WO2021015123A1

WO2021015123A1 - Method for producing allergen-immobilized carrier, and method for detecting allergen-specific antibody

Info

Publication number: WO2021015123A1
Application number: PCT/JP2020/027835
Authority: WO
Inventors: 和美山下; 正照伊藤; 大河荒井; 翔若山
Original assignee: 東レ株式会社
Priority date: 2019-07-25
Filing date: 2020-07-17
Publication date: 2021-01-28
Also published as: JPWO2021015123A1

Abstract

Disclosed are: a method for producing an allergen-immobilized carrier which is for an allergy examination and with which an allergen-specific antibody can be detected with high sensitivity; and a method for detecting an allergen-specific antibody using said carrier. The method for producing an allergen-immobilized carrier for detecting an allergen-specific antibody comprises: step [1] for heating an allergenic extract to obtain a heat-treated material of the allergenic extract; and step [2] for immobilizing, on a carrier, the heat-treated material obtained in step [1]. The method for detecting an allergen-specific antibody in a specimen comprises a step for bringing a specimen into contact with the allergen-immobilized carrier produced by said method, and detecting a specific antibody, in the specimen, which forms a complex with the allergen immobilized on the carrier.

Description

Method for producing allergen-immobilized carrier and method for detecting allergen-specific antibody

The present invention relates to a method for producing an allergen-immobilized carrier for detecting an allergen-specific antibody and a method for detecting an allergen-specific antibody using the allergen-immobilized carrier.

When foreign substances such as bacteria and viruses invade the body, antibodies that bind to them are produced, and there is an immune system that eliminates the foreign substances and protects the body. However, even for foreign substances that are harmless to most people, such as pollen, food, and mites, antibodies that bind to them are produced, causing excessive immunity and causing allergic reactions that injure oneself. There is a person with a constitution.

When a foreign substance invades the body, various antibodies such as IgM, IgG, and IgA are produced, but it is the IgE antibody that is directly involved in the induction of allergies, and the IgE antibody that specifically binds to the allergen that causes allergies is specific. It is called an IgE antibody. When allergens that have invaded the body bind to specific IgE antibodies, various chemical messengers such as histamine are released from mast cells, etc., and have actions such as vasodilation, bronchial smooth muscle contraction, and increased permeability of capillaries. Allergic symptoms such as redness, cough, and nasal discharge appear. If the symptoms are severe, anaphylactic reactions due to shock symptoms such as dyspnea and decreased blood pressure may occur, leading to a life-threatening and dangerous condition. Therefore, it is necessary to identify the causative substance of allergic symptoms, connect it to treatment, and avoid contact with and ingestion of allergens to prevent allergic symptoms. Therefore, it is strongly desired that allergy tests be detected with high sensitivity and accuracy.

Allergy tests are mainly divided into "skin test", "oral stress test (food allergy)" and "blood test".

In the skin test, a dilute solution is prepared from extracts such as pollen, food, and mites, placed on the skin and pointed or scratched with a fine needle to the extent that it does not bleed (prick test, scratch test), or injected intradermally (prick test, scratch test). This is a method of observing wheals during an intradermal test). Although the skin test has high sensitivity, it has a problem of low specificity, and there is a problem of negative / positive discrimination performance.

In addition, for food allergies, the intradermal test has a high possibility of shock and a high false positive rate, so it is a test to confirm the presence or absence of allergic symptoms by ingesting a small amount of food that is suspected to be allergic. A load test is performed. However, even with this method, there is still a risk of shock, so it is limited to implementation only in facilities where emergency response is sufficient.

On the other hand, in the allergy test by blood test, the amount of histamine released from the basophils is measured by reacting the basophils in the collected blood with a dilute solution from extracts such as pollen, food and mites. (Histamine release test). It is a minimally invasive test called blood sampling, and has the advantage of being able to reflect in vivo reactions, but about 20% of cases have Low-Responders that show low reactions, and these are not applicable to the histamine release test. turn into. In addition, since it is necessary to test the refrigerated blood within 3 days, it is not widely used in clinical practice.

Currently, the allergy test that is generally performed in clinical practice is a method of measuring the amount of specific IgE antibody in blood.

For the detection of specific IgE antibody, an allergen-immobilized carrier in which the allergen is immobilized on the carrier is prepared, and a blood (serum / plasma) sample is reacted with the allergen-immobilized carrier to capture the specific IgE antibody that binds to the allergen. This is done by detecting a complex of an allergen and a specific antibody using an enzyme or a fluorescently labeled anti-IgE antibody. A specific example of a specific IgE antibody test method widely used in clinical practice is the CAP (capsulated hydrophilic carrier polymer) method of Thermo Fisher Scientific Co., Ltd.

Non-Patent Document 1 describes a method in which a carrier in which an allergen is immobilized on a porous cellulose sponge is used in order to improve the sensitivity of the CAP method, and the amount of allergen immobilized per carrier is increased by increasing the surface area. Has been done.

Further, Patent Document 1 describes a chip for allergy diagnosis in which allergens are extracted from a heated food (garlic) and an unheated food, and these extracted allergens are individually spotted.

Special Table 2004-533625

In recent years, the types of allergies have been subdivided, and the number of test items tends to increase. Therefore, there is a demand for a new method that can detect with high sensitivity even from a smaller amount of sample.

The present invention meets such needs, and provides a method for producing an allergen-immobilized carrier for an allergy test capable of detecting an allergen-specific antibody with high sensitivity, and a method for detecting an allergen-specific antibody using the carrier. To do.

As a result of diligent research to solve the above problems, the present inventors heat-treated the allergen extract extracted from the allergen raw material in the production of the allergen-immobilized carrier for allergy test, and heat-treated the allergen extract. It was found that by immobilizing the substance on the carrier, the amount of allergen immobilized is increased as compared with the carrier on which the allergen extract not subjected to the heat treatment is immobilized. Further, they have found that a specific antibody can be detected with higher sensitivity than before by using an allergen-immobilized carrier prepared by this method, and completed the present invention.

The present invention provides the following.

