JP2008170195A - Method of evaluating suppressive effect of allergen suppressor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of accurately evaluating a suppressive effect of an allergen suppressor by definitely eliminating an allergen from a sample containing the allergen suppressor by a simple operation. <P>SOLUTION: The method of evaluating a suppressive effect of an allergen suppressor comprises: contacting a sample containing an allergen suppressor in a base material with an allergen; treating the sample with an eliminating solution containing a surface-active agent or a buffer solution of pH 4-6 or 8.5-11; collecting into the eliminating solution the allergen that has not been inactivated in the sample; and measuring antigenicity of the allergen collected into the eliminating solution and that has not been inactivated. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for evaluating the ability of an allergen inhibitor contained in a substrate to suppress allergens such as mites and pollen.

  Allergens are antigenic substances that cause allergic diseases such as atopic dermatitis, bronchial asthma, and allergic rhinitis, and are well known for dust mite allergens (Der1, Der2) and cedar pollen allergens (Cryj1, Cryj2). It has been.

  The above allergens are indoor decoration materials such as tatami mats, floors (flooring) and wallpaper; bedding such as futons, pillows, blankets, mattresses, bed mats and sheets; covers for furniture and furniture such as sofas, chairs, and beds; air Filters for purifiers, air conditioners, vacuum cleaners, etc .; Car supplies such as car seats, car mats and child seats; Toys such as stuffed animals; Textile products such as carpets, curtains, clothes, towels, etc. Allergic diseases are developed by aspirating allergens accumulated in the place or aspirating allergens that have risen in the air.

  For this reason, it is required to remove allergens from the living space for the purpose of alleviating symptoms of allergic diseases or preventing new sensitization. In order to remove such allergens, allergens are denatured and inactivated. An allergen inhibitor to be used, an allergen scavenger for capturing and removing allergens, and the like are used. Further, in recent years, a substrate made of a fiber material such as cotton cloth or a porous material such as silica gel, which constitutes the allergen scavenger, is often used which contains an allergen inhibitor. .

  And as a method of evaluating the inhibitory ability of the allergen inhibitor contained in the base material, conventionally, a method for measuring the allergen removing ability of the allergen scavenger has been used. However, when evaluated by the above method, since the allergen-capturing material itself has the ability to capture allergens, the amount of allergen is reduced even if the allergen inhibitor itself does not have the ability to suppress allergens. As a result, there was a problem that an accurate evaluation result could not be obtained as a result that the inhibitor had a suppressive ability.

  Therefore, as described in Patent Document 1, the sample is immersed in a test solution in which a cedar pollen allergen is dissolved in a physiological saline solution, left to stand at room temperature for 1 hour, and then filtered using a pole filter having a pore diameter of 0.2 μm. Then, 0.5 mL of the obtained filtrate was added to 1.0 mL of serum of a subject with a history of cedar pollinosis, and the antibody (IgE antibody) in the serum was measured by the RAST method, whereby the allergen scavenger's ability to remove allergens was measured. In the evaluation method, using the sample containing the allergen inhibitor and the sample not containing the allergen inhibitor as the sample, the allergen removal ability is measured, and the results are compared. Thus, a method for evaluating the inhibitory ability of an allergen inhibitor has been performed. However, such an evaluation method has a problem that the operation is complicated and the reproducibility of the evaluation result is inferior, and the measurement sensitivity of the antibody is low, and an error is likely to occur in the result.

  In addition, 0.03 g of standard house dust containing mite allergen at a rate of 1,300 μg / g is placed evenly on a felt cut out into a circle having a diameter of about 6.6 cm as in Patent Document 2, Place a sample on the top, spray 1 to 2 g of distilled water containing 30% by weight of ethanol and 10% by weight of benzyl alcohol, and let it dry naturally, then remove the sample and place only the felt in a vinyl bag with a chuck. Further, 10 mL of phosphate buffer containing 10% by weight of skim milk is put in a vinyl bag to extract mite allergens on the felt, and the resulting extract is centrifuged (2000 rpm, 60 minutes), and the resulting supernatant is obtained. Disclosed is a method for measuring the amount of mite allergen in an enzyme-linked immunosorbent assay (ELISA) to evaluate the suppressive ability of an allergen inhibitor To have.

