WO2022158509A1 - アレルゲン固定化担体およびアレルゲン特異的抗体の検出方法 - Google Patents
アレルゲン固定化担体およびアレルゲン特異的抗体の検出方法 Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/686—Anti-idiotype
Definitions
- the present invention relates to an allergen-immobilized carrier for detecting allergen-specific antibodies and a method for detecting allergen-specific antibodies using the carrier.
- Allergy tests are usually performed by measuring the amount of allergen-specific IgE antibodies present in the blood and tears of the subject.
- a subject with a stronger allergic reaction to a specific allergen has a higher amount of specific IgE antibody to that allergen in the blood and tears, and thus the measured value is higher.
- a specific IgE antibody is detected by preparing an allergen-immobilized carrier in which an allergen is immobilized on the carrier, contacting the sample with this, and capturing the specific IgE antibody in the sample that binds to the allergen.
- a complex of an allergen and a specific antibody is detected using an enzyme or a fluorescently labeled anti-IgE antibody.
- ELISA Enzyme-Linked Immunosorbent Assay
- Patent Document 1 describes measurement of milk-specific IgE antibodies in milk-allergic patients by ELISA.
- Patent Document 2 describes immobilizing an allergen on a polystyrene carrier.
- Non-Patent Document 1 describes detection using a microarray in which proteins are immobilized on a glass carrier.
- Non-Patent Document 2 describes a carrier in which an allergen is immobilized on a porous cellulose sponge
- Patent Document 3 describes a carrier in which an allergen is immobilized on DLC (Diamond-like Carbon)
- Patent Document 4 describes a photoreaction detection using a carrier in which an allergen is immobilized on a synthetic resin is described.
- Immunocap (registered trademark). With reference to the value measured by this "Immunocap” (hereinafter also referred to as “Immunocap value”) and the probability curve (curve showing the relationship between the "Immunocap value” and the possibility of inducing allergic symptoms), allergy treatment policy, implementation of food challenge test, and determination of acquisition of allergy tolerance. In this way, "Immunocap” is standardly used in allergy test diagnosis, so it is expected that it will have a good correlation with “Immunocap value” for the development of new test agents for measuring specific IgE antibodies. is required.
- Immunocap is a product that can measure allergen-specific IgE antibodies for only one allergen in a single test. Since 40 microliters of serum is required for one measurement, several milliliters of blood must be collected to measure multiple items of allergen-specific IgE antibodies. Many of the test subjects are children with allergies, and collecting a few milliliters of blood from a very small child and having to puncture a child's extremely thin blood vessels with a needle poses a heavy physical burden. Simultaneous testing of multiple allergen-specific IgE antibodies used is difficult to implement.
- an allergen-immobilized carrier that can measure multiple allergen-specific IgE antibodies from a small amount of blood (approximately 100 ⁇ L or less, equivalent to one drop of blood) with high sensitivity and a good correlation with immunocap measurement values. is desired.
- the present inventors conducted extensive studies and found that simultaneous measurement of multiple allergens can be performed with high sensitivity by using a carrier on which allergens are immobilized at a density of 120 to 500 ng/cm 2 . It was found that it is possible to measure with high correlation with "Immunocap”.
- the present invention is configured in the following aspects (1) to (15).
- An allergen-immobilized carrier for detecting an allergen-specific antibody wherein the allergen is immobilized in the allergen-immobilized region, and the allergen is immobilized at a density of 120 ng/cm 2 or more and 500 ng/cm 2 or less. .
- R 1 represents an alkylene group having 1 to 4 carbon atoms
- R 2 represents R 3 , OR 4 or NHR 5
- R 3 and R 5 are each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms
- R 4 represents an alkyl group having 1 to 4 carbon atoms
- R 6 represents an alkylene group having 1 to 2 carbon atoms
- R 7 and R 8 are each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.
- (12) A step of contacting a sample with the allergen-immobilized carrier according to any one of (1) to (11) to form a complex between the allergen and the allergen-specific IgE antibody contained in the sample; and detecting the formed complex of the allergen and the allergen-specific IgE antibody.
- a method for producing a carrier wherein the carrier is provided with projections, and 1 or more and 100 or less allergens are fixed on the projections at a density of 120 ng/cm 2 or more and 500 ng/cm 2 or less, A method comprising the steps of: immobilizing an allergen on convex portions of a carrier; and quantifying the immobilized allergen to measure the immobilization density.
- the carrier of the present invention it is possible to simultaneously measure multiple allergen-specific IgE antibodies with a smaller amount of specimen than before, with high sensitivity and high correlation with "Immunocap".
- FIG. 2 is a diagram showing an example of a carrier provided with a reaction vessel having a plurality of projections on the upper surface of which allergens are fixed, according to the present invention.
- the allergen-immobilized carrier of the present invention is characterized in that the allergen is immobilized on the allergen-immobilized region of the carrier at a density of 120 ng/cm 2 or more and 500 ng/cm 2 or less.
- the allergens used in the present invention are proteins extracted from allergenic raw materials.
- the allergen may be an extracted crude protein, further separated and purified, or an isolated single protein.
- allergenic raw materials is a general term for materials that contain allergens that cause allergies. Specific examples include microorganisms, plants, animals, insects and mites, house dust, and foods containing these. Whether or not it causes an allergy depends on one's physical constitution.
- microorganisms that contain allergens include Alternaria, Aspergillus, Candida, and Malassezia.
- plants containing allergens include Japanese cedar, Japanese cypress, Japanese alder, white birch, Dactylis, ragweed, mugwort and ragweed, and their pollen.
- animals containing allergens include cats, dogs, mice, rats, hamsters, rabbits, parakeets, ducks, and the like.
- allergen-containing insects include moths, cockroaches, bees, and the like.
- Examples of foods containing allergens include eggs, milk, wheat, peanuts, soybeans, buckwheat, sesame, rice, shrimp, crab, squid, octopus, kiwi, bananas, apples, peaches, tomatoes, garlic, tuna, salmon, and mackerel. , beef, pork and chicken.