(1) A method for producing an allergen-immobilized carrier for detecting an allergen-specific antibody.
A step of heating the allergen extract to obtain a heat-treated product of the allergen extract [1], and a step of immobilizing the heat-treated product obtained in the above step [1] on a carrier [2].
A method for producing a carrier, including.
(2) The production method according to (1), wherein the heating temperature in the step [1] is 60 ° C. or higher and lower than 100 ° C.
(3) The production method according to (1) or (2), wherein the specific antibody is a specific IgE antibody.
(4) The method according to any one of (1) to (3), wherein in the step [2], the heat-treated product is immobilized on the carrier by a covalent bond.
(5) A sample is brought into contact with an allergen-immobilized carrier produced by any of the methods (1) to (4), and a specific antibody in the sample forming a complex with the allergen immobilized on the carrier is obtained. A method for detecting an allergen-specific antibody in a sample, which comprises a step of detecting.

In the production of an allergen-immobilized carrier for allergy testing, by immobilizing the allergen extract after heat treatment, it is possible to obtain an allergen-immobilized carrier in which more allergens are immobilized than when used without heating. Become. Further, by using the carrier, it becomes possible to detect an allergen-specific antibody with high sensitivity even with a small amount of sample.

The allergen extract used in the present invention is an extract of a protein contained in an allergic raw material.

Allergy raw materials are a general term for materials containing allergens that cause allergies, and specific examples thereof include microorganisms, plants, animals, insects and house dust, and foods containing these. Whether or not it causes allergies depends on the constitution.

Examples of microorganisms containing allergens include Alternaria, Aspergillus, Candida, Malassezia, and mites.

Examples of plants containing allergens include sugi, hinoki, alder, birch, Dactylis, ragweed, mugwort and timothy, and pollen of these.

Examples of animals containing allergens include cats, dogs, mice, rats, hamsters, rabbits, inco, ducks and the like. Examples of insects containing allergens include moths, cockroaches and bees.

Examples of foods containing allergens include eggs, milk, wheat, peanuts, soybeans, soba, sesame, rice, shrimp, crab, squid, octopus, kiwi, banana, apple, peach, tomato, garlic, tuna, salmon, mackerel. , Beef, pork, chicken and so on.

As the allergen extract, a commercially available one may be used, or one extracted from allergen raw materials by oneself may be used. Commercially available allergen extracts can be purchased from ITEA Inc., Cosmo Bio Co., Ltd., and the like. The method for extracting the allergen extract is not particularly limited, but it may be extracted by a known method. Specifically, it can be extracted by the following method.

First, the allergen raw material is finely chopped or crushed after freeze-drying, homogenized by adding a solvent, extracted overnight at 4 ° C., and then centrifuged to obtain a supernatant. Subsequently, the supernatant is filtered with a filter paper, gauze, a filter or the like several times, and then centrifuged to obtain a supernatant. Further, the supernatant obtained by dialyzing the supernatant with a buffer solution or further freeze-drying can be used as the allergen extract of the present invention.

In the above extraction method, the solvents used for extraction include ion-exchanged water, phosphate buffered saline (PBS), sodium phosphate buffer, sodium hydrogen carbonate aqueous solution, Tris buffer, acetate buffer, and citrate buffer. A carbonate buffer, Good's buffer, or other solution suitable for dissolving proteins may be used. Among these, sodium phosphate buffer solution and sodium hydrogen carbonate aqueous solution are preferably used as solvents. Further, in order to stabilize the allergen, saccharides such as Dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), alcohols such as glycerol, methanol or ethanol, glucose, fructose and trehalose are added at the time of extraction. You may.

The allergen extract used in the present invention may be a crude extract or a purified product obtained by purifying the crude extract. An example of a method for purifying a crude extract is, for example, in the case of an allergic raw material containing a large amount of lipid, a method of removing lipid by freeze-drying, pulverizing, treating with hexane, obtaining pellets and drying. Examples thereof include a method of removing impurities by ion exchange chromatography, gel chromatography, ultrafiltration and the like, and isolation of a specific protein.

The present invention is characterized in that the allergen extract is heat-treated and then immobilized on a carrier. The heat treatment temperature of the allergen extract is preferably 60 ° C. or higher and lower than 100 ° C., more preferably 80 ° C. or higher and lower than 100 ° C., and most preferably 80 ° C. or higher and 95 ° C. or lower. The heating time can be appropriately set according to the amount of the allergen extract to be treated, but is preferably 1 minute or more. There is no particular upper limit to the heating time, and the heating time is, for example, 60 minutes or less, particularly 30 minutes or less, but usually 10 minutes is sufficient.

The heat treatment of the allergen extract is preferably performed by dissolving the allergen extract in a liquid. As the solution of the allergen extract, the solvent used in the above allergen extraction can be used. When the solvent used for extracting the allergen is unknown, such as when a commercially available allergen extract is used, the liquid for dissolving the allergen extract is phosphate buffered saline (PBS), ion-exchanged water, or sodium phosphate. A solvent suitable for dissolving a buffer solution, an aqueous sodium hydrogen carbonate solution, a Tris buffer solution, an acetate buffer solution, a citrate buffer solution, a carbonate buffer solution, Good's buffer, and other proteins can be used.

The heat treatment of the allergen extract may be carried out in the presence of a surfactant. Surfactants are preferably used because they have a protein solubilizing effect and a coagulation preventing effect during heat treatment. Further, when the heat-treated allergen extract is immobilized on the carrier, the presence of the surfactant improves the wettability and enables uniform immobilization. The effect of improving wettability and enabling uniform immobilization can be obtained even when a surfactant is added after the heat treatment, so that the heat treatment is performed in the absence of the surfactant. Even in this case, it is also preferable to add a surfactant after the heat treatment.

There are various types of surfactants such as anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants, all of which can be preferably used. Examples of the anionic surfactant include sodium dodecyl sulfate (SDS) and the like. Examples of amphoteric tenside agents include 3-[(3-Cholamidopropyl) dimethyramminio] propanesulfonate (CHAPS) and 3-[(3-Cholamidoplopyl) dimethyramminio) -2-hydropypropanesulfone. Nonionic surfactants include Triton X-100 (trademark), Triton X-114 (trademark), NP-40, Brij-35, Brij-58, Tween20 (trademark), Tween80 (trademark), Octyl Glucside, Octyl thioglucoside, etc. Can be mentioned. Among these, SDS and Tween 20 ™ are particularly preferably used. If the concentration of the surfactant is too high, the heat-treated allergen extract may be denatured to the extent that it cannot bind to the specific IgE antibody. Therefore, when SDS or Tween 20 (trademark) is used, it is preferable to add the allergen extract to the solution to be heat-treated so that the final concentration is 0.001 to 0.1% by volume. It is preferable to add the mixture in an amount of 0.003 to 0.05% by volume.

