serum, specific IgE test, blood eosinophil and eosinophil cationic protein test, skin reaction test, and antigen-induced test. It is a simple, economical and diagnostic value that can be done, or it is often burdensome or painful to the subject.

As an allergy test that does not burden the subject, a method of measuring IgE antibodies in serum has been preferred. For example, a person with atopic dermatitis may increase the amount of the total IgE antibody by up to 10 times. Therefore, it is a method of determining the presence of allergy by measuring the amount of the total IgE antibody in the serum. By the way, total IgE antibody is present in the smallest concentration among the total immunoglobulin isotypes and is only 0.05%. Therefore, conventional techniques for detecting allergy by detecting total IgE antibodies in serum have problems in terms of sensitivity. Recently, many studies for using allergen-specific IgE antibodies in serum have been developed, but the amount of allergen-specific IgE antibodies is significantly smaller than that of total IgE antibodies. Therefore, the method of identifying allergen-specific IgE antibody also has a problem in terms of sensitivity. Such a prior art is an "antibiotic allergy diagnostic kit comprising an antibiotic-human serum protein conjugate" (Korean Patent Publication No. 10-2012-0058842). In addition, regardless of the method using the IgE antibody, when an allergic reaction occurs, there have been attempts to diagnose the allergy by using the expression of genes or proteins that increase in the body specifically. Such a prior art is "use of CTCF gene in atopic and allergic diseases" (Korean Patent Publication No. 10-2016-0115645).

On the other hand, among the methods frequently used for diagnosis, there is a flow immunoassay chip test method. In this test, a method of labeling gold nanoparticles is mainly used as a coloring material, and the gold nanoparticles are colored red by the intrinsic plasmon phenomenon and are visually identified and used for diagnosis. However, since gold nanoparticles have low analytical sensitivity, they can be applied only to analytes that are present in large amounts in body fluids. To improve this, attempts to amplify a signal by binding a first antibody or a first binding site (ligand) to gold nanoparticles and then binding a second antibody or second specific binding material containing a fluorescent material (Korea Patent Publication No. 2010-118550).

Among the present inventors, Jinwoo improves the gold nanoparticle method and the signal amplification method, so that "a highly sensitive lateral flow immunochromic chip using an enzyme-inspired inorganic nanoparticle and a detection method using the same" (Korea Patent Publication No. 10-1597413) ) Was invented. It is a method of improving sensitivity by immobilizing an antibody capable of detecting an analyte on nanoparticles using iron oxide (Fe3O4) and platinum (Pt) instead of gold, and amplifying a color signal using an enzyme-substrate reaction. As a result, the analyte can be grasped and diagnosed with the naked eye.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

It is an object of the present invention to provide an allergy diagnostic enzyme-mimicking nanoparticle that can be used in place of the enzyme-based composition used in the conventional allergy diagnosis.

Another object of the present invention is to provide an allergic diagnostic side flow immunochromic chip using allergic diagnostic enzyme-mimicking nanoparticles.

In addition, another object of the present invention is to provide a method for manufacturing a side flow immunochromic chip for allergy diagnosis.

It is another object of the present invention to provide a method for detecting total IgE antibodies in a sample and a method for detecting allergen-specific IgE antibodies using an allergic diagnostic side flow immunochromic chip.

It is another object of the present invention to provide a method for providing information on allergen-specific allergic disease diagnosis or allergen-specific allergic disease diagnosis using a side flow immunochromic chip for allergic diagnosis.

It is another object of the present invention to provide an allergy diagnostic Immunoplate method using allergen diagnostic nanoparticles.

It is another object of the present invention to provide an allergy diagnostic Immunoplate kit containing allergen diagnostic nanoparticles.

It is another object of the present invention to provide a method for providing information on allergen-specific allergic disease diagnosis or allergen-specific allergic disease diagnosis using an allergy diagnostic Immunoplate kit.

In order to achieve the above object, an aspect of the present invention for diagnosing allergy, characterized in that the first anti-IgE antibody is labeled on the surface by a physical bond, monodisperse, and comprises an inorganic metal catalyst for oxidizing the chromogenic substrate Enzyme mimic nanoparticles.

In a specific embodiment of the present invention, the inorganic metal catalyst is characterized in that it has an enzyme mimic activity of peroxidase or oxidase, the inorganic metal catalyst is characterized in that to precipitate or insolubilize the color substrate. It is preferable.

In one specific embodiment of the present invention, the inorganic metal catalyst preferably includes iron oxide (Fe 3 O 4), and more preferably, the inorganic metal catalyst has magnetic properties. .

In one specific embodiment of the present invention, the inorganic metal catalyst is preferably characterized by containing platinum (Pt), and more specifically, the inorganic metal catalyst is synthesized in the form of a layered structure. More preferably.

In one specific embodiment of the present invention, it is preferable that the allergic diagnosing enzyme-mimicking nanoparticle having the monodispersity is synthesized monodisperse by controlling the size uniformly using a surfactant in an organic solvent environment. .

In one specific embodiment of the present invention, the allergy diagnostic enzyme-mimicking nanoparticle replaces the surface of the nanoparticle with carboxylic acid, and the first anti-IgE antibody reacts with the carboxyl group of the carboxylic acid to react with the nanoparticle. It is preferable that the inorganic nanoparticles and the substituted anti-IgE antibodies are bound to the particles and uniformly dispersed and fixed in physiological saline.

In order to achieve the above object, another aspect of the present invention is an allergy diagnostic enzyme mimic nanoparticles comprising a first anti-IgE antibody at the junction, there is a fixed substance capable of binding to the target material of the sample at the reaction portion Side flow immunochromic chip for allergy diagnosis, characterized in that.

In one specific embodiment of the present invention, the target material of the sample is a total IgE antibody, and the fixed material is preferably a second anti-IgE antibody, more specifically, the allergy diagnostic enzyme-mimicking nanoparticles The first anti-IgE antibody labeled on the surface of the allergen binds to the total IgE antibody which is a target substance of the sample and the total IgE antibody binds to the second anti-IgE antibody which is a fixed substance of the reaction part. More preferably, the diagnostic enzyme-mimicking nanoparticles are immobilized on the reaction part and react with the color substrate to amplify the detection signal.

In one specific embodiment of the present invention, the target substance of the sample is an allergen-specific IgE antibody, and the immobilizing substance is allergen. More specifically, the allergy diagnostic enzyme The first anti-IgE antibody labeled on the surface of the mimic nanoparticles binds to the allergen-specific IgE antibody which is a target material of the sample, and the allergen-specific IgE antibody binds to the allergen, which is a fixed substance of the reaction part. It is more preferable that the allergy diagnostic enzyme-mimicking nanoparticles are immobilized on the reaction part and react with the color substrate to amplify the detection signal.