- Other examples of allergens that cause indoor allergies include dust mites and house dust.
- examples including allergens that cause occupational allergies include latex and the like.
- allergens may be used, or those extracted from allergenic raw materials may be used.
- a method for extracting an allergen from an allergenic raw material is not particularly limited, but it may be extracted by a known method.
- Commercially available allergens can be purchased from Stallergenes Greer (GREER), Torii Pharmaceutical Co., Ltd., ITEA Co., Ltd., Cosmo Bio Co., Ltd., Biosta Co., Ltd. (Biosta Co.), and the like.
- Raw materials for the allergen-immobilized carrier of the present invention include, for example, resins, glass, metals, silicon wafers, ceramics, cellulose, nitrocellulose, etc. Resins that are inexpensive and excellent in moldability are desirable.
- resins a resin containing an aliphatic structure has lower autofluorescence than a resin having an aromatic structure, and is therefore preferable because it can reduce noise when used as a carrier for fluorescence detection analysis.
- a resin containing an ester bond is preferable because it can generate a carboxyl group by hydrolysis treatment and can be immobilized by covalent bonding with an amino group contained in an allergen protein.
- acrylic resins are excellent in processability, and are suitable for various molding methods such as injection molding, extrusion molding, and compression molding, and are also suitable for cutting, cutting, bonding, bending, and the like, and are therefore more preferable.
- the acrylic resin include polyacrylic acid ester, polymethacrylic acid ester and modifications thereof, and polymethacrylic acid ester is preferred.
- polymethacrylate include polymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA) and polypropyl methacrylate (PAMA) such as polyalkyl methacrylate (PAMA), preferably by injection molding or hot embossing.
- PMMA is easy to microfabricate.
- a copolymer with an acrylic resin can also be used as the acrylic resin.
- copolymers containing polymethacrylate include methyl methacrylate/acrylonitrile/butadiene/styrene copolymer (MABS resin), methyl methacrylate/butadiene/styrene copolymer (MBS resin), methyl methacrylate/ Styrene copolymer (MS resin) and the like can be mentioned.
- a black substance may be contained in the acrylic resin.
- Preferred black substances include carbon black, graphite, titanium black, aniline black, oxides of Ru, Mn, Ni, Cr, Fe, Co and Cu, carbides of Si, Ti, Ta, Zr and Cr, and the like. Available.
- the shape of the carrier may be appropriately selected from plate-like, thin film, particulate, porous and the like, preferably plate-like.
- the carrier preferably has projections as immobilization regions where the allergen is immobilized.
- the projections as allergen-immobilized regions are present on the upper surface of the carrier when placed horizontally. With this surface, it is possible to provide a large number of protrusions, which serve as immobilization regions, on a single carrier.
- the allergen-immobilized carrier of the present invention is used to detect allergen-specific IgE antibodies.
- the allergen-specific IgE antibody bound to the carrier of the present invention is detected with a fluorescence-labeled secondary antibody, a method of scanning the carrier using a scanner or the like can be employed.
- a carrier with an allergen immobilized on the upper surface of the convex portion of the carrier is used, the effect that it is difficult to detect the fluorescence (noise) of the fluorescence-labeled secondary antibody that non-specifically adsorbs to the surface other than the convex portion is difficult to detect.
- Demonstrate This is because the irradiation light is focused on the upper surface of the convex portion, so that the irradiation light is defocused on areas other than the upper surface of the convex portion.
- the convex part preferably has a flat upper surface.
- the upper surface of the projection is flat, it is possible to obtain a good signal (fluorescence) without causing unevenness in the intensity of fluorescence detected within one upper surface of the projection.
- the areas of the upper surfaces of the protrusions are substantially the same.
- the phrase "the areas of the upper portions of the protrusions are substantially the same” means that the value obtained by dividing the largest top surface area of the protrusions by the smallest top surface area is 1.2 or less.
- the area of the upper surface of the projection is not particularly limited, but is preferably 4 mm 2 or less and 10 ⁇ m 2 or more from the viewpoints of reducing the amount of allergens and ease of handling.
- the height of the convex portion is preferably 0.01 mm or more and 1 mm or less.
- the plate-shaped carrier preferably has an immobilization region where the allergen is immobilized and a reaction vessel which is a vessel capable of holding a specimen such as a body fluid and allowing it to react with the allergen.
- a reaction vessel which is a vessel capable of holding a specimen such as a body fluid and allowing it to react with the allergen.
- the reaction tank include a concave tank provided on the carrier and a tank surrounded by a convex partition on the carrier.
- Fig. 1 shows an example of a carrier with a reaction tank.
- the carrier 1 has a reaction vessel 2 for reacting the specimen, and can react with the specimen in the reaction vessel 2 . Since the outside of the reaction chamber does not come into contact with the specimen, it is possible to prevent contamination of the outside of the reaction chamber with the specimen.
- the reaction vessel may be present singly in one carrier as illustrated in A of FIG. 1, or may have a plurality of reaction vessels 2 in one carrier as shown in B of FIG. good.
- a plurality of reaction chambers are provided, a plurality of specimens can be reacted individually in each reaction chamber of the same carrier. Therefore, accurate measurement of a plurality of specimens can be performed on the same carrier while preventing contamination between specimens. above at the same time.
- An allergen-immobilized region in which allergens are immobilized is present in the reaction tank, and the allergens immobilized in the reaction tank are brought into contact with the specimen for reaction.
- One or more allergens may be immobilized on the allergen-immobilized region.
- the reaction tank may have a single allergen-immobilized region or may have a plurality of allergen-immobilized regions. In the case of having a plurality of allergen-immobilized regions, the same allergen may be immobilized in all the allergen-immobilized regions, or two or more allergens may be immobilized in separate allergen-immobilized regions.
- the carrier that can detect one type of allergen-specific antibody in one reaction tank, the latter can detect a plurality of allergen-specific antibodies in one reaction tank. It has the advantage of reducing the amount of specimen required for specific antibody testing.
- Fig. 2 shows an example in which a plurality of projections are provided in the reaction tank and allergens are fixed on the upper surfaces of the projections.