The allergen-immobilized carrier produced by the method of the present invention is contained in the sample, which forms a complex with the allergen on the surface of the carrier by contacting the sample with the carrier on which the heat-treated product of the allergen extract is immobilized. It is possible to detect a specific antibody such as a specific IgE antibody. That is, by using the allergen-immobilized carrier provided by the present invention, it is possible to detect a specific antibody that forms a complex with the allergen in the sample.

If heat-treated products of a plurality of allergen extracts are immobilized on a plurality of locations on one carrier, and each heat-treated product can be contacted with a sample in one reaction field (described later), a smaller amount is required. It is more preferable because a plurality of allergen-specific IgE antibodies can be simultaneously tested from the sample.

Examples of the carrier material for immobilizing the heat-treated product of the allergen extract include glass, resin, metal, metal oxide, gel, cellulose, carbon material, etc., but any carrier capable of immobilization can be used. Not limited. As the resin used as the material of the carrier, polystyrene, polycarbonate, polyethylene, polypropylene, polymethylmethacrylate, polyethylene terephthalate, polyether sulfone, polybutylene terephthalate, polybutylene succinate, polylactic acid, polyamide resin, ABS and the like are used. be able to. As the metal used as the material of the carrier, gold, silver, copper, platinum, titanium, palladium, iron, aluminum, nickel, alloys thereof and the like can be used. As the metal oxide used as the material of the solid phase carrier, silicon dioxide, aluminum oxide, cerium oxide, zirconium oxide, composite oxide and the like can be used.

The shape of the carrier may be appropriately selected from plate-like, thin film, particle-like, porous, etc., but is preferably plate-like. As the material of the plate-shaped carrier, a resin material such as polymethylmethacrylate, polystyrene, or polypropylene, which can be produced by injection molding, is preferable from the viewpoint of productivity.

As a method of immobilizing the heat-treated product of the allergen extract on the carrier, there are physical adsorption and chemical bond, but the method of chemically immobilizing is preferable. Further, among the chemical immobilizations, a bond by a covalent bond is preferable. In order to immobilize the heat-treated product of the allergen extract on the carrier by covalent bonding, a carrier having a functional group capable of binding to the heat-treated product of the allergen extract is used. For example, by generating a carboxyl group on the carrier by a known method, it can be covalently immobilized with the amino group of the heat-treated product of the allergen extract. Specifically, when the carrier is an acrylic resin such as polymethylmethacrylate, the following methods can be mentioned. The surface of the carrier is hydrolyzed with an alkaline aqueous solution such as caustic soda to generate a carboxyl group on the resin surface, and then the carboxyl group and the amino group contained in the heat-treated product of the allergen extract are directly covalently bonded (amide). It can be fixed by binding). In addition, a maleimide group is introduced into the carboxyl group generated on the carrier by an ester bond, and the maleimide group is covalently bonded to the thiol group derived from the cysteine residue contained in the heat-treated product of the allergen extract. It can also be fixed by. The method itself for covalently bonding a protein to a carboxyl group on a carrier is well known, and is specifically described in the following examples.

Further, when the heat-treated product of the allergen extract is chemically immobilized by a covalent bond, the heat-treated product of the allergen extract can be dissolved in a solvent. Specific examples of the solvent include phosphate buffered saline (PBS), ion-exchanged water, sodium phosphate buffer, sodium hydrogen carbonate aqueous solution, Tris buffer, acetate buffer, citrate buffer, carbonate buffer, Good. A solvent suitable for dissolving's buffer and other proteins can be used. If the same solvent can be used consistently from dissolving, heating, and immobilizing the allergen extract to the carrier, no solvent replacement operation is required on the way, and the immobilization carrier of the heat-treated product of the allergen extract is not required. It is preferable because a simple process can be adopted for producing the above. As the solvent in this case, PBS, sodium phosphate buffer solution, and sodium hydrogen carbonate aqueous solution are preferably used.

As a method of bringing the solution of the heat-treated product of the allergen extract into contact with the carrier, a method of immersing the carrier in the solution or a method of dropping the solution onto the carrier can be used. If the allergen and the sample are immobilized so that they can come into contact with each other at the same time in one reaction field, multiple allergies can be detected from a smaller number of samples at the same time. Therefore, on the same plate-like carrier using a known spotting device. It is preferable to spot and immobilize the solutions of the heat-treated products of the plurality of allergen extracts. Here, "contacting the allergen and the sample in one reaction field" means that the sample is added and brought into contact with a carrier in which heat-treated products of a plurality of allergen extracts are independently immobilized without partition walls. This means that the specific antibody in the sample can be freely contacted in the same space as the heat-treated product of any of the immobilized allergen extracts. By detecting the specific antibody bound to the heat-treated product of each allergen extract, it is possible to simultaneously detect a plurality of specific antibodies that cause allergies. Compared to a system that can detect one type of specific antibody in one reaction field, if the reaction and detection can be performed in one reaction field without a partition wall, the amount of sample required for testing per allergy item will be reduced. be able to.

The amount of immobilized allergen can be determined by a method of directly quantifying the amount of protein of the allergen on the surface of the carrier, or by reacting the immobilized allergen with a solution containing an acid or a reducing agent to dissociate the allergen from the carrier. Any method of quantifying the amount of protein present in the protein by the BCA method, the Bradford method, the ultraviolet absorption altitude method, or the like can be used. Of these, since it is not easy to disperse an allergen covalently bonded to the carrier, for example, an amide bond from the carrier, a method of directly quantifying the amount of allergen protein on the surface of the carrier is preferable. Examples of the method for quantifying the protein amount of the allergen on the surface of the carrier include observation with a high-speed atomic force microscope and surface plasmon resonance method. When the carrier is made of resin, time-of-flight secondary ion mass spectrometry is used. The quantitative method used is preferable.