  However, in the above evaluation method, allergens remain in the felt, so the amount of allergens extracted by the extract is less than the amount of allergens that are not actually denatured or adsorbed by the allergen inhibitor. The problem that the inhibitory ability of the allergen inhibitor could not be accurately evaluated occurred.

JP-A-6-158494 JP 2001-214367 A

  The present invention provides a method for accurately evaluating and suppressing the allergen inhibitor's inhibitory ability by reliably desorbing and recovering all inactive allergens from a sample containing the allergen inhibitor by a simple operation. To do.

  The method for evaluating the inhibitory ability of the allergen inhibitor of the present invention is obtained by treating the sample with an allergen inhibitor and treating the sample with a desorbing solution after contacting the sample with the allergen inhibitor in the base material. The allergen that maintains the reactivity to the specific antibody is desorbed and collected in the desorbed solution, and the antigenicity of the allergen in the desorbed solution is measured.

  The allergen referred to in the present invention is not particularly limited as long as it is an antigenic substance that causes allergic diseases. For example, dust mite allergens (Der1, Der2), cedar pollen allergens (Cryj1, Cryj2), dogs and cats, etc. Examples include allergens derived from pets (Can f1, Fel d1).

  In the method for evaluating the inhibitory ability of the allergen inhibitor of the present invention, a sample comprising an allergen inhibitor contained in a substrate is used. The base material constituting the sample is not particularly limited. For example, a base material made of cellulose such as woven fabric, non-woven fabric, knitted fabric or Japanese paper such as cotton, hemp or silk, clay mineral, activated carbon And a base material made of a porous material such as silica gel, a base material made of cellulose is preferable, and a cloth made of natural fibers is more preferable.

  And the said allergen inhibitor means what has the effect which denatures or adsorb | sucks the said allergen, and suppresses the reactivity with respect to the specific antibody of an allergen, ie, the inactivation of an allergen. Examples of such allergen inhibitors include aromatic hydroxy compounds such as tannic acid, catechins, polyphenols, lignophenol derivatives, and aromatic polyether compounds.

  First, the aromatic hydroxy compound will be described. The aromatic hydroxy compound is not particularly limited as long as it has an aromatic hydroxy group and has an allergen suppressing effect. For example, the linear hydroxy polymer is represented by the following formulas (1) to (6). Fragrance formed by polymerizing or copolymerizing a monomer containing at least one substituent represented by the formulas (1) to (6), such as an aromatic hydroxy compound having at least one of the substituents Polymers of monomers having an aromatic heterocyclic hydroxyl group, such as aromatic hydroxy compounds; aromatic heterocyclic hydroxy compounds; aromatic hydroxy compounds having an aromatic heterocyclic hydroxy group as a substituent on a linear polymer Alternatively, an aromatic hydroxy compound obtained by copolymerization, an aromatic hydroxy compound having an alicyclic structure in the main chain of the chain polymer, and the like can be given.

  First, an aromatic hydroxy compound obtained by polymerizing or copolymerizing a monomer containing at least one substituent represented by formulas (1) to (6) will be described. The substituents of this aromatic hydroxy compound are represented by the following formulas (1) to (6).

  In the formulas (1) to (6), n is an integer of 0 to 5. This is because when n is 6 or more, the allergen suppression effect expressed by the substituents represented by formulas (1) to (6) becomes insufficient.

In the plurality of substituents R ¹ , R ¹ , R ¹ ... In each formula, each substituent R ¹ is hydrogen or a hydroxyl group. Furthermore, in each formula, at least one of the plurality of substituents R ¹ needs to be a hydroxyl group in order for the aromatic hydroxy compound to exhibit an allergen suppressing effect. However, if the number of hydroxyl groups is too large, the sample is likely to be colored or discolored, so one hydroxyl group is preferable. That is, in each formula, it is preferable that only ^one of a plurality of substituents R ¹ is a hydroxyl group, while all the substituents R ¹ other than this substituent are hydrogen.