- a plurality of projections 3 are provided in a reaction vessel 2 of a carrier 1, and regions 4 to which allergens are fixed are formed on the upper surfaces of the projections.
- One or more allergens may be fixed.
- a resin carrier having a complicated structure as shown in FIG. 2 can be manufactured by injection molding.
- a method for immobilizing the allergen on the carrier either a method of immobilization by physical adsorption or a method of chemical immobilization by chemical bonding may be used, but the method of chemical immobilization is preferable because it is strong.
- immobilization by physical adsorption include methods utilizing van der Waals force and hydrogen bonding.
- the allergen can be physically adsorbed onto the carrier by dropping an allergen solution onto the carrier and allowing the carrier to stand until the moisture evaporates.
- examples of chemical immobilization include methods utilizing covalent bonds, ionic bonds, coordinate bonds, etc. Among these, methods of binding by covalent bonds are preferred.
- Allergens can be immobilized on resin carriers containing esters such as acrylic resins by covalent bonding by forming functional groups that can bind to allergens on the surface of the carrier.
- esters such as acrylic resins
- it can be immobilized by forming a carboxyl group on the carrier by a known method and covalently bonding it to an amino group contained in the allergen protein.
- the surface of an acrylic resin carrier such as PMMA is hydrolyzed with an alkaline aqueous solution such as caustic soda to generate carboxyl groups on the resin surface, and then the carboxyl groups and a heat-treated product of an allergen extract are combined. It can be immobilized by direct covalent bonding (amide bond) with the contained amino group.
- a maleimide group may be introduced via an ester bond to the carboxyl group formed on the carrier, and the maleimide group may be immobilized by covalently bonding the thiol group derived from the cysteine residue contained in the allergen.
- crosslinkers such as DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DSP (dithiobis (succinimidyl propionate)) and their water-soluble analogues, and PEG (polyethylene glycol) linkers
- the polymers shown below may be immobilized on a carrier such as an acrylic resin.
- Specific polymers that can be used for immobilizing allergens include polymers containing units represented by formula (Ia) or formula (Ib).
- R 1 in formulas (Ia) and (Ib) represents an alkylene group having 1 to 4 carbon atoms.
- R 1 is preferably a methylene group and an ethylene group, more preferably a methylene group.
- R2 in formula (Ia) represents R3 , OR4 or NHR5 .
- R 3 and R 5 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R 4 represents an alkyl group having 1 to 4 carbon atoms.
- R 3 is preferably a methyl group and an ethyl group, more preferably a methyl group.
- R 4 is preferably a methyl group and an ethyl group, more preferably a methyl group.
- R5 is preferably a hydrogen atom.
- R 6 in formula (Ib) represents an alkylene group having 1 to 2 carbon atoms.
- R6 is preferably an ethylene group.
- R 7 and R 8 in formula (Ib) each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.
- R 7 and R 8 are preferably a hydrogen atom and a methyl group, more preferably a hydrogen atom.
- a heteropolymer when the polymer is immobilized on the substrate surface by covalent bonding, units other than the units represented by formula (Ia) or (Ib) have reactive functional groups. is preferred. Examples of reactive functional groups include amino groups, carboxyl groups, hydroxyl groups, halogeno groups, tosyl groups, epoxy groups, acyl groups, and azide groups, with amino groups being preferred.
- the ratio of units represented by formula (Ia) or (Ib) (hereinafter sometimes collectively referred to as formula (I) for convenience) to all units is preferably 5 mol % or more and 95 mol % or less, more preferably 10 mol % or more and 90 mol % or less, and still more preferably 30 mol % or more and 70 mol % or less.
- R1 in formula (II) represents an alkylene group having 1 to 4 carbon atoms, like R 1 in formula (Ia) or (Ib).
- the ratio of the unit represented by formula (I) to the total of the unit represented by formula (I) and the unit represented by formula (II) is preferably 5 mol% or more and 95 mol% or less. , more preferably 10 mol % or more and 90 mol % or less, and still more preferably 30 mol % or more and 70 mol % or less.
- Application (spotting) of the allergen solution to the allergen-immobilized region of the carrier can be performed using a known spotting device, dispenser, printer, or the like.
- a carrier provided with multiple independent projections in the reaction vessel is used in order to avoid contamination of adjacent allergen-immobilized regions with different allergens.
- the allergen solution is applied to the upper surface of the projection as an allergen-immobilized region, the allergen solution can be retained on the upper surface of the projection by surface tension, which is preferable. In this case, it is preferable that the heights of the plurality of protrusions are substantially the same.
- substantially the same height means that the same amount of the test substance is bound to the surfaces of the protrusions with slightly different heights, and the amount thereof is measured by fluorescence or the like with a scanner or the like. In this case, it means the height at which differences in measured values such as fluorescence intensity do not pose a problem. Specifically, “substantially the same height” means that the difference in height is less than 100 ⁇ m.
- the allergen is immobilized on the allergen-immobilized region at a density of 120 ng/cm 2 or more and 500 ng/cm 2 or less.
- the allergen immobilization density is preferably 180 ng/cm 2 or more and 300 ng/cm 2 or less. It is more preferably 190 ng/cm 2 or more and 280 ng/cm 2 or less, still more preferably 195 ng/cm 2 or more and 270 ng/cm 2 or less.
- the amount of allergen required for the surface area of the immobilization region is calculated, and the required amount of allergen solution with a known concentration is applied to the entire immobilization region. is mentioned.
- the allergen protein concentration, reaction temperature, and reaction time can be appropriately selected in consideration of the reaction efficiency of immobilizing the allergen on the amino group of the protein.
- the immobilization efficiency is almost 100%.
- the immobilization efficiency is almost 100%.
- the amount of allergen that provides a desired immobilization density with respect to the area of the upper surface of the convex portion is adjusted. After applying the calculated allergen solution containing the required amount, it is allowed to stand overnight to dry the moisture, whereby a carrier having a desired allergen immobilization density can be produced.