The sample used in the present invention is preferably derived from a body fluid. Body fluids include blood, sweat, urine, tears, saliva, sputum / airway secretions, breast milk, amniotic fluid, cerebrospinal fluid, ascites, pleural fluid, joint fluid, semen, vaginal discharge, etc., but specific IgE antibodies. Blood containing a large amount of specific antibodies such as the above is preferable. Further, the sample may be pretreated if necessary. For example, in the case of blood, serum or plasma can also be preferably used.

In the present invention, the complex of the allergen on the surface of the carrier and the specific antibody can be detected by the following method. For example, a detection method using a surface plasmon resonance method based on a difference in refractive index due to coupling or a quartz crystal microbalance method using a difference in resonance frequency as a detection principle can be mentioned. Further, a method using a secondary antibody such as a fluorescent substance (fluorescent dye), an enzyme that produces a color-developing / luminescent substance, and an anti-IgE antibody modified (labeled) with a radioisotope element or the like can also be mentioned. Among these, a method using an anti-IgE antibody modified with a fluorescent substance, which is safe and easy to handle, is preferable.

When detecting a complex formed by an allergen and a specific antibody using the allergen-immobilized carrier provided by the present invention, not only the detection of the specific antibody but also the amount of the specific antibody should be quantified. You can also. For the quantification of the specific IgE antibody, it is preferable to use an anti-IgE antibody modified with a fluorescent substance. Specific methods for quantifying the amount of specific IgE antibody contained in a sample using the method of the present invention include the following methods.

Quantitative immunoassays in specific allergy diagnosis usually use or refer to the World Health Organization IgE International Standard (WHO International Standard immunoglobulin (IgE), human serum) to construct a calibration system. This international standard product is a freeze-dried sample of total IgE antibody derived from human serum (The 3rd International Standard for serum IgE: report of the international collaborative group 21 BS / 2013.2220), for example, can be used to prepare total IgE antibody standard solutions of various concentrations and quantified by comparing the measured values of the standard and the sample.

The present invention will be described in more detail with reference to the following examples.

<Allergen extract>
The extracts of each allergen used in the following Examples and Comparative Examples were obtained from the following purchasers or extracted from allergen raw materials.

The crab allergen extract was purchased from Torii Pharmaceutical Co., Ltd. The cockroach allergen extract was purchased from Biosuta. The alder pollen allergen extract was purchased from ITEA. The ragweed allergen extract was purchased from Torii Pharmaceutical Co., Ltd. The tomato allergen extract was purchased from Torii Pharmaceutical Co., Ltd. The birch pollen allergen extract was purchased from ITEA. The cypress pollen allergen extract was purchased from ITEA. The banana allergen extract was prepared by the method of Reference Example 1 using banana as an allergic raw material. The heated garlic allergen extract was prepared by heating raw garlic at 95 ° C. for 20 minutes as an allergen raw material by the method of Reference Example 1. The soybean allergen extract was purchased from Stallergenes Greer.

Reference Example 1 Preparation of allergen extract The allergen raw material was finely ground, 3 times the weight of PBS was added and stirred, and the mixture was extracted overnight at 4 ° C. and then centrifuged to obtain a supernatant. Subsequently, the supernatant was filtered through a filter paper, and the supernatant obtained by centrifugation was dialyzed against PBS to obtain an allergen extract.

Reference Example 2 Preparation of allergen-immobilized carrier for specific antibody detection In the following Examples and Comparative Examples, an allergen-immobilized carrier for specific antibody detection prepared by the following method was used.

A polymethylmethacrylate (PMMA) resin substrate (manufactured by Kuraray, outer diameter 75 mm × 25 mm × 1 mm, average molecular weight 30,000) was used as a carrier. The substrate was immersed in 10 specified sodium hydroxide at 70 ° C. for 14 hours, washed with pure water three times, and dried. In this way, the side chain of PMMA on the surface of the substrate was hydrolyzed to generate a carboxyl group. Then, the substrate was washed 3 times with 50 mM MES buffer (pH 6), and 1-ethyl-3- (3-dimethylaminopropyl) was added to 50 mM MES buffer (pH 6) containing 20% N, N-dimethylformamide (DMF). ) Carbodiimide hydrochloride (EDC) (manufactured by Wako Junyaku Co., Ltd.) and sulfo-N-hydroxysuccinimide (Sulfo-NHS) (manufactured by Wako Junyaku Co., Ltd.) are immersed in a solution prepared to have a concentration of 5% by weight at room temperature for 1 hour. Then, it was washed 3 times with 0.5 mM hydrochloric acid to obtain an NHS esterified PMMA resin substrate.

Next, the same NHS produced above by using a spotting robot (Nippon Laser Electronics Co., Ltd., GTMASStammp-2) for the heat-treated product (Example) or non-heat-treated product (Comparative Example) of each allergen extract. Four spots were spotted on the esterified PMMA resin substrate. The substrate was then placed in a closed plastic container and incubated overnight at 37 ° C. and 100% humidity to immobilize heat-treated or non-heat-treated products of each allergen extract. Subsequently, the cells were washed 3 times with PBS (PBST) containing 0.05% Tween 20 ™ and dried to obtain an allergen-immobilized carrier for detecting specific antibodies.

Reference Example 3 Detection of Allergen-Specific Antibody In the following Examples and Comparative Examples, in order to detect the allergen-specific antibody present in the sample by the following method, a fluorescent dye-labeled anti-IgE antibody was used to detect the fluorescence intensity. Was measured.

The allergen-immobilized carrier for detecting specific antibodies prepared in Reference Example 2 was immersed in a PBS solution containing 1 wt% bovine serum albumin (BSA) (manufactured by Sigma-Aldrich) at 4 ° C. overnight for blocking treatment. , Washed 3 times with PBST.

A solution diluted 3-fold with a PBS aqueous solution containing 1 wt% BSA for 10 μL of each sample was added dropwise to the portion of the carrier on which the allergen was immobilized, and a gap cover glass (manufactured by Matsunami Glass Industry Co., Ltd .: 24 mm) was added dropwise. It was sealed with a cover (× 25 mm, gap size 20 μm). After reacting at 37 ° C. for 2 hours, the gap cover glass was removed and washed 3 times with PBST. Next, a 30 μL substrate was added with a Dilight-650 dye-labeled anti-human IgE goat polyclonal antibody (manufactured by Novus biologicals) diluted 1000-fold with PBST containing 1 wt% BSA, covered with a gap cover glass, and reacted at room temperature for 1 hour. It was. Then, the gap cover glass was removed, washed with PBST three times, dried, and the fluorescence intensity was measured with a scanner device (3D-GeneScanner 3000 manufactured by Toray Industries, Inc.) under the following conditions.