  Furthermore, the position of the hydroxyl group is preferably bonded to the position having the least steric hindrance. For example, in the formula (1), the hydroxyl group is preferably bonded to the para position.

  The monomer containing at least one substituent represented by the above formulas (1) to (6) is not particularly limited as long as it has a substituent represented by the formula (1) to (6). Examples thereof include monomers having a monovalent phenol group such as vinylphenol, tyrosine, and 1,2-di (4-hydroxyphenyl) ethene (formula (7)).

  Furthermore, the monomer containing at least one substituent represented by the formulas (1) to (6), preferably one or more monovalent phenol groups within a range not inhibiting the allergen suppressing effect of the aromatic hydroxy compound. You may copolymerize the monomer which can be copolymerized with this monomer with this monomer.

  And as a monomer copolymerizable with the monomer containing at least one substituent shown by said formula (1)-(6), ethylene, acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, for example , Hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, styrene and the like.

  And as a linear polymer to which the substituent shown by Formula (1)-(6) has couple | bonded, it does not specifically limit, A vinyl polymer, polyester, polyamide, etc. are mentioned. The chemical bond between the linear polymer and the substituents represented by formulas (1) to (6) is not particularly limited, and examples thereof include a carbon-carbon bond, an ester bond, an ether bond, and an amide bond. .

  Here, as an aromatic hydroxy compound having at least one of the substituents represented by the formulas (1) to (6) in the linear polymer, for example, (1) formulas (1) to (6) A polymer or copolymer of a monomer containing at least one substituent represented by formula (1), a monomer containing at least one substituent represented by formulas (1) to (6), Examples thereof include a copolymer of a monomer and a copolymerizable monomer.

  Specific examples of the aromatic hydroxy compound having at least one of the substituents represented by the formulas (1) to (6) on the linear polymer include poly (3,4,5- Preferred are vinyl hydroxybenzoate), polyvinylphenol, polytyrosine, poly (1-vinyl-5-hydroxynaphthalene), poly (1-vinyl-6-hydroxynaphthalene), and poly (1-vinyl-5-hydroxyanthracene).

  The molecular weight of the aromatic hydroxy compound obtained by polymerizing the monomer is not particularly limited, but is preferably an aromatic hydroxy compound obtained by polymerizing two or more monomers, and includes five monomers. An aromatic hydroxy compound obtained by polymerization as described above is more preferable.

  In addition, the aromatic heterocyclic hydroxy compound is not particularly limited as long as it has an allergen suppressing effect, and examples thereof include 2-hydroxyfuran, 2-hydroxythiophene, hydroxybenzofuran, and 3-hydroxypyridine.

  Next, an aromatic hydroxy group obtained by polymerizing or copolymerizing a monomer having an aromatic heterocyclic hydroxy group such as an aromatic hydroxy compound having an aromatic heterocyclic hydroxy group as a substituent on a linear polymer. The compound will be described.

  Examples of the aromatic heterocyclic hydroxy group include those in which a hydroxy group is bonded to a heterocyclic skeleton such as thiophene and furan (formulas (8) and (9)), and a skeleton having a heterocyclic ring and an aromatic ring. Are bonded to each other (formula (10)), a heterocyclic skeleton is bonded to a hydroxy group and an alkyl group having 5 or less carbon atoms, and a skeleton having a heterocyclic ring and an aromatic ring is bonded to a hydroxy group and 5 or less carbon atoms. And those having an alkyl group bonded thereto.

  The linear polymer to which the aromatic heterocyclic hydroxy group is bonded is not particularly limited, and examples thereof include vinyl polymers, polyesters, and polyamides. The chemical bond between the linear polymer and the aromatic heterocyclic hydroxy group is not particularly limited, and examples thereof include a carbon-carbon bond, an ester bond, an ether bond, and an amide bond.