- the efficiency of the immobilization reaction depends on the amount of lysine possessed by the protein contained in each allergen.
- the allergen concentration, reaction temperature, and reaction time are selected as appropriate, since the content varies depending on the content and the three-dimensional structure of the protein.
- a reaction vessel and a convex portion are provided in the interior thereof, and an acrylic resin carrier having the upper surface of the convex portion as an immobilization region is used, carboxyl groups are generated on the surface of the carrier, and the allergen is immobilized by covalent bonding. In that case, it can be produced as follows.
- a resin carrier containing an ester such as an acrylic resin is prepared and immersed in a 10N sodium hydroxide aqueous solution at 70° C. for 14 to 48 hours to generate carboxyl groups.
- This carboxyl group is NHS-esterified by a known method such as reacting N-hydroxysuccinimide (NHS) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and by dropping an allergen solution, It is condensed with the amino group of the allergen protein.
- the NHS ester is inactivated by hydrolysis at the same time as the condensation reaction with the amino group of the protein, a large excess amount, specifically 100-fold equivalent or more of the allergen protein is usually used, and the allergen protein is used overnight at room temperature. react. Thereafter, by washing unbound allergen proteins with physiological saline or the like, a carrier on which allergens are immobilized at a density of 120 ng/cm 2 or more and 500 ng/cm 2 or less can be produced. Prepare a plurality of allergen solutions with the amount of allergen protein appropriately ranging from 0.01 to 50 mg/mL and incubate for 1 to 48 hours under conditions of 1 to 37°C, and prepare while estimating the reaction efficiency. Better to
- Quantification of allergens immobilized on a carrier can be done by an indirect method in which the allergens immobilized on the carrier are detached from the carrier and recovered in a liquid or the like for measurement, or by direct quantification of the allergens immobilized on the carrier. It can be implemented by a method to do.
- the allergen immobilized on the carrier is detached from the carrier and recovered in a liquid, and the allergen concentration in the liquid is measured by ultraviolet absorptiometry, BCA method, Bradford method, and Lowry method.
- a method of measuring by a known protein quantification method can be mentioned.
- the following method of directly quantifying allergens on carriers is preferred in such cases. .
- Methods for directly quantifying allergens immobilized on a carrier include labeling the allergen protein on the carrier with a fluorescent substance, radioactive isotope, or the like and measuring the intensity thereof, but the labeling efficiency varies from protein to protein. , it may not be easy to accurately calculate the labeling efficiency.
- time-of-flight secondary ion mass spectrometry TOF-SIMS is used to determine the intensity ratio between the peak derived from the allergen protein and the peak derived from the carrier resin in the obtained mass spectrum. It is preferable to apply a method of calculating the immobilized amount.
- the method for detecting an allergen-specific antibody of the present invention includes the step of contacting a sample with the allergen-immobilized carrier to form a complex between the allergen and the allergen-specific IgE antibody contained in the sample; detecting complexes formed between allergens and allergen-specific IgE antibodies. After the step of forming a complex between the allergen and the allergen-specific IgE antibody, a step of washing the carrier to remove excess specimen components may be performed.
- the step of detecting the formed complex can be performed by reacting a fluorescence-labeled secondary antibody that binds to the allergen-specific IgE antibody and measuring the fluorescence intensity.
- a fluorescence-labeled secondary antibody that binds to the allergen-specific IgE antibody
- enzymes that produce colored or luminescent substances, radioactive isotopes, etc. can also be used as secondary antibody labels.
- a possible method using a secondary antibody modified with a fluorescent substance is preferred.
- the specimen used in the present invention is preferably derived from body fluid.
- Body fluids include blood, sweat, urine, tears, saliva, sputum/respiratory secretions, breast milk, amniotic fluid, cerebrospinal fluid, ascites, pleural effusion, synovial fluid, semen, vaginal secretions, etc. Allergen-specific Blood or tears that contain a large amount of specific antibodies such as IgE antibodies are preferred.
- serum and plasma can also be preferably used.
- the sample may be pretreated as necessary.
- Pretreatment includes sample dilution and removal of components contained in the sample. If a diluted specimen is used, or a specimen from which proteins and lipids other than the allergen-specific IgE antibody, which is the test substance, is removed from the specimen, it is possible to suppress the non-specific adsorption reaction, and the allergen-specific The detection sensitivity of IgE antibodies can be improved.
- IgG antibodies, IgM antibodies, IgA antibodies, and IgD antibodies in body fluid specimens specifically bind to allergens and cause competitive inhibition when detecting allergen-specific IgE antibodies, resulting in lower detection sensitivity for allergen-specific IgE antibodies.
- At least one antibody selected from the group consisting of IgG antibody, IgM antibody, IgA antibody and IgD antibody (hereinafter sometimes referred to as "non-IgE antibody" for convenience) is removed. is preferably used.
- IgG antibodies are the antibodies with the highest content in body fluid specimens
- pretreatment to remove IgG antibodies is preferable in order to eliminate competition with allergen-specific IgE antibodies.
- sample pretreatment include filtration using a membrane and chromatography such as affinity chromatography and ion exchange chromatography.
- non-IgE antibodies in the sample can be adsorbed and removed by using affinity chromatography in which a known ligand such as protein L from Jacques et al., jacalin derived from jackfruit is fixed to a carrier.
- the allergen-immobilized carrier of the present invention is produced by a method comprising a step of immobilizing an allergen on the projections of the carrier and a step of quantifying the immobilized allergen to measure the immobilization density.
- the step of immobilizing the allergen on the projections of the carrier can be performed by the method described above. That is, a method of immobilization by physical adsorption or a method of chemical immobilization by chemical bonding may be used, but the method of chemical immobilization is preferable because it is strong. Examples of chemical immobilization include methods utilizing covalent bonding, ionic bonding, and coordinate bonding, and among these, methods involving covalent bonding are preferred.
- the step of immobilizing the allergen on the convex portion of the carrier can be performed by applying (spotting) the allergen solution to the allergen-immobilized region of the carrier.