Fluorescence intensity measurement condition Device: 3D-GeneScanner3000 (manufactured by Toray Industries, Inc.)
Excitation light wavelength: 635 nm
Detection wavelength: 670 nm
PMT: 30.

Example 1 Preparation of immobilized carrier of heat-treated product of canial allergen extract, measurement of allergen-specific antibody The canial allergen extract was added to 20 mM sodium phosphate buffer (pH 7.0) so as to have a final concentration of 1 mg / mL. It was dissolved and the solution was prepared to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of a crab allergen was prepared by the method described in Reference Example 2.

Using this allergen-immobilized carrier, the fluorescence intensities of positive and negative samples were measured by the method described in Reference Example 3, and allergen-specific antibodies were detected.

As a positive sample, crab allergy-positive plasma was purchased from PlasmaLab and used. As a negative sample, plasma confirmed to be negative as a result of a crab allergy test by SRL, Inc. was used.

Table 1 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Comparative Example 1 Preparation of an immobilized carrier of a non-heat-treated product of a crab allergen extract and measurement of an allergen-specific antibody The same method as in Example 1 was carried out except that the heat treatment of the allergen extract was not carried out. .. The measurement results are shown in Table 1.

Example 2 Preparation of immobilized carrier of heat-treated product of canial allergen extract, measurement of allergen-specific antibody SDS as a surfactant during heat treatment of allergen extract was added to 20 mM sodium phosphate buffer (pH 7.0). The procedure was the same as in Example 1 except that the mixture was added so as to have a final concentration of 0.02% by volume. The measurement results are shown in Table 1.

Comparative Example 2 Preparation of immobilized carrier of non-heat-treated product of crab allergen extract, measurement of allergen-specific antibody The same method as in Example 2 was carried out except that the heat treatment of the allergen extract was not carried out. .. The measurement results are shown in Table 1.

As shown in Table 1, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the crab allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against crab can be detected with high sensitivity and high specificity by using the heat-treated product of the crab allergen extract.

Example 3 Preparation of immobilized carrier of heat-treated product of gokibrial allergen extract, measurement of allergen-specific antibody The gokibrial allergen extract was added to 20 mM sodium phosphate buffer (pH 7.0) to a final concentration of 1 mg / mL. It was dissolved and the solution was prepared to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of a cockroach allergen was prepared by the method described in Reference Example 2.

As a positive sample, cockroach allergy positive plasma was purchased from PlasmaLab and used. As a negative sample, plasma confirmed to be negative as a result of a cockroach allergy test by SRL, Inc. was used.

Table 2 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Example 4 Preparation of immobilized carrier of heat-treated product of cockroach allergen extract, measurement of allergen-specific antibody The same method as in Example 3 was carried out except that the heat treatment condition of the allergen extract was set to 60 ° C. .. The measurement results are shown in Table 2.

Comparative Example 3 Preparation of a non-heated immobilized carrier of cockroach allergen extract and measurement of allergen-specific antibody The same method as in Example 3 was carried out except that the allergen extract was not heat-treated. .. The measurement results are shown in Table 2.

Example 5 Preparation of immobilized carrier of heat-treated product of gokibrial allergen extract, measurement of allergen-specific antibody SDS as a surfactant during heat treatment of allergen extract was added to 20 mM sodium phosphate buffer (pH 7.0). The procedure was the same as in Example 3 except that the mixture was added so as to have a final concentration of 0.02% by volume. The measurement results are shown in Table 2.

Example 6 Preparation of immobilized carrier of heat-treated product of cockroach allergen extract, measurement of allergen-specific antibody The same method as in Example 5 was carried out except that the heat treatment condition of the allergen extract was set to 60 ° C. .. The measurement results are shown in Table 2.

Comparative Example 4 Preparation of a non-heated immobilized carrier of cockroach allergen extract and measurement of allergen-specific antibody The same method as in Example 5 was carried out except that the allergen extract was not heat-treated. .. The measurement results are shown in Table 2.

As shown in Table 2, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the cockroach allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against cockroach can be detected with high sensitivity and high specificity by using the heat-treated product of the cockroach allergen extract.

Example 7 Preparation of immobilized carrier of heat-treated product of Hannoki pollen allergen extract, measurement of allergen-specific antibody 20 mM sodium phosphate buffer (pH 7.0) so that the final concentration of Hannoki pollen allergen extract is 1 mg / mL. ) To prepare the solution to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added to a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of alder pollen allergen was prepared by the method described in Reference Example 2.

As a positive sample, alder pollen allergy-positive plasma was purchased from PlasmaLab and used. As a negative sample, plasma confirmed to be negative as a result of the alder pollen allergy test of SRL, Inc. was used.

Table 3 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Example 8 Preparation of immobilized carrier of heat-treated product of alder pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 7 was carried out except that the heat treatment condition of the allergen extract was set to 60 ° C. did. The measurement results are shown in Table 3.

Comparative Example 5 Preparation of a non-heated immobilized carrier of alder pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 7 was carried out except that the allergen extract was not heat-treated. did. The measurement results are shown in Table 3.

Example 9 Preparation of immobilized carrier of heat-treated product of Hannoki pollen allergen extract, measurement of allergen-specific antibody SDS was used as a surfactant during heat treatment of allergen extract, and 20 mM sodium phosphate buffer (pH 7.0). It was carried out in the same manner as in Example 7 except that it was added and used so as to have a final concentration of 0.02% by volume. The measurement results are shown in Table 3.

Example 10 Preparation of immobilized carrier of heat-treated product of alder pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 9 was carried out except that the heat treatment condition of the allergen extract was set to 60 ° C. did. The measurement results are shown in Table 3.

Comparative Example 6 Preparation of non-heated immobilized carrier of alder pollen allergen extract, measurement of allergen-specific antibody Performed in the same manner as in Example 9 except that the allergen extract was not heat-treated. did. The measurement results are shown in Table 3.

As shown in Table 3, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the alder pollen allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against alder pollen can be detected with high sensitivity and high specificity by using the heat-treated product of the alder pollen allergen extract.