In addition, in one specific embodiment of the present invention, the allergen is grass, bark, animal epithelium or hair, insect, mite, house dust, parasite, microorganism, bee venom, drug, occupational allergen, fruit, vegetable, seed, Nuts, spices, fish, shellfish, eggs, poultry, meat, dairy products, food additives and the like is preferably at least one selected from the group consisting of, the sample is tissue, cells, whole blood, serum, plasma, It is preferred to include saliva, sputum, cerebrospinal fluid or urine.

In addition, in one specific embodiment of the present invention, the allergy diagnostic enzyme-mimicking nanoparticles are monodisperse inorganic nanoparticles, and the allergic diagnostic enzyme-mimetic nanoparticles having the monodispersity are sized using a surfactant in an organic solvent environment. It is preferable to synthesize | combine it to be uniformly adjusted and monodisperse.

In one specific embodiment of the present invention, the allergy diagnostic enzyme-mimicking nanoparticle comprising the first anti-IgE antibody replaces the surface of the nanoparticle with carboxylic acid, and the first anti-IgE antibody is It is preferable that the carboxyl group of the carboxylic acid reacts with the nanoparticles. In addition, it is preferable that the first anti-IgE antibody labeled on the surface of the allergy diagnostic enzyme-mimicking nanoparticle comprising the first anti-IgE antibody is bound to the immobilization material immobilized on the support of the reaction part. . In addition, the allergy diagnostic enzyme-mimetic nanoparticles comprising the first anti-IgE antibody is preferably characterized in that it comprises any one or more selected from the group consisting of iron oxide, platinum, allergy diagnostic enzymes containing platinum More preferably, the nanoparticles are synthesized in a hierarchical structure.

In one specific embodiment of the present invention, the allergy diagnostic enzyme-mimicking nanoparticles have enzyme-simulating activity of peroxidase or oxidase and are preferably oxidation catalysts of the chromogenic substrate, and more specifically, The allergy diagnostic enzyme-mimicking nanoparticles are more preferably characterized by precipitation or insolubilization of the chromogenic substrate, wherein the chromogenic substrate is 3-amino-9-ethylcarbazole. It is more preferable to set it as.

In addition, in a specific embodiment of the present invention, the allergic diagnostic side flow immunochromic chip includes a sample portion into which a sample containing a target substance is injected, and an allergic diagnostic enzyme-mimicking nanoparticle including a first anti-IgE antibody, Wherein the first anti-IgE antibody of the nanoparticles conjugated with the target material can be conjugated to bind, the reaction part is present a fixed material that can bind to the target material of the sample, the control material is present to confirm the error A control unit includes an absorption unit capable of absorbing a liquid sample by capillary action, and the sample moves from the sample unit to the absorption unit through the reaction unit and the control unit on the support by capillary action. Preferably, it is characterized in that, more specifically, the control unit Ig as a control material More preferably characterized in that the E antibody is immobilized, the immobilizing material is allergen, the target material contained in the sample is an allergen-specific IgE antibody, the reaction part is divided into several sections each section It is characterized in that different types of allergens are immobilized each time, and that the presence of the allergen-specific IgE antibody contained in the sample for each allergen in the various sections can be detected simultaneously by one test. It is more preferable to characterize.

In one specific embodiment of the present invention, the allergic edema, anaphylaxis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergy, food allergy Allergies), allergic asthma (allergic asthma) or allergic disease, characterized in that any one selected from the group consisting of drug allergy, and the like.

In addition, in one specific embodiment of the present invention, (1) detecting the total IgE antibody as a target substance in a sample using the allergy diagnostic side flow immunochromic chip; And (2) amplifying a detection signal by reacting the allergy diagnostic enzyme-mimicking nanoparticles bound to the total IgE antibody with a chromogenic substrate; It provides a total IgE detection method comprising a.

In addition, in a specific embodiment of the present invention, (1) detecting the allergen-specific IgE antibody as a target substance in a sample by using the allergy diagnostic side flow immunochromic chip; And (2) amplifying a detection signal by reacting the allergen-diagnosing enzyme-mimicking nanoparticles associated with the allergen-specific IgE antibody with a chromogenic substrate; It provides an allergen-specific IgE detection method comprising a.

In addition, in a specific embodiment of the present invention, (1) detecting the allergen-specific IgE antibody as a target substance in a sample by using the allergy diagnostic side flow immunochromic chip; (2) amplifying a detection signal by reacting the allergen-diagnosing enzyme-mimicking nanoparticles associated with the allergen-specific IgE antibody with a chromogenic substrate; (3) identifying, from the amplified detection signal, an allergen that is a fixed substance bound to the allergen-specific IgE antibody; (4) determining an allergic disease by confirming identity of the allergen; It provides a method for providing information on the diagnosis of allergen-specific allergic disease or allergen-specific allergic disease comprising a.

In one specific embodiment of the present invention, (1) an allergy diagnostic enzyme-mimicking nanoparticle, wherein the first anti-IgE antibody is labeled with a physical bond and contains an inorganic metal catalyst for oxidizing a chromogenic substrate. Synthesizing particles; (2) a sample portion into which a sample containing a target substance is introduced, a junction at which the allergic diagnostic enzyme-mimetic nanoparticle including the first anti-IgE antibody is located, and a target material of the sample introduced into the sample portion Assembling a reaction part having a fixed substance present therein, a control part having an IgE antibody fixed as a control material for checking errors on the side of the reaction part, and an absorbing part capable of absorbing a liquid sample by capillary action on a support; It provides a method for producing a side flow immunochromatic chip for allergy diagnosis comprising a.

In order to achieve the above object, another aspect of the present invention comprises the steps of (1) fixing a fixing material capable of binding with the target material of the sample to the well of the immunoplate; (2) dispensing a sample in which the target material is present into each well of the Immunoplate to bind the fixed material and the target material; (3) binding the allergy diagnostic enzyme-mimicking nanoparticles labeled with the first anti-IgE antibody to the target material dispensed into each well of the Immunoplate; And (4) after the first anti-IgE antibody binds to the fixation material through the target material, the allergy diagnostic enzyme-mimicking nanoparticle reacts with a chromogenic substrate to amplify the detection signal; Immunoplate method for allergic diagnosis comprising a.

In one specific embodiment of the present invention, the target material of the sample is a total IgE antibody, and the fixed material is preferably a second anti-IgE antibody, wherein the target material of the sample is an allergen-specific IgE. Preferably, the fixing substance is allergen, and more specifically, the allergen may be grasses, bark, animal epithelium or hair, insects, mites, house dust, parasites, microorganisms, bee venom, drugs, More preferably, it is at least one substance selected from the group consisting of occupational allergens, fruits, vegetables, seeds, nuts, spices, seafood, eggs, poultry, meat, dairy products, food additives and the like. Further, in one specific embodiment of the present invention, different types of allergens are immobilized in each well of the immunoplate so that the presence of the allergen-specific IgE antibody against each allergen can be detected simultaneously by one test. More preferred. In addition, the sample preferably contains tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

In a specific embodiment of the present invention, the allergy diagnostic enzyme-mimicking nanoparticles are monodisperse inorganic nanoparticles, and the monodisperse inorganic nanoparticles are synthesized monodispersely with a uniform size controlled using a surfactant in an organic solvent environment. It is preferable to be characterized by being. In addition, the allergy diagnostic enzyme-mimetic nanoparticles comprising the first anti-IgE antibody replace the surface of the nanoparticle with carboxylic acid, and the first anti-IgE antibody reacts with the carboxyl group of the carboxylic acid. It is preferable to be characterized in that it is bonded to the nanoparticles.