- Application of the allergen solution can be performed using a known spotting device, dispenser, printer, or the like.
- the step of quantifying the immobilized allergen and measuring the immobilization density can be performed by the method described above. That is, it can be carried out by an indirect method in which the allergen immobilized on the carrier is detached from the carrier and collected in a liquid or the like for measurement, or by a method in which the allergen immobilized on the carrier is directly quantified.
- a method for direct quantification in addition to a method of labeling the allergen protein on the carrier with a fluorescent substance or a radioactive isotope and measuring the intensity thereof, the TOF-SIMS method can be used, with the TOF-SIMS method being preferred.
- the manufacturing method of the present invention it is possible to determine whether or not the allergen is immobilized at a predetermined immobilization density, and quality control can be performed in the manufacturing process of the allergen-immobilized carrier.
- Example 1 to 6 (1) Production of allergen-immobilized carrier (carrier A) (1)-1 Production of carrier made of NHS-esterified PMMA As schematically shown in FIG. A substrate (75 mm ⁇ 25 mm ⁇ 1.0 mm) made of polymethyl methacrylate (PMMA) provided with one reactor (12.5 mm ⁇ 10 mm ⁇ 0.15 mm depth) having 256 (16 ⁇ 16, pitch 560 ⁇ m) was immersed in a 10 N sodium hydroxide aqueous solution at 70° C. for 14 hours. Then, it was washed with pure water, 0.1N HCl aqueous solution, and pure water in that order. In this way, the side chains of PMMA on the substrate surface were hydrolyzed to generate carboxyl groups.
- PMMA polymethyl methacrylate
- N-hydroxysuccinimide NHS
- EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
- MES 2-morpholinoethanesulfonic acid monohydrate
- the egg white allergen used in Examples 1 and 2 and the milk allergen used in Examples 4 and 5 were allergen scratch extracts obtained from Torii Pharmaceutical. Since the allergen scratch extract contains 50% concentrated glycerin, dialysis (dialysis membrane: Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, dialysate: phosphate buffered saline) is performed to remove glycerin. After that, the concentration shown in Table 1 was prepared with phosphate buffered saline and used.
- the egg white allergen and cow's milk allergen from the allergen manufacturer GREER used in Examples 3 and 6 are both freeze-dried powders. board.
- TOF-SIMS measurement was performed on the allergen-immobilized region of the standard substrate to obtain a positive secondary ion mass spectrum (device: TOF.SIMS5 (manufactured by ION-TOF), primary ion species: Bi 3 ++ , Primary ion acceleration voltage: 25 kV, pulse width: 11.7 ns, bunching: yes, degree of vacuum measured: ⁇ 4 ⁇ 10 ⁇ 7 Pa, charge neutralization: yes, post-acceleration: 10 kV).
- a calibration curve was prepared for calculating the immobilization density of the egg white allergen from the peak intensity ratio of 8 N + and C 4 H 5 O + .
- the allergen-immobilized region of the carrier A prepared in (1) was subjected to TOF-SIMS measurement under the same conditions, and from the peak intensity ratio of the obtained C 4 H 8 N + and C 4 H 5 O
- the immobilization density of the egg white allergen immobilized on the upper surface of each convex portion of the carrier A was calculated using the method (Examples 1 to 3).
- the allergen-specific antibody (fluorescence intensity) was measured as follows.
- Carrier A prepared in (1) was immersed in Blocking Reagent For ELISA (Sigma-Aldrich) overnight at 4°C for blocking treatment, and then washed three times with PBST.
- 50 ⁇ L of a solution obtained by diluting the specimen 3-fold with phosphate buffered saline is dropped onto the portion of the carrier where the allergen is immobilized after the above treatment, and a gap cover glass (manufactured by Matsunami Glass Industry Co., Ltd.: 24 mm ⁇ 25 mm) is added dropwise. , gap size 20 ⁇ m). After reacting at 37° C. for 2 hours, the gap cover glass was removed and washed 3 times with PBST.
- Dylight-650 dye-labeled anti-human IgE goat polyclonal antibody (manufactured by Novus Biologicals) diluted 1000-fold with PBST containing 1 wt% BSA was added to the substrate, covered with a gap cover glass and allowed to react at room temperature for 1 hour. . Thereafter, the gap cover glass was removed, washed with PBST three times, dried, and fluorescence intensity was measured with a scanner (3D-GeneScanner 3000 manufactured by Toray Industries, Inc.) under the following conditions.
- a scanner 3D-GeneScanner 3000 manufactured by Toray Industries, Inc.
- All samples were plasma samples purchased from PlasmaLab.
- a specimen negative for all egg white, milk, peanut, and mite allergies that is, a negative specimen A of class 0 for egg white allergy class, milk allergy class, peanut allergy class, and mite allergy class (egg white-specific IgE concentration: 0 .1 IU/mL, milk-specific IgE concentration: less than 0.1 IU/mL, peanut-specific IgE concentration: less than 0.1 IU/mL, mite-specific IgE concentration: less than 0.1 IU/mL) and egg white allergy class 1
- positive sample B of milk allergy class 1 egg white-specific IgE concentration: 0.38 IU/mL, milk-specific IgE concentration: 0.62 IU/mL was used.
- Allergy classes are divided into 7 stages of negative class 0 and positive classes 1 to 6, and the higher the specific IgE concentration, the higher the allergy class.
- a specific IgE antibody concentration less than 0.35 is negative and class 0.
- a specific IgE antibody concentration of 0.35 or more is positive, class 1 is 0.35 or more and less than 0.70, and the higher the specific IgE antibody concentration, the larger the class.
- Table 1 shows the results of measuring the fluorescence intensity of negative specimen A and positive specimen B and calculating the ratio of the fluorescence intensity of positive specimen B to the fluorescence intensity of negative specimen A (positive specimen/negative specimen).
- Examples 7 to 11 Allergen items, allergen manufacturers, allergen concentrations, and incubation times are set as shown in Table 1, and egg white and milk allergens are placed in one reaction tank in the same manner as in Examples 1 to 6. "Carrier B” immobilized on the upper surface of each different convex portion was prepared, and the allergen immobilization density was measured, the correlation with "Immunocap” was evaluated, and the allergen-specific IgE was measured.