  Examples of the compound obtained by polymerizing or copolymerizing a monomer having an aromatic heterocyclic hydroxy group include, for example, (1) a polymer or copolymer of a monomer having an aromatic heterocyclic hydroxy group. And (2) a copolymer of a monomer having an aromatic heterocyclic hydroxy group and a monomer copolymerizable with this monomer.

  Examples of the monomer copolymerizable with the monomer having an aromatic heterocyclic hydroxy group include ethylene, acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxy Examples include propyl acrylate, hydroxypropyl methacrylate, and styrene.

  Subsequently, an aromatic hydroxy compound having an alicyclic structure in the main chain of the chain polymer will be described. The aromatic hydroxy compound having an alicyclic structure in the main chain of the chain polymer has an alicyclic structure in the main chain of the chain polymer and a phenol group in the main chain or side chain of the chain polymer. The alicyclic structure is preferably Formula (11) or Formula (12) from the viewpoint that the texture of the sample is not impaired.

Here, in Formula (11) (12), R < ² > -R < ⁹ > is hydrogen, a hydrocarbon group, or a phenol group. Examples of the hydrocarbon group include alkyl groups such as methyl, ethyl, propyl, and butyl groups, cycloalkyl groups such as cyclobutyl, cyclopentyl, and cyclohexyl groups, alkenyl groups such as vinyl groups, and cycloalkenyl groups such as cyclobutenyl groups. Group, an aryl group such as a phenyl group and an ethylphenyl group, and an aralkyl group.

Further, the alicyclic structure portion preferably has a substituent represented by the formula (13), and at least one substituent of R ^{2 to} R ^{5 in} the formula (11) is represented by the formula (13). More preferably, it is a substituent.

Here, in Formula (13), R ^{10 to} R ¹⁴ are hydrogen or a hydroxy group, and at least one of R ^{10 to} R ¹⁴ is a hydroxy group. This is because, when at least one of R ^{10 to} R ¹⁴ is not a hydroxy group, the allergen inhibitory effect of the allergen inhibitor may be reduced. However, since the colorability of the allergen inhibitor may increase as the number of hydroxy groups increases, only one substituent out of R ^{10 to} R ¹⁴ in formula (13) is a hydroxy group and the remainder. preferably the substituents are hydrogen, it is less steric hindrance, it is more preferable and R ¹² is a hydroxy group R ^10, R ^11, R ¹³ and R ¹⁴ are hydrogen.

  In Formula (13), X is a direct bond or a hydrocarbon group, and examples of such a hydrocarbon group include a methylene group, an ethylene group, a propylidene group, and a butylidene group.

  The aromatic hydroxy compound having an alicyclic structure represented by the formula (11) in the main chain of the chain polymer and having a substituent represented by the formula (13) in the alicyclic structure portion is a liquid. It can be synthesized using polybutadiene and phenol as raw materials, and is commercially available, for example, under the trade name “PP series” from Shin Nippon Oil Co., Ltd., and the fat represented by the formula (12) in the main chain of the chain polymer Aromatic hydroxy compounds having a cyclic structure can be synthesized using dicyclopentadiene and phenol as raw materials, and are commercially available, for example, under the trade names “DPP series” and “DPA series” from Nippon Oil Corporation. .

  Moreover, the said lignophenol derivative means the compound by which a phenol derivative is added to lignin, and the partial structural formula which showed an example of the lignophenol derivative in following formula (14) is shown.

  As the lignin constituting the lignophenol derivative, lignin existing in any lignocellulosic material can be used. Examples of such lignocellulosic materials include woody parts of plants such as wood and processed products (for example, paper).

  When wood is used as the lignocellulosic material, the type of tree is not particularly limited, and may be a conifer or a broad-leaved tree. In addition, various herbaceous plants such as rice, corn, and sugarcane may be used as lignocellulosic materials. Furthermore, the lignocellulosic material may be a waste material or scrap of lignocellulosic material, or feed or agricultural waste.