Example 11 Preparation of immobilized carrier of heat-treated product of ragweed pollen allergen extract, measurement of allergen-specific antibody 20 mM sodium phosphate buffer (pH 7.0) so that the final concentration of ragweed pollen allergen extract is 1 mg / mL. ) To prepare the solution to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of ragweed pollen allergen was prepared by the method described in Reference Example 2.

As a positive sample, ragweed pollen allergy-positive plasma was purchased from Abbaltis and used. As a negative sample, plasma confirmed to be negative as a result of the ragweed pollen allergy test of SRL, Inc. was used. Table 4 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Comparative Example 7 Preparation of immobilized carrier of non-heat-treated product of ragweed pollen allergen extract, measurement of allergen-specific antibody Performed in the same manner as in Example 11 except that the heat treatment of the allergen extract was not performed. did. The measurement results are shown in Table 4.

Example 12 Preparation of immobilized carrier of heat-treated product of ragweed pollen allergen extract, measurement of allergen-specific antibody SDS was used as a surfactant during heat treatment of allergen extract, and 20 mM sodium phosphate buffer (pH 7.0). It was carried out in the same manner as in Example 11 except that it was added and used so as to have a final concentration of 0.02% by volume. The measurement results are shown in Table 4.

Comparative Example 8 Preparation of immobilized carrier of non-heat-treated product of ragweed pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 12 was carried out except that the heat treatment of the allergen extract was not carried out. did. The measurement results are shown in Table 4.

As shown in Table 4, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the ragweed pollen allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against ragweed pollen can be detected with high sensitivity and high specificity by using the heat-treated product of the ragweed pollen allergen extract.

Example 13 Preparation of immobilized carrier of heat-treated product of tomato allergen extract, measurement of allergen-specific antibody Put the tomato allergen extract in 20 mM sodium phosphate buffer (pH 7.0) so as to have a final concentration of 1 mg / mL. It was dissolved and the solution was prepared to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of tomato allergen was prepared by the method described in Reference Example 2.

As a positive sample, tomato allergy-positive plasma was purchased from PlasmaLab and used. As a negative sample, plasma confirmed to be negative as a result of a tomato allergy test by SRL, Inc. was used.

Table 5 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Comparative Example 9 Preparation of immobilized carrier of non-heat-treated tomato allergen extract, measurement of allergen-specific antibody The same method as in Example 13 was carried out except that the allergen extract was not heat-treated. ..

Example 14 Preparation of immobilized carrier of heat-treated product of tomato allergen extract, measurement of allergen-specific antibody SDS was added as a surfactant during heat treatment of allergen extract. The procedure was the same as in Example 13 except that SDS was added to a 20 mM sodium phosphate buffer solution (pH 7.0) so as to have a final concentration of 0.02% by volume.

Comparative Example 10 Preparation of an immobilized carrier of a non-heat-treated tomato allergen extract and measurement of allergen-specific antibody The same method as in Example 14 was carried out except that the allergen extract was not heat-treated. ..

As shown in Table 5, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the tomato allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against tomato can be detected with high sensitivity and high specificity by using the heat-treated product of the tomato allergen extract.

Example 15 Preparation of immobilized carrier of heat-treated product of Shirakamba pollen allergen extract, measurement of allergen-specific antibody 20 mM phosphorus containing 0.02% by volume SDS of Shirakamba pollen allergen extract to a final concentration of 1 mg / mL. It was dissolved in sodium acid buffer (pH 7.0) to prepare 100 μL of the solution. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of the birch pollen allergen was prepared by the method described in Reference Example 2.

Using the specific allergen-immobilized carrier of the birch pollen allergen, the fluorescence intensity of the positive sample and the negative sample was measured by the method described in Reference Example 3, and the allergen-specific antibody was detected.

As a positive sample, birch pollen allergy-positive plasma was purchased from PlasmaLab and used. As a negative sample, plasma confirmed to be negative as a result of the birch pollen allergy test of SRL, Inc. was used.

Table 6 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Example 16 Preparation of immobilized carrier of heat-treated product of birch pollen pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 15 was carried out except that the heat treatment condition of the allergen extract was set to 60 ° C. did. The measurement results are shown in Table 6.

Comparative Example 11 Preparation of immobilized carrier of non-heat-treated birch pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 15 was carried out except that the allergen extract was not heat-treated. did. The measurement results are shown in Table 6.

As shown in Table 6, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the birch pollen allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against birch pollen can be detected with high sensitivity and high specificity by using the heat-treated product of the birch pollen allergen extract.

Example 17 Preparation of immobilized carrier of heat-treated product of hinoki pollen allergen extract, measurement of allergen-specific antibody 20 mM sodium phosphate buffer (pH 7.0) so that the final concentration of hinoki pollen allergen extract is 1 mg / mL. ) To prepare the solution to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added to a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of hinoki pollen allergen was prepared by the method described in Reference Example 2.

As a positive sample, hinoki pollen allergy-positive plasma was purchased from Abbaltis and used. As a negative sample, plasma confirmed to be negative as a result of the hinoki pollen allergy test of SRL, Inc. was used.

Table 7 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Example 18 Preparation of immobilized carrier of heat-treated product of hinoki pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 17 was carried out except that the heat treatment condition of the allergen extract was set to 60 ° C. did. The measurement results are shown in Table 7.

Comparative Example 12 Preparation of immobilized carrier of non-heat-treated hinoki pollen allergen extract, measurement of allergen-specific antibody The same method as in Example 17 was carried out except that the heat treatment of the allergen extract was not carried out. did. The measurement results are shown in Table 7.

As shown in Table 7, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the hinoki pollen allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against hinoki cypress pollen can be detected with high sensitivity and high specificity by using the heat-treated product of the hinoki cypress pollen allergen extract.

Example 19 Preparation of immobilized carrier of heat-treated product of banana allergen extract, measurement of allergen-specific antibody The banana allergen extract was diluted with PBS to a final concentration of 1 mg / mL, and the solution was prepared to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of the allergen extract, an allergen-immobilized carrier for detecting a specific antibody of a banana allergen was prepared by the method described in Reference Example 2.

Using the banana allergen-specific allergen-immobilized carrier, the fluorescence intensities of positive and negative samples were measured by the method described in Reference Example 3, and allergen-specific antibodies were detected.