- Example 12 Comparative Example 1
- Allergen items, allergen manufacturers, allergen concentrations, and incubation times are set as shown in Table 1, and egg white and milk allergens are placed in one reaction tank in the same manner as in Examples 1 to 6.
- "Carrier C” immobilized on the upper surface of each different convex portion was prepared, and the allergen immobilization density was measured, the correlation with "Immunocap” was evaluated, and the allergen-specific IgE was measured.
- Examples 13-15, Comparative Examples 2-4 Allergen items, allergen manufacturers, allergen concentrations, and incubation times were set as shown in Table 2, and each allergen of egg white, milk, peanuts, and mites was added to one piece in the same manner as in Examples 1 to 6. "Carrier D" immobilized on the upper surface of different protrusions provided in the reaction tank was prepared, and allergen immobilization density was measured, correlation with "Immunocap” was evaluated, and allergen-specific IgE was measured. did.
- the peanut allergen used in Example 13 was obtained from Torii Pharmaceutical as an allergen scratch extract. Since the allergen scratch extract contains 50% concentrated glycerin, dialysis (dialysis membrane: Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, dialysate: phosphate buffered saline) is performed to remove glycerin. After that, the concentrations shown in Table 2 were prepared with phosphate buffered saline and used.
- Positive specimen C is a plasma specimen purchased from PlasmaLab, and is class 1 for all of the egg white allergy class, milk allergy class, peanut allergy class, and mite allergy class (egg white specific IgE concentration: 0.39 IU / mL, milk specific IgE concentration: 0.50 IU/mL, peanut-specific IgE concentration: 0.39 IU/mL, mite-specific IgE concentration: 0.38 IU/mL).
- Table 2 shows the results. [Examples 16 to 20, Comparative Examples 5 and 6] Allergen items, allergen manufacturers, allergen concentrations, and incubation times were set as shown in Table 2, and egg white, milk, peanuts, and mites were treated in the same manner as in Examples 13-15 and Comparative Examples 2-4. Allergens are immobilized on different convex upper surfaces provided in one reaction vessel to prepare "carrier E", measure allergen immobilization density, evaluate correlation with "immunocap”, allergen specific Target IgE measurements were performed.
- the peanut allergen from GREER, an allergen manufacturer used in Example 17 is a freeze-dried powder, so it was dissolved in phosphate-buffered saline and adjusted to the concentration shown in Table 2 before use.
- Table 2 shows the results.
- Allergen items, allergen manufacturers, allergen concentrations, and incubation times were set as shown in Table 2, and egg white, milk, peanuts, and mites were treated in the same manner as in Examples 13-15 and Comparative Examples 2-4. Allergens are immobilized on different convex upper surfaces provided in one reaction vessel to prepare "carrier F", measure allergen immobilization density, evaluate correlation with "immunocap”, allergen-specific IgE measurements were performed.
- the allergen-immobilized carrier of the present invention was shown to have a correlation coefficient R of 0.7 or more for all allergens and to have a high correlation with "Immunocap".
- allergen-immobilized carriers in allergy test drugs are extracted proteins contained in allergen raw materials, but allergen raw materials are often obtained from the natural world. For this reason, it has been difficult to realize a test that is highly correlated with "Immunocap” due to differences in individual allergen raw materials, sampling methods, and extraction conditions. According to the allergen-immobilized carrier of the present invention, even if allergen manufacturers are different, such as Torii Pharmaceutical, GREER, and Biosta, and individual allergic raw materials and extraction conditions are different, it has a high correlation with "Immunocap”. was shown.
- the fluorescence intensity ratio of the class 0 and class 1 specimens which are the boundaries between negative and positive allergens, was 1.5 times or more. It was shown that highly sensitive and accurate allergy testing can be performed.
- the correlation coefficient R is 0.7 or more, which is highly correlated with "Immunocap", and the fluorescence intensity ratio between class 0 and class 1 specimens is 1.5 times or more, so high sensitivity and accurate measurement are possible. Furthermore, as shown in Examples 1 to 8, 14, 17, 18, and 24, the correlation coefficient R is 0.9 or more when allergens are fixed at a density of 180 ng/cm 2 or more and 300 ng/cm 2 or less. As a result, the correlation with "Immunocap" is even higher, and the fluorescence intensity ratio between class 0 and class 1 specimens is more than double, enabling even higher sensitivity and more accurate measurement.
- carriers A, B, D, E, and F used in Examples 1 to 11, 13 to 24 each contained a plurality of allergens (egg white allergen, milk allergen, peanut allergen, and mite allergen) are immobilized, and multiple allergen-specific IgE measurements are performed simultaneously using this carrier.
- allergens egg white allergen, milk allergen, peanut allergen, and mite allergen
- carrier of the present invention it was shown that multiple allergens can be measured at the same time with high correlation with "Immunocap” and with high sensitivity and accuracy.