In one specific embodiment of the present invention, the allergy diagnostic enzyme-mimicking nanoparticle is preferably characterized in that it comprises any one or more selected from the group consisting of iron oxide and platinum, and more specifically, the allergy diagnostic enzyme. More preferably, the nanoparticles containing platinum of the mimic nanoparticles are synthesized in a hierarchical structure.

In one specific embodiment of the present invention, the allergy diagnostic enzyme-mimicking nanoparticles have enzyme-simulating activity of peroxidase or oxidase and are preferably oxidation catalysts of the chromogenic substrate, and more specifically, The allergy diagnostic enzyme-mimicking nanoparticles are more preferably characterized by precipitation or insolubilization of the chromogenic substrate, wherein the chromogenic substrate is 3-amino-9-ethylcarbazole. It is more preferable to set it as.

In one specific embodiment of the present invention, the allergic edema, anaphylaxis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergy, food allergy allergies, allergic asthma, drug allergy, and the like.

In one specific embodiment of the present invention, an allergy comprising the composition comprising the allergic diagnostic enzyme-mimicking nanoparticles for performing the allergy diagnostic Immunoplate method and the immobilizing material capable of binding to a target material of a sample It is desirable to provide a diagnostic Immunoplate kit. More specifically, different types of the allergens are immobilized in each well of the immunoplate so that the presence of the allergen-specific IgE antibody against each allergen can be detected simultaneously by a single test. It is more preferable to further include.

In addition, in one specific embodiment of the present invention, (1) detecting the allergen-specific IgE antibody as a target substance in a sample using the allergy diagnostic Immunoplate kit; (2) amplifying a detection signal by reacting the allergen-diagnosing enzyme-mimicking nanoparticles associated with the allergen-specific IgE antibody with the chromogenic substrate; (3) identifying, from the amplified detection signal, an allergen that is a fixed substance bound to the allergen-specific IgE antibody; (4) determining an allergic disease by confirming identity of the allergen; It provides a method for providing information on the diagnosis of allergen-specific allergic disease or allergen-specific allergic disease comprising a.

According to the present invention as described above, it is possible to provide an allergy diagnostic enzyme mimic nanoparticles that can be used in place of the enzyme-based composition used in the conventional allergy diagnosis, side flow immunochromatic chip for allergy diagnosis, Immunoplate kit for allergy diagnosis Can be provided.

According to the present invention as described above, the stability of the diagnostic kit is increased by using an inorganic metal catalyst-based diagnostic immunoassay that improves the instability and variability of the enzyme caused by temperature change and pH change, which are problematic in the conventional enzyme-based diagnostic immunoassay. In addition, it is possible to provide a side flow immunochromic chip for allergy diagnosis and an Immunoplate kit for allergy diagnosis, which improves the economics by reducing the time required for inspection, and reducing the cost of manufacturing, transport, storage, and inspection.

In FIG. 1, a schematic diagram (above) of a conventional allergy diagnostic procedure is compared with a schematic diagram (bottom) of a diagnostic procedure according to an embodiment of the present invention.
2 is a transmission electron microscope (TEM) photograph of H-Pt nanoparticles mimicking peroxidase activity according to an embodiment of the present invention.
Figure 3 is a graph showing the results of comparing the stability of the temperature and pH conditions by confirming the enzyme activity of the organic peroxidase (HRP) and H-Pt nanoparticles according to an embodiment of the present invention.
4 is a graph and a photograph showing the results of color development diagnosis of IgE antibody concentration using an anti-IgE antibody conjugated with organic peroxidase (HRP) and H-Pt nanoparticles according to an embodiment of the present invention.
5 is a graph showing a result of measuring a calibration curve for total IgE concentration and specific IgE concentration according to an embodiment of the present invention.
FIG. 6 is a table listing data comparing the concentration values obtained by detecting and quantitatively analyzing total IgE in human serum with actual IgE concentration values provided by Gachon University Gil Hospital.
FIG. 7 is a table summarizing data comparing concentration values obtained by detecting and quantitating specific IgE in human serum with actual IgE concentration values provided by Gachon University Gil Hospital, according to an embodiment of the present invention.
8 is a schematic diagram showing the configuration and principle of the side flow allergy diagnostic kit, according to an embodiment of the present invention.

Hereinafter, with reference to the accompanying drawings will be described an embodiment of the present invention in more detail. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention relates to an in vitro diagnostic technology that can analyze specific indicators using samples derived from the human body, such as blood, urine, saliva, and more specifically, using an immune response (antigen-antibody response). The present invention relates to a method for detecting and quantifying IgE in blood to diagnose allergies. The diagnostic technique using the antigen-antibody reaction shows that there is a high sensitivity of the antigen-antibody reaction by the reaction between the substrate and the enzyme when the antibody to which the enzyme is bound reacts with a specific antigen. Technology. Over the last few decades, research on immunodiagnostic technology using protein-based organoenzyme activity has been extensively conducted, and various products have been commercialized. Such organoenzyme-based diagnostic techniques have the advantage of being able to diagnose specific and sensitive target substances, but the enzyme's activity is gradually changed depending on the reaction environment and storage time, which may cause problems in the reliability of the diagnostic results. There is this. For example, Phadia's ImmunoCAP test, the most commonly used diagnostic method for allergy, is a proven immunodiagnostic method that can clinically measure allergy by quantitatively measuring IgE in human serum or plasma samples. In addition, the use of the organic enzyme β-galactosidase in the test process has the disadvantages that the enzyme activity is reduced according to the surrounding environment and storage time and the diagnostic method is limited to the fluorescence detection method. Therefore, the demand for alternative materials that can supplement the disadvantages of the organic enzyme and maintain the enzyme activity has increased rapidly, and many studies have been continued until recently.

The present invention maximizes stability and catalytic activity through a strategy of binding enzyme-mimicking nanoparticles to anti-IgE globulinE antibodies instead of organic enzymes, and a high sensitivity allergy to specifically detect and quantitate IgE antibodies. Suggest a diagnostic. The first anti-IgE to be used for allergy diagnosis is immobilized on the nanoparticles of iron oxide (Fe ₃ O ₄ ) and platinum (Pt) in the inorganic metal.