As a positive sample, banana allergy-positive plasma was purchased from PlasmaLab and used. As a negative sample, plasma confirmed to be negative as a result of a banana allergy test by SRL, Inc. was used.

Table 8 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Comparative Example 13 Preparation of immobilized carrier of non-heat-treated banana allergen extract, measurement of allergen-specific antibody The same method as in Example 19 was carried out except that the heat treatment of the allergen extract was not carried out. .. The measurement results are shown in Table 8.

Example 20 Preparation of immobilized carrier of heat-treated banana allergen extract, measurement of allergen-specific antibody SDS was added to 20 mM sodium phosphate buffer (pH 7.0) as a surfactant during heat treatment of allergen extract. The procedure was the same as in Example 19 except that the mixture was added so as to have a final concentration of 0.02% by volume. The measurement results are shown in Table 8.

Comparative Example 14 Preparation of an immobilized carrier of a non-heat-treated banana allergen extract and measurement of allergen-specific antibody The same method as in Example 20 was carried out except that the allergen extract was not heat-treated. .. The measurement results are shown in Table 8.

As shown in Table 8, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the banana allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against banana can be detected with high sensitivity and high specificity by using the heat-treated product of the banana allergen extract.

Example 21 Preparation of immobilized carrier of heat-treated product of canial allergen extract, measurement of allergen-specific antibody The canial allergen extract was added to 20 mM sodium phosphate buffer (pH 7.0) so as to have a final concentration of 1 mg / mL. It was dissolved and the solution was prepared to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

As positive samples, allergy class 4 crab allergy positive plasma and allergy class 5 crab allergy positive plasma were obtained from PlasmaLab and used. As a negative sample, plasma confirmed to be negative (allergy class 0) as a result of a crab allergy test by SRL, Inc. was used.

The ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample and the result of measuring the fluorescence intensity by the method described in Reference Example 3 (positive sample (class 4) / negative sample, positive sample (class 5) / negative sample) , Fluorescence intensity ratio between positive samples (positive sample (class 5) / positive sample (class 4)) is shown in Table 9. Each measured value is an average value of the measured values of the four spots.

Example 22 Preparation of immobilized carrier of heat-treated product of crab allergen extract, measurement of allergen-specific antibody The same method as in Example 21 was carried out except that the heat treatment condition of the allergen extract was set to 90 ° C. .. The measurement results are shown in Table 9.

Example 23 Preparation of immobilized carrier of heat-treated product of crab allergen extract, measurement of allergen-specific antibody The same method as in Example 21 was carried out except that the heat treatment conditions of the allergen extract were set to 80 ° C. .. The measurement results are shown in Table 9.

Comparative Example 15 Preparation of immobilized carrier of non-heat-treated product of crab allergen extract, measurement of allergen-specific antibody The same method as in Example 21 was carried out except that the heat treatment of the allergen extract was not carried out. .. The measurement results are shown in Table 9.

As shown in Table 9, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the canylergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against crab can be detected with high sensitivity and high specificity by using the heat-treated product of the crab allergen extract.

In addition, the value of the fluorescence intensity ratio (positive sample (class 5) / positive sample (class 4)) of the allergy class 5 positive sample to the allergy class 4 positive sample is also the value when the heat-treated allergen extract is used. It was shown to be larger than when the non-heat-treated product was used. That is, it was shown that the allergen class can be discriminated with high accuracy and the concentration of the specific IgE antibody can be measured by using the allergen extract after heat treatment.

Example 24 Preparation of immobilized carrier of heat-treated product of canial allergen extract, measurement of allergen-specific antibody SDS was added as a surfactant during heat treatment of allergen extract to a final concentration of 20 mM sodium phosphate buffer pH 7.0. It was carried out in the same manner as in Example 21 except that it was added and used so as to be 0.02% by volume. The measurement results are shown in Table 10.

Example 25 Preparation of immobilized carrier of heat-treated product of crab allergen extract, measurement of allergen-specific antibody The same method as in Example 24 was carried out except that the heat treatment conditions of the allergen extract were set to 90 ° C. .. The measurement results are shown in Table 10.

Example 26 Preparation of immobilized carrier of heat-treated product of crab allergen extract, measurement of allergen-specific antibody The same method as in Example 24 was carried out except that the heat treatment conditions of the allergen extract were set to 80 ° C. .. The measurement results are shown in Table 10.

Comparative Example 16 Preparation of immobilized carrier of non-heat-treated product of crab allergen extract, measurement of allergen-specific antibody The same method as in Example 24 was carried out except that the heat treatment of the allergen extract was not carried out. .. The measurement results are shown in Table 10.

As shown in Table 10, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the crab allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that the specific IgE antibody against crab can be detected with high sensitivity and high specificity by using the heat-treated product of the crab allergen extract.

Reference Example 4 Measurement of the amount of allergen immobilized on the carrier A standard substrate in which a fixed amount of protein is immobilized on a PMMA resin substrate (protein amount per unit area between 0 mg / μm ² and 9.5E- ¹² mg / μm ² ) 7 levels) were prepared, and a mass spectrum was obtained by the flight time type secondary ion mass analysis method. Of these, a calibration curve was created to calculate the fixed amount of protein per unit area from the intensity ratio of the protein-derived peak and the PMMA-derived peak.

Time-of-flight secondary ion mass spectrometry was performed on the test carrier, and the amount of protein immobilized per unit area was calculated from the intensity ratio of the peak derived from the allergen protein and the peak derived from PMMA using the calibration curve. ..

Measurement conditions Device: TOF. SIMS 5 (manufactured by ION-TOF)
Primary ion: Bi ₃ ⁺⁺
Secondary ion polarity: Positive ions only.

Example 27 Measurement of allergen-immobilized amount of a carrier on which a heat-treated product of a crab allergen extract is immobilized The amount of allergen immobilized on an allergen-immobilized carrier for detecting a specific antibody of a crab allergen prepared in Example 1 , Measured by the method described in Reference Example 4. The results are shown in Table 11.

Comparative Example 17 Measurement of allergen-immobilized amount of a carrier on which a non-heat-treated product of a crab allergen extract was immobilized The amount of allergen immobilized on an allergen-immobilized carrier for detecting a specific antibody of a crab allergen prepared in Comparative Example 1. Was measured by the method described in Reference Example 4. The results are shown in Table 11.