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Abstract
Description
(1)アレルゲンがアレルゲン固定化領域に固定化された担体であって、アレルゲンが密度120ng/cm2以上500ng/cm2以下で固定された、アレルゲン特異的抗体を検出するためのアレルゲン固定化担体。
(2)前記アレルゲンが密度180ng/cm2以上300ng/cm2以下で固定された、(1)に記載のアレルゲン固定化担体。
(3)前記担体に凸部が設けられており、当該凸部が前記アレルゲン固定化領域である、(1)または(2)に記載のアレルゲン固定化担体。
(4)2種類以上100種類以下のアレルゲンがアレルゲン固定化領域に固定化された、(1)~(3)のいずれかに記載のアレルゲン固定化担体。
(5)前記アレルゲンが式(Ia)又は(Ib)で示されるユニットを含むポリマーを介して固定化された、(1)~(4)のいずれかに記載のアレルゲン固定化担体。
(6)前記凸部を含む反応槽が設けられた、(3)に記載のアレルゲン固定化担体。
(7)前記アレルゲンのうち少なくとも1種類が卵白、牛乳、落花生またはダニのいずれかである、(1)~(6)のいずれかに記載のアレルゲン固定化担体。
(8)前記担体が脂肪族構造を含む樹脂製である、(1)~(7)のいずれかに記載のアレルゲン固定化担体。
(9)前記担体がエステルを含む樹脂製である、(1)~(8)のいずれかに記載のアレルゲン固定化担体。
(10)前記担体がアクリル系樹脂製である、(1)~(9)のいずれかに記載のアレルゲン固定化担体。
(11)前記担体がポリアクリル酸エステル製またはポリメタクリル酸エステル製である、(1)~(10)のいずれかに記載のアレルゲン固定化担体。
(12)(1)~(11)のいずれかに記載のアレルゲン固定化担体に検体を接触させて、当該アレルゲンと検体中に含まれる当該アレルゲン特異的IgE抗体との複合体を形成させる工程、および当該形成されたアレルゲンとアレルゲン特異的IgE抗体との複合体を検出する工程、とを含む、アレルゲン特異的抗体の検出方法。
(13)前記検体は、IgG抗体、IgM抗体、IgA抗体およびIgD抗体から成る群より選ばれる少なくとも1種の抗体が除去されている検体である、(12)に記載のアレルゲン特異的抗体の検出方法。
(14)担体に凸部が設けられており、前記凸部に1種類以上100種類以下のアレルゲンが密度120ng/cm2以上500ng/cm2以下で固定されている担体の製造方法であって、アレルゲンを担体の凸部に固定化する工程および当該固定化されたアレルゲンを定量して固定化密度を測定する工程を含む、方法。
(15)前記アレルゲンが密度180ng/cm2以上300ng/cm2以下で前記アレルゲン固定化領域に固定化された、(14)に記載の方法。
反応槽には、単一のアレルゲン固定化領域を有していても、複数のアレルゲン固定化領域を有していてもよい。複数のアレルゲン固定化領域を有する場合、全てのアレルゲン固定化領域に同一のアレルゲンが固定化されていても、2種以上のアレルゲンが別個のアレルゲン固定化領域にそれぞれ固定されていてもよい。1つの反応槽で1種類のアレルゲン特異的抗体が検出できる担体と比較して、後者の場合、1つの反応槽で複数のアレルゲン特異的抗体の検出を行うことができるので、アレルゲン1項目あたりの特異的抗体検査に必要な検体量を減らすことができる利点がある。
(1)アレルゲン固定化担体(担体A)の作製
(1)-1 NHSエステル化PMMA製担体の作製
図2に模式的に示すような、上部表面が直径150μmである円錐台形状の凸部を256個(16×16、ピッチ560μm)有する反応槽(12.5mm×10mm×深さ0.15mm)が1個設けられたポリメチルメタクリレート(PMMA)製の基板(75mm×25mm×1.0mm)を、10N水酸化ナトリウム水溶液に70℃で14時間浸漬した。次いで、純水、0.1N HCl水溶液、純水の順で洗浄した。このようにして、基板表面のPMMAの側鎖を加水分解して、カルボキシル基を生成した。
実施例1および2で使用した卵白アレルゲン、実施例4および5で使用した牛乳アレルゲンは、いずれもアレルゲンスクラッチエキスとして鳥居薬品から入手した。アレルゲンスクラッチエキスには濃グリセリンが50%含まれているため、透析(透析膜:Slide-A-Lyzer MINI Dialysis Device, 10K MWCO、透析液:リン酸緩衝生理食塩水)を実施してグリセリンを除去した後、リン酸緩衝生理食塩水で表1に示す濃度に調製して用いた。
(1)-2で調製した各アレルゲン溶液を、スポッティング用ロボット(日本レーザー電子(株)、GTMASStamp-2)を用いて、(1)-1で作製したNHSエステル化PMMA樹脂基板のそれぞれ異なる凸部上面にスポットした。次いで、その基板を密閉したプラスチック容器に入れて、37℃、湿度100%の条件で24時間インキュベートして固定化した。インキュベーション後、0.05% Tween20を含むリン酸緩衝生理食塩水で洗浄し、未固定のアレルゲンを除去した。以上のようにして、卵白及び牛乳の各アレルゲンが、1個の反応槽に設けられた異なる凸部上面にそれぞれ固定化された「担体A」が得られた。
卵白アレルゲン溶液を水・ホルムアミド混合溶媒で段階希釈して標準液を調製し、非接触ジェットディスペンサーCyber jet2(武蔵エンジリアリング製)を用いて、(1)-1で使用したのと同一のPMMA製基板上に液滴を吐出し、溶媒が蒸発するまで室温で自然乾燥させた。吐出液量と吐出液の付着面積からアレルゲン固定化密度を求め、密度0~400ng/cm2の範囲内で21水準の標準基板を作成した。標準基板のアレルゲン固定化領域に対し、TOF-SIMS測定を実施して正2次イオン質量スペクトルを取得し(装置:TOF.SIMS5(ION-TOF社製)、1次イオン種:Bi3 ++、1次イオン加速電圧:25kV、パルス幅:11.7ns、バンチング:あり、測定真空度:<4x10-7Pa、帯電中和:あり、後段加速:10kV)、取得したスペクトルのうち、C4H8N+とC4H5O+のピーク強度比から卵白アレルゲンの固定化密度を算出できる検量線を作成した。
(1)で作製した担体Aを用いて、以下の方法により、アレルギー陽性検体およびアレルギー陰性検体の蛍光強度を測定し、各検体中のアレルゲン特異的抗体を測定した。検体は、PlasmaLab社より購入したイムノキャップ値が既知の卵白アレルギー陰性検体および陽性検体を計7検体(血漿)用いた。
装置:3D-GeneScanner3000(東レ製)
励起光波長:635nm