Nanoparticles provided by the present invention can be carried out by two or more methods. The first is to diagnose allergic reactions to specific allergens. Each allergen is immobilized on the underside of the reaction section of the diagnostic kit, and a blood sample is passed over it to bind allergen-specific IgE antibodies (allergen-specific IgE). In addition, the first anti-IgE antibody, to which the nanoparticles are bound, binds to the allergen-specific IgE antibody, and the chromogenic substrate is colored by the enzyme mimic function of the nanoparticles, thereby the presence of the allergen-specific IgE antibody. Can be identified. The second is to diagnose allergic reactions by detecting an increase in total IgE antibody in the blood. The second anti-IgE antibody is immobilized on the bottom of the reaction section of the diagnostic kit, and passing the blood sample thereon causes the total IgE antibody in the blood sample to bind to the second anti-IgE antibody. In addition, the first anti-IgE antibody having nanoparticles bound thereon binds to the total IgE antibody, and the coloring substrate is colored by the enzyme mimic function of the nanoparticles, thereby identifying an allergic increase in the amount of the total IgE antibody. Can be diagnosed.

Using the diagnostic methods provided by the present invention, compared to conventional diagnostic methods using organic enzymes, in which structural degeneration and reduced activity are inevitably exhibited over time, the present invention provides a platinum structure in a hierarchical form that mimics enzyme activity. Since nanoparticles (H-Pt nanoparticles) are used, more stable immunodiagnosis can be provided. The present invention is the first case of applying an enzyme-mimicking nanoparticle to a conventional organoenzyme-based allergy diagnostic method.

Hereinafter, terms used in the present invention will be described.

In one embodiment of the invention “allergic disease” means edema, anaphylaxis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergy, food allergies ), Allergic asthma (allergic asthma) and drug allergy is preferably any one selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention "allergen" refers to an allergen that causes allergy, grasses, bark, animal epithelium or hair, insects, mites, house dust, parasites, microorganisms, bee venom, drugs, occupational Allergens, fruit, vegetables, seeds, nuts, spices, fish, shellfish, eggs, poultry, meat, dairy products, food additives, etc. is preferably any one selected from the group consisting of, but not limited to. The four representative allergens in our country are house dust mite, wormwood pollen, cat hair and alternaria fungus. The types of allergens are summarized in Table 1 below.

Types of allergens Classification Allergen herb
Grass pollens Reed, forsythia, sageweed, oat, broadleaf laver, wheat, bahiagrass, barley, oat, syria, wild rye grass, mud barley, duck, orchardgrass, saltweed, corn, kingpofoam, Umbrella grass, canary grass, black beetle, big fern, scented grass, rye, rye grass, white eared, white haired bird, fake woodworm, dog flower, long eared greens, maple leaf, pips, oxtail, ragweed, French chrysanthemum, Marguerite, tusks, seaweed, dandelion, lupine, amaranth, sugar beet, ragweed, grasshopper, pine needles, nettle, mugwort, bitter orange, baby swimming, rapeseed, alfalfa, plantain, chamomile, pellet, fern, sunflower Marsh Elder Tree tree
(Tree pollens) Spruce, hazel, Savior, oil palm, larch, beech, four-leaf maple, elm, jujube, rib oak, linden, mesquite, veneer, ash, aspen, american poplar, migraine, Chestnut, cedar, cedar, white birch, kayuput, willow, red mulberry, poison mulberry, mulberry, desert berry, cypress, cypress, cypress, oak, cypress, pine, strobe pine, cedar elm, Acacia, Lamb's Eye, Queen's Palm, Alder, Olive, Eucalyptus, Japanese Cedar, Birch, Red Cedar, Privet, Mediterranean Cypress, Oak, Horse Chestnut, Marronnier, Hackberry, Peppertree, Pelobuddy, Prunus, Sycamore, Pecan, juniper, walnut, australian pine, hickory animal
(Epidermal and animal proteins) Dog Dandruff, Goose Down, Guervilose Rat Epithelium, Cat Dandruff, Guinea Pig Epithelium, Green Macaw Feces, Green Macaw Feathers, Chicken Hair, Chicken Feces, Chicken Serum Protein, Ferret Epidermis, Pork Epithelium, Horse Dandruff, Horse Serum Protein, Mink Epithelium, pigeon droppings, pigeon feathers, deer epithelium, bovine dandruff, reindeer epithelium, mouse epithelium, mouse urine protein, mouse serum protein, sagittal epithelium, sagittal urine protein, sagittal serum protein, sheep epithelium, fox hair, goat epithelium, duck down , Parrot feathers, macaw feathers, macaw feces, macaw serum protein, chinchilla epithelium, turkey feathers, canary feces, canary feathers, rabbit epithelium, rabbit urine protein, rabbit serum protein, pinch feathers, hamster epithelium insect
(Insects) Grain weevil, midget, moth, beetle, horsefly, iron, mosquito, cockroach, fire ant, earthworm, mediterranean flour moth mite
(Mites) House dust mite, storage mite House dust House dust helminth
(Parasites) Whale roundworm, roundworm Bee venom
(Venoms) Bees, bumblebees, wasps (White-faced hornet), wasps (Yellow hornet), wasps (European hornet), paper wasps, European paper wasp, bumble bees drug
(Drugs) Amoxicillin, Ampicillin, Sephachlor, Chlorhexidine, Chimopapine, Gelatin, Insulin, Penicillin, Polcodine, Morphine, Schisametonium Professionalism
(Occupational)
Bougainvillea, Castor Seed, Cotton Seed, Raw Coffee Bean, Polyurethane (Isocyanate (TDI / MDI / HDI)), Latex, Silk, Silken Crumb, Sunflower Seed, Abachi wood dust, Alkalase, Maxatase, Savinase, Chloramin T, Ispaghula,
Ethylene oxide, Formaldehydee / Formalin, Hexahydrophtalic anhydrid, Methyltetrahydrophtalic anhydrid, Phtalic anhydrid, Trimellitic anhydride Fruits and Vegetables
Friuts, Vegetables Eggplant, Persimmon, Potato, Sweet Potato, Guava, Chard, Carrot, Jujube, Wild Rose Fruit, Strawberry, Lime, Lemon, Garlic, Mandarin, Mango, Melon, Sesame Seed, Fig, Mini Cabbage, Banana, Baramil, Pear, Cherry, Peach, Red currant, broccoli, blackberry, blueberry, apple, raspberry, apricot, lettuce, celery, watermelon, spinach, avocado, asparagus, cabbage, onion, rich, orange, cinnamon, cucumber, olive, plum, grapefruit, bamboo shoot, Cauliflower, Cranberry, Kiwi, Tomato, Pineapple, Papaya, Passion Fruit, Grape, Pumpkin, Fennel Seeds and nuts
(Seeds, nuts) Hazelnut / Hazelnut, Oat, Gluten, Gliadin, Haricot Bean, Peanut, Lentil, Lupine Seed, Lima Bean, Macadamia Nut, Buckwheat, Wheat, Chestnut, Chickpea, Barley, Red Kidney Bean, Brazil Nut, Beet Seed, Sorghum, millet, spelled wheat, flax seed, almond, poppy seed, corn, pea, rapeseed seed, indian pea, pine nut, crude, sesame, cashew nut, coconut, soybean, quinoa, pistachio, pecan, walnut, fenugreek, Rye, pumpkin seeds, white kidney beans Spice
(Spices) Mustard, cinnamon, coriander, elf, rubbish, marjoram, mace, vanilla, basil, peppermint, white nut, thyme, saffron, cedar, ginger, sage, anise, oregano, allspice, laurel leaf, cloves, chili, curry, cara Whey, parsley, paprika, bell pepper, fennel seeds, pepper Seafood
(Fish, shellfish, mollusks) Scallop, flounder, flounder, crayfish, skewer, crab, flatfish, oyster, perch, mackerel, snail, cod, lobster, clam, catfish, anchovy, pollock, octopus, lobster, red sea bream, roe, shrimp, trout, salmon , Orange nappy, squid, eel, horse mackerel, abalone, sardines, tuna, herring, tilapia, mussels, swordfish Egg, poultry
(Egg, fowl) Egg yolk, egg white, chicken, turkey meat
(Meat) Pork, Horse, Venison, Beef, Lamb, Rabbit dairy product
(Milk) Goat milk, cow whey, mare milk, sheep milk, sheep whey, milk, cheddar cheese, mold cheese Food additives
(Additives) Guar gum, gum arabic, carob, cochineal, tragacanth Other foods
(Miscellaneous) Honey, malt, mushroom, mushroom, tea, cacao, coffee, hops, yeast Microorganisms
(Microorganisms) Acremonium kiliense, Alternaria alternata, Aspergillus rim piece, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Aureobasidium pullulans, Botrytis cinerea, Candida albicans, Chaetomium globosum, Cladosporium herbarum, Curvularia lunata, Epicoratum spurasta s purosa prosa Mucor racemo, Penicillium chrysogenum, Penicillium glabrum, Phoma betae, Pityrosporum orbiculare, Rhizopus nigricans, Staphylococcal enterotoxins, Stemphylium herbarum, Tilletia tritici, Trichoderma viride, Trichophyton mentagrophytes var. goetzii, Trichophyton mentagrophytes var. interdigitale, Trichophyton rubrum, Trichosporon pullulans, Ulocladium chartarum