Example 28 Measurement of allergen-immobilized amount of a carrier on which a heat-treated product of a crab allergen extract is immobilized The amount of allergen immobilized on an allergen-immobilized carrier for detecting a specific antibody of a crab allergen prepared in Example 2 , Measured by the method described in Reference Example 4. The results are shown in Table 11.

Comparative Example 18 Measurement of allergen-immobilized amount of a carrier on which a non-heat-treated product of a crab allergen extract was immobilized The amount of allergen immobilized on an allergen-immobilized carrier for detecting a specific antibody of a crab allergen prepared in Comparative Example 2. Was measured by the method described in Reference Example 4. The results are shown in Table 11.

As shown in Table 11, it was shown that the amount of allergen immobilized on the carrier was increased by heat-treating the crab allergen extract. That is, by heat-treating and immobilizing the allergen extract, more allergens are immobilized on the carrier as compared with the case where the non-heat-treated product is used, so that the allergen-specific antibody is detected with higher sensitivity. I found that I could do it.

Example 29 Preparation of immobilized carrier of heat-treated product of heated garlic allergen extract, measurement of allergen immobilization amount The heated garlic allergen extract is diluted with PBS to a final concentration of 0.2 mg / mL, and the solution is 100 μL. Prepared in. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of the heated garlic allergen was prepared by the method described in Reference Example 2, and the amount of the immobilized allergen was shown in Reference Example 4. It was measured by the method described. The results are shown in Table 12.

Comparative Example 19 Preparation of an immobilized carrier of a non-heat-treated product of a heated garlic allergen extract and measurement of the amount of allergen-immobilized allergen extract by the same method as in Example 29 except that the heat treatment of the allergen extract was not performed. An allergen-immobilized carrier for detecting a specific antibody of a heated garlic allergen was prepared, and the amount of the immobilized allergen was measured by the method described in Reference Example 4. The results are shown in Table 12.

Example 30 Preparation of immobilized carrier of heat-treated product of heated garlic allergen extract, measurement of allergen-immobilized amount SDS was added as a surfactant during heat treatment of allergen extract. For detection of specific antibody of heated garlic allergen by the same method as in Example 29 except that it was added to 20 mM sodium phosphate buffer (pH 7.0) so as to have a final concentration of 0.02% by volume. The allergen-immobilized carrier of No. 1 was prepared, and the amount of the immobilized allergen was measured by the method described in Reference Example 4. The results are shown in Table 12.

As shown in Table 12, the heated garlic allergen extract has already been heat-treated at the extraction stage (corresponding to the cooked garlic disclosed in Patent Document 1), but by the method of the present invention. It was shown that the amount of allergen immobilized on the carrier is increased by further heat-treating this and then immobilizing it.

Example 31 Preparation of immobilized carrier of heat-treated product of soybean allergen extract, measurement of allergen-specific antibody Soybean allergen extract was added to 20 mM sodium phosphate buffer (pH 7.0) so as to have a final concentration of 1 mg / mL. It was dissolved and the solution was prepared to 100 μL. Subsequently, the solution was heat-treated for 10 minutes under heating conditions of 95 ° C. Then, Tween 20 ™ was added so as to have a final concentration of 0.003% by volume to obtain a heat-treated product of the allergen extract.

Using the heat-treated product of this allergen extract, an allergen-immobilized carrier for detecting a specific antibody of soybean allergen was prepared by the method described in Reference Example 2.

As a positive sample, soybean allergy-positive plasma was purchased from PlasmaLab and used. As a negative sample, plasma confirmed to be negative as a result of a soybean allergy test by SRL, Inc. was used.

Table 13 shows the ratio of the fluorescence intensity of the positive sample to the fluorescence intensity of the negative sample (positive sample / negative sample) and the result of measuring the fluorescence intensity by the method described in Reference Example 3. Each measured value is an average value of the measured values of the four spots.

Comparative Example 20 Preparation of immobilized carrier of non-heat-treated soybean allergen extract, measurement of allergen-specific antibody The same method as in Example 31 was carried out except that the heat treatment of the allergen extract was not carried out. .. The measurement results are shown in Table 13.

Example 32 Preparation of immobilized carrier of heat-treated product of soybean allergen extract, measurement of allergen-specific antibody SDS was added to 20 mM sodium phosphate buffer (pH 7.0) as a surfactant during heat treatment of allergen extract. The procedure was the same as in Example 31 except that the mixture was added so as to have a final concentration of 0.02% by volume. The measurement results are shown in Table 13.

Comparative Example 21 Preparation of immobilized carrier of non-heat-treated soybean allergen extract, measurement of allergen-specific antibody The same method as in Example 32 was carried out except that the heat treatment of the allergen extract was not carried out. .. The measurement results are shown in Table 13.

As shown in Table 13, it was found that the fluorescence intensity of the positive sample was significantly increased by heat-treating the soybean allergen extract. In addition, for negative samples, no significant change was observed with or without heat treatment of the allergen extract. Furthermore, it was also confirmed that by heat-treating the allergen extract, the fluorescence intensity ratio (positive sample / negative sample) of the positive sample to the negative sample became larger than that in the case where the heat treatment was not performed. As described above, it was shown that specific IgE antibody against soybean can be detected with high sensitivity and high specificity by using a heat-treated product of soybean allergen extract.

Claims

A method for producing an allergen-immobilized carrier for detecting an allergen-specific antibody.
A step of heating the allergen extract to obtain a heat-treated product of the allergen extract [1], and a step of immobilizing the heat-treated product obtained in the above step [1] on a carrier [2].
A method for producing a carrier, including.
The method according to claim 1, wherein the heating temperature in the step [1] is 60 ° C. or higher and lower than 100 ° C.
The method according to claim 1 or 2, wherein the allergen-specific antibody is an IgE antibody.
The method according to any one of claims 1 to 3, wherein in the step [2], the heat-treated product is immobilized on the carrier by a covalent bond.
The step includes contacting a sample with an allergen-immobilized carrier produced by the method according to any one of claims 1 to 4, and detecting a specific antibody in the sample forming a complex with the allergen immobilized on the carrier. , A method for detecting an allergen-specific antibody in a sample.