検出波長:670nm
PMT:30
(4)陰性検体、陽性検体中のアレルゲン特異的IgEの測定
(1)で作製した担体Aを用いて、上記(3)と同様の方法により、以下の陰性検体Aおよび陽性検体Bの蛍光強度を測定して卵白アレルゲン特異的抗体(実施例1~3)、牛乳アレルゲン特異的抗体(実施例4~6)を検出した。
アレルゲンの項目、アレルゲンのメーカー、アレルゲンの濃度、インキュベート時間を表1に示すとおりに設定し、実施例1~6と同様の方法で、卵白及び牛乳の各アレルゲンが、1個の反応槽に設けられた異なる凸部上面にそれぞれ固定化された「担体B」を作製し、アレルゲン固定化密度の測定、「イムノキャップ」との相関性の評価、アレルゲン特異的IgEの測定を実施した。
アレルゲンの項目、アレルゲンのメーカー、アレルゲンの濃度、インキュベート時間を表1に示すとおりに設定し、実施例1~6と同様の方法で、卵白及び牛乳の各アレルゲンが、1個の反応槽に設けられた異なる凸部上面にそれぞれ固定化された「担体C」を作製し、アレルゲン固定化密度の測定、「イムノキャップ」との相関性の評価、アレルゲン特異的IgEの測定を実施した。
アレルゲンの項目、アレルゲンのメーカー、アレルゲンの濃度、インキュベート時間を表2に示すとおりに設定し、実施例1~6と同様の方法で、卵白、牛乳、落花生、ダニの各アレルゲンが、1個の反応槽に設けられた異なる凸部上面にそれぞれ固定化された「担体D」を作製し、アレルゲン固定化密度の測定、「イムノキャップ」との相関性の評価、アレルゲン特異的IgEの測定を実施した。
[実施例16~20、比較例5、6]
アレルゲンの項目、アレルゲンのメーカー、アレルゲンの濃度、インキュベート時間を表2に示すとおりに設定し、実施例13~15、比較例2~4と同様の方法で、卵白、牛乳、落花生、ダニの各アレルゲンが、1個の反応槽に設けられた異なる凸部上面にそれぞれ固定化された「担体E」を作製し、アレルゲン固定化密度の測定、「イムノキャップ」との相関性の評価、アレルゲン特異的IgEの測定を実施した。
[実施例21~24、比較例7~9]
アレルゲンの項目、アレルゲンのメーカー、アレルゲンの濃度、インキュベート時間を表2に示すとおりに設定し、実施例13~15、比較例2~4と同様の方法で、卵白、牛乳、落花生、ダニの各アレルゲンが、1個の反応槽に設けられた異なる凸部上面にそれぞれ固定化された「担体F」作製し、アレルゲン固定化密度の測定、「イムノキャップ」との相関性の評価、アレルゲン特異的IgEの測定を実施した。
2 反応槽
3 凸部
4 アレルゲン固定化領域
Claims (15)
- アレルゲンがアレルゲン固定化領域に固定化された担体であって、アレルゲンが密度120ng/cm2以上500ng/cm2以下で固定された、アレルゲン特異的抗体を検出するためのアレルゲン固定化担体。
- 前記アレルゲンが密度180ng/cm2以上300ng/cm2以下で固定された、請求項1に記載のアレルゲン固定化担体。
- 前記担体に凸部が設けられており、当該凸部が前記アレルゲン固定化領域である、請求項1または2に記載のアレルゲン固定化担体。
- 2種類以上100種類以下のアレルゲンがアレルゲン固定化領域に固定化された、請求項1~3のいずれかに記載のアレルゲン固定化担体。
- 前記凸部を含む反応槽が設けられた、請求項3に記載のアレルゲン固定化担体。
- 前記アレルゲンのうち少なくとも1種類が卵白、牛乳、落花生またはダニのいずれかである、請求項1~6のいずれかに記載のアレルゲン固定化担体。
- 前記担体が脂肪族構造を含む樹脂製である、請求項1~7のいずれかに記載のアレルゲン固定化担体。
- 前記担体がエステルを含む樹脂製である、請求項1~8のいずれかに記載のアレルゲン固定化担体。
- 前記担体がアクリル系樹脂製である、請求項1~9のいずれかに記載のアレルゲン固定化担体。
- 前記担体がポリアクリル酸エステル製またはポリメタクリル酸エステル製である、請求項1~10のいずれかに記載のアレルゲン固定化担体。
- 請求項1~11のいずれかに記載のアレルゲン固定化担体に検体を接触させて、当該アレルゲンと検体中に含まれる当該アレルゲン特異的IgE抗体との複合体を形成させる工程、および当該形成されたアレルゲンとアレルゲン特異的IgE抗体との複合体を検出する工程、とを含む、アレルゲン特異的抗体の検出方法。
- 前記検体は、IgG抗体、IgM抗体、IgA抗体およびIgD抗体から成る群より選ばれる少なくとも1種の抗体が除去されている検体である、請求項12に記載のアレルゲン特異的抗体の検出方法。
- 担体に凸部が設けられており、前記凸部に1種類以上100種類以下のアレルゲンが密度120ng/cm2以上500ng/cm2以下で固定されている担体の製造方法であって、アレルゲンを担体の凸部に固定化する工程および当該固定化されたアレルゲンを定量して固定化密度を測定する工程を含む、方法。
- 前記アレルゲンが密度180ng/cm2以上300ng/cm2以下で前記アレルゲン固定化領域に固定化された、請求項14に記載の方法。
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KR (1) | KR20230134467A (ja) |
CN (1) | CN116724232A (ja) |
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- 2022-01-20 CN CN202280010366.XA patent/CN116724232A/zh active Pending
- 2022-01-20 JP JP2022505257A patent/JPWO2022158509A1/ja active Pending
- 2022-01-20 US US18/271,493 patent/US20240060976A1/en active Pending
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EP4283307A1 (en) | 2023-11-29 |
US20240060976A1 (en) | 2024-02-22 |
TW202246775A (zh) | 2022-12-01 |
KR20230134467A (ko) | 2023-09-21 |
JPWO2022158509A1 (ja) | 2022-07-28 |
CN116724232A (zh) | 2023-09-08 |
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