In one embodiment of the present invention "diagnosis" (diagnosis) means to confirm the presence or characteristics of the pathological state, for the purpose of the present invention, the diagnosis is to determine whether the allergic disease. In the present invention, allergic disease diagnosis may be made by a method of confirming the presence of 'allergen-specific IgE' or 'total IgE' included in blood samples collected from a test subject.

In the present invention, "enzyme-mimicking nanoparticles" are iron (Fe), copper (Cu), aluminum (Al), cobalt (Co), cerium (Ce), nickel (Ni), manganese (Mn), zinc (Zn), It may be made by a method of synthesizing nanoparticles including one or more selected from the group consisting of metals such as platinum (Pt), and selecting those having peroxidase or oxidase activity. More specifically, the nanoparticles are preferably one or more selected from the group consisting of iron oxide (Fe ₃ O ₄ ) and platinum (Pt).

As used herein, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to the marker of the present invention, and more specifically, an antibody that specifically binds to IgE (Immunoglobulin E, immunoglobulin E). The monoclonal antibody against the protein is cloned into an expression vector according to a monoclonal antibody production method conventional in the art to obtain a protein encoded by the marker gene, and then produced and used by a conventional method from the obtained protein. You may use it and a commercially available thing can be used. In addition, a polyclonal antibody that recognizes the protein may be used instead of the monoclonal antibody, which may be manufactured and used through a conventional antiserum production method in the art. In addition, the antibody of the present invention also includes a partial peptide that can be made from the protein, and the partial peptide of the present invention includes at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. do. The form of the antibody of the present invention is not particularly limited and, if it is a polyclonal antibody, a monoclonal antibody or antigen-binding, a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included. Aptamers consisting of non-base sequences are also included in the concept of antibodies in the present invention. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody. Antibodies used for IgE detection of the present invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.

In the present invention, "IgE" (Immunoglobulin E) is an antibody that is involved in type 1 hypersensitivity reactions in mammalian immune reactions. It is a monomer antibody having four domains and plays an important role in allergic reactions. Used as marker protein for diagnosis of disease.

In the present invention, "allergen-specific IgE" refers to an immunoglobulin that is expressed against a specific antigen during type 1 hypersensitivity, and an IgE-class antibody specific for the antigen is bound to mast cells or basophil cells. These reactions can occur locally or systemically, depending on the type and amount of mediator these cells produce, which is also called immediate hypersensitivity because they occur within an hour after the antigen has been introduced. This systemic manifestation is called anaphylaxis and can cause severe asphyxiation and circulatory death, but even when exposed to the same allergen, this reaction may or may not be present in some people. Also called atopy of meaning .

In the present invention, "sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and the like, which differ in the expression level of the IgE antibody. In a specific embodiment of the present invention, the serum of blood collected from the test subject is used, but is not limited thereto.

In the present invention, "antigen-antibody complex" means a combination of a marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formation can be quantitatively measured through the size of a signal of a detection label.

In a specific embodiment of the present invention, in addition to the method of visually confirming to measure the quantitative level of IgE, ELISA can be used. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. For example, an allergen is attached to a solid support (bottom of the reaction part of the diagnostic kit) and the sample is reacted, and then labeled onto an enzyme-mimicking nanoparticle that recognizes the antigen (allergen-specific IgE contained in the sample) of the antigen-antibody complex. The first anti-IgE antibody conjugated to develop an enzymatic color or attach the second anti-IgE antibody to a solid support and react the sample to recognize the antigen of the antigen-antibody complex (total IgE contained in the sample). The first anti-IgE antibody labeled with the enzyme-mimicking nanoparticles can be detected and developed by the enzyme catalytic reaction. Allergic diseases can be diagnosed by confirming the detected degree of antigen-antibody complex formation.

In one embodiment of the present invention, "diagnosis kit" refers to a set of compositions and accessories necessary for a specific purpose. For the purposes of the present invention, the diagnostic kit of the present invention is to determine whether the allergic disease. The kit of the present invention includes an enzyme-mimicking nanoparticle having an enzymatic activity including a first anti-IgE antibody for determining an allergic disease, a solid support including a reaction portion to which each allergen or a second anti-IgE antibody is attached, Reagents capable of measuring enzymatic activity, such as peroxidase, may be included, in addition to one or more other component compositions, solutions or devices suitable for analytical methods.

In one embodiment of the present invention, "side flow immunochromic chip" is a kit showing a color signal upon detection so that the liquid sample containing the target material is fixed while moving through the porous medium and reacts with the detection material. Means.

As used herein, the term "nano-particle" refers to any particle in nano units having a diameter of less than 1000 nm. Specifically, in the embodiment of the present invention, the nanoparticles preferably have a diameter of 10 to 90 nm.

In one embodiment of the present invention, "inorganic nanoparticles" means nanoparticles containing an inorganic component. The inorganic material may be an inorganic material, an oxide of an inorganic material, or an inorganic composite. The inorganic composite may be, for example, a composite material in which nonmetals, ceramics, plastics, polymers, biological materials, semiconductors, and quantum dots are combined with metals, and the composite material may be, for example, ceramics or polymers. The same non-metallic nucleating agent is contained and the outside may be particles coated with an inorganic material, and a reactive functional group or a molecular sieve may be present on the surface of the inorganic particles.

 In one embodiment of the present invention, "enzyme-mimicking nanoparticles" are inorganic nanoparticles capable of oxidizing a chromogenic substrate, preferably particles consisting of iron oxide (Fe3O4), platinum (Pt), mixtures thereof, and the like. In the present invention, the chromogenic substrate amplifies a detection signal while the enzyme mimics the ksh particles as an oxidation catalyst to precipitate and insoluble at the position where the inorganic nanoparticles are present after oxidation. In a specific embodiment of the present invention, the chromogenic substrate may be 3-amino-9-ethylcarbazole (AEC) when the peroxidase mimic nanoparticles.

 In one embodiment of the present invention, functional groups capable of immobilizing the detection antibody may be present on the surface of the nanoparticles. The functional group present on the surface of the inorganic nanoparticles is preferably a carboxylic acid group (Carboxylic acid group) capable of providing covalent bonds or ionic electrical attraction as a means of fixing the detection antibody. In this case, an amine group (-NH2, amino group) which is present in abundance in the IgE antibody can be used.

 In one embodiment of the present invention, the nanoparticles are preferably mono-dispersed nanoparticles contained in the liquid phase to be able to move to the junction of the lateral flow immunochromic chip through a capillary diameter of several micrometers. In the present invention, "monodispersibility" refers to the degree of uniformity of the size and structure of the nanoparticles, J. Am. Chem. Soc. (Shouheng Sun et al., Monodisperse MFe2O4 (M = Fe, Co, Mn) nanoparticles: 2004, Vol. 126, 273-279) can be prepared using the prior art. In a specific embodiment, the synthesis of the monodisperse nanoparticles may be prepared using a synthesis method to improve the monodispersity using a surfactant in an organic solvent.

Hereinafter, preferred embodiments will be presented to aid in understanding the nickname. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Experimental Example

Experimental Materials and Methods

In order to secure a stationary phase to which an allergen or anti-IgE antibody is bound, ImmunoCAP of Phadia was used, and an actual patient serum sample was used as an IgE antibody as a detection target. In addition, high sensitivity to selectively detect and quantitate IgE antibodies by conjugating hierarchical platinum nanoparticles (Advanced Healthcare Materials, 2015, 4 (9), 1311-1316) with high catalytic activity and stability to anti-IgE antibodies. Allergy diagnostics were implemented. For quantitative analysis, sandwich immunoassay was adopted.

Place ImmunoCAP on a 96-well plate, dispense 200 μL of phosphate buffered saline to remove the coating of ImmunoCAP and wait for 30 minutes. Then, the sample to be measured (human serum sample) is dispensed into each well and allowed to react for 1 hour. The unreacted material is washed three times with phosphate buffered saline containing 0.05% tween-20. Thereafter, the anti-IgE antibody-H-Pt nanoparticle conjugate prepared in Example 1 is dispensed and reacted for 1 hour. Unreacted anti-IgE antibody-H-Pt nanoparticle conjugate was washed three times with phosphate buffered saline. Dispense 250 μL of a colorant consisting of hydrogen peroxide (H ₂ O ₂ ), 3,3 ′, 5,5′-Tetramethylbenzidine (TMB) and sodium acetate buffer solution (pH 4.0) and react for 5 minutes. After removal of ImmunoCAP, 200 μL of each solution is transferred to a 96-well plate and the absorbance of each well is measured with a 96-well plate reader.

[Experiment result]

The total IgE antibody and specific IgE antibody concentration data in serum provided by the actual site (Gachon University Gil Hospital) were compared with the data obtained through immunodiagnosis based on the nanozyme of the present invention, and nearly identical blood IgE concentration data were obtained. . In addition, standard deviation and coefficient of variation were analyzed to verify the diagnostic reliability of each data. The coefficient of variation, which represents the measurement accuracy, was low (2.3-10.9%) for five different blood samples, and the recovery value was also reliable (95.4-104.7%) (Figs. 6 and 7). .

EXAMPLE

Example 1 Method of Making Nanoparticles Containing Anti-IgE Antibody

(1) Synthesis of Iron Oxide Nanoparticles

Iron oxide nanoparticles were synthesized using a co-precipitation method. 0.4 g of ferrous chloride and 1.1 g of ferric chloride were added to 20 mL of distilled water, mixed in a hot stirrer and heated up to 80 ° C. After adding 5 mL of ammonium hydroxide solution of 28% at 80 ° C it was reacted for one hour. The produced magnetic nanoparticles were attracted using a magnet, the supernatant was removed, and washed three times with ethanol and distilled water, respectively.

Magnetic nanoparticles synthesized by the above method has the advantage of being magnetic and easily separated and recovered by using a magnet without a separate centrifugation process. In addition, it has the property of oxidizing peroxidase substrate when H2O2 is present, such as organic peroxidase (HRP) such as horseradish peroxidase (HRP), which can effectively replace organic enzymes used in enzyme immunoassay methods using existing color substrates. Is in the spotlight.

(2) Synthesis of Hierarchically Structured Pt Nanoparticles

Platinum nanoparticles were synthesized via seed-growth synthesis. 18 mL of 0.2% chloroplatinic acid hexahydrate was added to 232 mL of boiling distilled water. After 1 minute, 5.5 mL of a solution containing 1% sodium citrate and 0.05% citric acid was added. After 30 seconds, 2.75 mL of a solution containing 0.08% sodium borohydrate, 1% sodium citrate, and 0.05% citric acid was added thereto, and reacted for 10 minutes. A seed solution containing 5 nm platinum nanoparticle seed was obtained. Mix the above seed solution with distilled water 1: 9 ratio (10 mL: 90 mL), add 0.05 mL of 0.4 M chloroplatinic acid hexahydrate, 1% sodium citrate, and 0.5 mL of lascorbic acid to the boiling point at 10 ° C / min. Keep for minutes and let cool. The particles were allowed to settle for 20 minutes by centrifugation (8000 rpm), and then the supernatant was removed to remove the remaining reactants and washed three times with ethanol and distilled water.

Platinum nanoparticles synthesized by the above method have oxidase activity capable of oxidizing color substrate only with dissolved oxygen even when H 2 O 2 is not present. Above all, it has the strongest peroxidase activity among the metal nanoparticles found so far, and the color development can be visually confirmed when applied to various color diagnostic methods. In addition, it can be synthesized into nanoparticles that show stronger activity than when used alone by synthesizing in a hierarchical structure.

(3) H-Pt nanoparticle binding to anti-IgE antibodies

Allergy diagnostic enzyme-mimicking nanoparticles can be made by physically binding an anti-IgE antibody to a nanoparticle comprising iron oxide or a nanoparticle comprising platinum.

H-Pt nanoparticles (1.0 mg / mL) are washed twice with distilled water and dispersed in the same concentration in 1 mL of distilled water. 250 μL of H-Pt nanoparticle solution (1 mg / mL) dispersed in distilled water and 250 μL of anti-IgE antibody (200 μg / mL) are mixed. 500 μL of phosphate buffered saline solution containing blocking agents (0.1% Tween-20 and 3% bovine albumin) is then added to the mixture. 4 ° C. to promote physical binding between the anti-IgE antibody and the H-Pt nanoparticles. React in a shaker for 24 hours at rinse three times with storage solution (phosphate buffered saline solution containing 0.1% Tween-20 and 3% bovine serum albumin), disperse in the same storage solution and store at 4 ℃.

In order to determine how effectively the anti-IgE antibody was conjugated to the H-Pt nanoparticles by the above method, centrifugation was performed before separating the H-Pt nanoparticles and the unmodified anti-IgE antibody. The supernatant in which the anti-IgE antibody was dissolved was analyzed by BCA protein assay, and it was confirmed that about 90% of the anti-IgE antibody was modified in H-Pt nanoparticles.

Example 2 Use as Lateral Flow Immunodiagnostic Kit

(1) Preparation of membrane coated with antigen and anti-IgE antibody

A lateral flow immunoassay chip was prepared as shown in the schematic diagram of FIG. 8. The allergen antigen protein (fixed material, 620) was used as a test line of the reaction part, and IgE antibody was used as a control line of the control part. The allergen antigen protein was diluted to 1 mg / mL in phosphate buffered saline (pH 7.4) and coated using a dispenser to the reaction portion of the nitrocellulose membrane, which is the support 600. The allergen antigen protein may be an anti-IgE antibody when the target substance is a total IgE antibody, and an allergen may be used when the target substance is an allergen-specific IgE antibody. Here, the anti-IgE antibody bound to the nanoparticles and the anti-IgE antibody used as the immobilizing material have different epitopes with respect to the IgE antibody and can simultaneously bind to each other with IgE interposed therebetween. In order to distinguish from each other, the anti-IgE antibody in the nanoparticles will be referred to as the first anti-IgE antibody, and the anti-IgE antibody used as the immobilizing material will be referred to as the second anti-IgE antibody.

The sprayed nitrocellulose membrane was dried for 12 hours in a desiccator equipped with a dehumidifier. In addition, IgE antibody solution diluted to a concentration of 1 mg / mL in the control line was coated and dried in the same manner as above.

(2) Preparation of anti-IgE antibody-H-Pt nanoparticle junction (conjugate pad, 500)

The anti-IgE antibody-H-Pt nanoparticles 520 prepared in Example 1 were diluted to a concentration of 1 mg / mL in phosphate buffered saline (pH7.4) containing sucrose and bovine serum albumin, and After coating, it was dried to prepare a joint. At the junction, the nanoparticle 520 and the total IgE antibody or allergen-specific IgE antibody, which are the target material 720 in the sample 700, are conjugated to each other.

(3) Preparation of sample part (sample pad 300) and absorbent part (absorption pad 400)

The sample pad 300 and the absorbent pad 400 were manufactured by drying so that the reaction solution could be absorbed well. After the sample 700 is injected into the sample pad 300, the sample 700 passes from the sample part 300 to the reaction part on the support 600 by capillary action through the junction part 500. The absorber 400 is moved to the absorber 400 through the checker.

(4) Assembling side flow immunochromic chip for allergy diagnosis

The nitrocellulose membrane 600 and the pads coated with the test line and the control line prepared above are bonded to the support (backing pad) in the order of overlapping sample pads, nitrocellulose membranes, and finally absorbing pads, and the plastic lower plate and the upper plate. Assembled in a configured housing to prepare a side flow immunochromic chip for allergy diagnosis.

(5) IgE antibody detection and diagnosis process

The allergy diagnostic lateral flow immunochromic chip is used to detect total IgE antibodies or allergen-specific IgE antibodies as target substances in the sample. An allergic diagnostic enzyme mimic nanoparticle binds to the total IgE antibody or allergen-specific IgE antibody in the sample through a first anti-IgE antibody labeled on the surface of the nanoparticle, and the total IgE antibody or allergen-specific IgE antibody When combined with the second anti-IgE antibody or allergen, which is an immobilization material in the reaction portion of the immunochromic chip, the enzyme-mimicking nanoparticles react with the chromogenic substrate to amplify the detection signal. From the amplified detection signal, the presence of total IgE antibody or allergen-specific IgE antibody in the sample is provided and information on allergic disease diagnosis is provided. If the IgE is fixed as a control material in addition to the control part, even if there is no IgE antibody in the sample, the first anti-IgE antibody of the nanoparticles is combined with the IgE, which is a control material, so that the signal is amplified and displayed. You can check whether the lateral flow immunochromic chip is working properly or not.

In addition, the reaction part of the allergic diagnostic side flow immunochromic chip is divided into several sections on the immunochromic chip, and a different allergen is placed in each compartment to examine the presence of allergen-specific IgE antibodies against several allergens at once. can do. According to this method, the allergen-specific IgE antibody can be identified easily and easily in place of the painful skin test, thereby providing information on the diagnosis of the allergic disease in the subject. Done.

Example 3 Use as Enzyme Immunoassay

(1) Preparation of Immunoplate Kit for Allergy Diagnosis with Allergen or Anti-IgE Antibody

An allergen-immobilized Immunoplate was prepared for enzyme-immunoassay to diagnose specific IgE antibodies in blood.

In order to prepare an allergen-immobilized Immunoplate, allergens (Allergen, 620) of various concentrations were dispensed at 100 μL per well, and stored at 4 ° C. for 12-16 hours to sufficiently react. After storage, Immunoplate was washed three times with phosphate buffered saline containing 0.05% tween-20 to remove unbound antigen. Thereafter, 100 μL of phosphate-buffered saline containing 1% bovine serum albumin was dispensed at each well for reaction for 2 hours to block the unbound antigen.

(2) Anti-IgE Antibody-H-Pt Nanoparticle Solution Preparation

Anti-IgE antibody-H-Pt nanoparticle conjugate that binds the target material (allergen-specific IgE, 720) in the sample 700 to the immobilizing material 620 attached to the Immunoplate and then selectively binds to the IgE antibody. (520) was prepared by diluting to a concentration of 1 mg / mL. The sample (serum or plasma, etc.) was dispensed on an Immunoplate and then reacted at 37 ° C. for about 6 hours and washed, and then the prepared solution was dispensed and reacted at 37 ° C. for about 6 hours.

(3) Preparation of color diagnostic reagent

In order to remove the anti-IgE antibody-H-Pt nanoparticles that are not bound to IgE, washing was performed, and reagents for color diagnosis were prepared.

TMB (3,3 ', 5,5'-Tetramethylbenzidine liquid substrate system for ELISA, Sigma) and 1M hydrogen peroxide (H ₂ O ₂ ) for enzyme immunoassay and sodium acetate buffer solution (pH 4.0) and 7: 1: Mix the mixture at 2 ratios to prepare a color diagnostic reagent, and dispense 100 μL into each well. Finally, after reacting for 20 minutes at 45 ℃ visually confirmed the color signal, the color signal was confirmed by measuring the absorbance of the ELISA meter.

(4) IgE antibody detection and diagnosis process

The allergy diagnostic Immunoplate kit is used to detect total IgE antibodies or allergen-specific IgE antibodies as targets in the sample. The total IgE antibody or allergen-specific IgE antibody in the sample binds to the second anti-IgE antibody or allergen, which is a fixed substance in each well of the Immunoplate, and the allergy diagnostic enzyme-mimicking nanoparticles are labeled on the nanoparticle surface. When combined with the total IgE antibody or allergen-specific IgE antibody in the sample through the first anti-IgE antibody, the enzyme-mimicking nanoparticles react with a chromogenic substrate to amplify the detection signal. From the amplified detection signal, the presence of total IgE antibody or allergen-specific IgE antibody in the sample is provided and information on allergic disease diagnosis is provided. If the IgE antibody is immobilized in place of the sample as a control substance, even if there is no IgE antibody in the sample, the first anti-IgE antibody of the nanoparticles is combined with the IgE antibody as the control substance and the signal is amplified so that the allergy diagnostic Immunoplate kit You can check whether it is working correctly or not.

In addition, different allergens may be placed in each well of the allergy diagnostic Immunoplate to examine the presence or absence of allergen-specific IgE antibodies against several allergens at once. According to this method, the allergen-specific IgE antibody can be identified easily and easily in place of the painful skin test, thereby providing information on the diagnosis of the allergic disease in the subject. Done.

The embodiments of the present invention have been described in detail above. For those of ordinary skill in the art, it will be apparent that such specific techniques are only preferred embodiments, and thereby the scope of the present invention is not limited. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

In the test method using the allergy diagnostic Immunoplate,
The inspection method
(1) immobilizing a fixing material capable of binding to the target material of the sample to the well of the immunoplate;
(2) dispensing a sample in which the target material is present into each well of the Immunoplate to bind the fixed material and the target material;
(3) binding the allergy diagnostic enzyme-mimicking nanoparticles labeled with the first anti-IgE antibody to the target material dispensed into each well of the Immunoplate; And
(4) after the first anti-IgE antibody binds to the fixation material through the target material, the allergy diagnostic enzyme-mimicking nanoparticle reacts with a color substrate to amplify a detection signal; The allergy diagnostic enzyme-mimicking nanoparticles include a platinum nanoparticle in a hierarchical structure,
The target material of the sample is total IgE antibody or allergen-specific IgE, the fixative material is a second anti-IgE antibody or allergen,
The allergy diagnostic enzyme-mimicking nanoparticles are monodisperse inorganic nanoparticles, and the monodisperse inorganic nanoparticles are synthesized monodisperse by uniformly adjusting the size using a surfactant in an organic solvent environment.
The allergy diagnostic enzyme-mimicking nanoparticles labeled with the first anti-IgE antibody replace the surface of the nanoparticles with carboxylic acid,
The first anti-IgE antibody reacts with the carboxyl group of the carboxylic acid to bind to the nanoparticles,
The test method is a test method using the allergy diagnostic Immunoplate, characterized in that the diagnosis is possible from pH 2 to 13.
delete delete The method of claim 1,
The allergens include grasses, bark, animal epidermis or hair, insects, mites, house dust, parasites, microorganisms, bee venom, drugs, occupational allergens, fruits, vegetables, seeds, nuts, spices, seafood, eggs, poultry, meat , Dairy products, food additives, the test method using the allergy diagnostic Immunoplate, characterized in that at least any one selected from the group consisting of.
The method of claim 1,
A test method using an allergy diagnostic Immunoplate, wherein different types of allergens are immobilized in each well of the immunoplate so that the presence of the allergen-specific IgE antibody against each allergen can be detected simultaneously with a single test. .
delete The method of claim 1,
The sample is a test method using an allergy diagnostic Immunoplate comprising tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.
delete delete delete The method of claim 1,
The allergy diagnostic enzyme-mimicking nanoparticles have an enzyme mimic activity of peroxidase or oxidase and are an oxidation catalyst of the chromogenic substrate.
The method of claim 11,
The allergy diagnostic enzyme-mimicking nanoparticles test method using allergy diagnostic Immunoplate, characterized in that to precipitate or insolubilize the chromogenic substrate.
The method of claim 12,
The chromogenic substrate is 3-amino-9-ethylcarbazole (3-amino-9-ethylcarbazole), characterized in that the test method using allergy diagnostic Immunoplate.
The method of claim 1,
The allergy is edema, anaphylaxis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergies, food allergies, allergic asthma or An allergy diagnosis method characterized in that the allergic disease, characterized in that any one selected from the group consisting of drug allergies.
An allergy diagnostic Immunoplate kit comprising a composition comprising the allergic diagnostic enzyme-mimicking nanoparticle for performing the test method using the allergy diagnostic Immunoplate according to claim 1, and the immobilizing material capable of binding to a target material of a sample.
The method of claim 15,
A different type of allergen is immobilized in each well of the immunoplate so that the presence of an allergen-specific IgE antibody for each allergen can be detected simultaneously with a single test. .
(1) detecting an allergen-specific IgE antibody as a target substance in a sample by using the allergy diagnostic Immunoplate kit of claim 15;
(2) amplifying a detection signal by reacting the allergen-diagnosing enzyme-mimicking nanoparticles associated with the allergen-specific IgE antibody with a chromogenic substrate;
(3) identifying, from the amplified detection signal, an allergen that is a fixed substance bound to the allergen-specific IgE antibody; And
(4) determining an allergic disease by confirming the identity of the allergen;
Method for providing information regarding the diagnosis of allergen-specific allergic diseases comprising a.

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