  In addition, it does not specifically limit as a form of a lignocellulose type material, Although a powder form, a chip | tip form, etc. are mentioned, Since a lignophenol derivative can be extracted efficiently, a powder form is preferable.

  And it does not specifically limit as a phenol derivative added to lignin, For example, a monohydric phenol derivative, a bivalent phenol derivative, a trihydric phenol derivative etc. are mentioned, A phenolic hydroxyl group is concerned in allergen suppression ability. Therefore, divalent phenol derivatives and trivalent phenol derivatives having a large number of phenolic hydroxyl groups are preferable, and catechol, pyrogallol, derivatives containing a catechol structure, and derivatives containing a pyrogallol structure are more preferable.

  A monohydric phenol derivative refers to a compound having one phenolic hydroxyl group, one phenolic hydroxyl group, a compound having another substituent on the benzene ring, and a derivative containing the structure of these compounds. Specifically, Examples include phenol and cresol.

  The dihydric phenol derivative means a compound having two phenolic hydroxyl groups, two phenolic hydroxyl groups, a compound having other substituents on the benzene ring, and a derivative containing the structure of these compounds. Specifically, catechol, resorcinol, hydroquinone and the like can be mentioned.

  The trivalent phenol derivative refers to a compound having three phenolic hydroxyl groups, three phenolic hydroxyl groups, a compound having other substituents on the benzene ring, and a derivative containing the structure of these compounds. Specifically, phloroglucinol, hydroxyhydroquinone, pyrogallol and the like can be mentioned.

  Although it does not specifically limit as a number average molecular weight of a lignophenol derivative, From viewpoints, such as a physical property, 500 or more are preferable and 1000 or more are more preferable. Moreover, since the handleability of a lignophenol derivative may fall when the molecular weight of a lignophenol derivative is too large, 100,000 or less is preferable and 10,000 or less is more preferable.

  Furthermore, examples of the aromatic polyether compound include epoxy resin, phenoxy resin, polyethersulfone, polysulfone, polyphenylsulfone, polyallylsulfone, polyetherketone, polyphenylene ether, polyetherimide, polyarylate, and the like. Epoxy resin, phenoxy resin, polyethersulfone, polysulfone, polyallylsulfone, and polyetherketone are preferable because coloring to the sample can be more effectively prevented, and epoxy resin, phenoxy resin, polyethersulfone, and polysulfone are preferable. More preferred.

Among the aromatic polyether compounds, an epoxy obtained by polymerization by a polycondensation or oxidative coupling reaction between a monomer such as bisphenol A, tetrachlorobisphenol A, bisphenol F, or bisphenol S and epichlorohydrin. Resin or phenoxy resin; aroma in which polycondensation is carried out by heating an alkali metal salt of bisphenol and an aromatic dihalide activated by an electron-withdrawing group such as —SO ₂ —, —CO—, or —CN in a polar solvent. Aromatic nucleophilic substitution polymerization method, and aromatic electrophilic substitution polymerization method using Friedel-Crafts sialylation reaction in which an aromatic compound rich in electrons such as diphenyl ether and aromatic diacid chloride are polymerized under a Lewis acid catalyst Obtained by polysulfone, polyallylsulfone, polyether Sulfone or polyether ketone is more preferable.

  Further, since the coloring of the sample can be particularly effectively prevented, the aromatic polyether compound contains the structural unit represented by the formula (15) and / or the formula (16) as a main repeating unit. Is preferred.

(R ^{15 to} R ²⁶ are hydrogen or a hydrocarbon group, which may be the same or different. X is selected from the group consisting of a methylene group, an ethylene group, a propylidene group, a butylidene group, and a sulfonyl group. A divalent organic group or a direct bond, and Z ¹ and Z ² are a divalent organic group selected from the group consisting of a methylene group, an ethylene group, a propylene group, a hydroxytrimethylene group, and a butylene group, and a phenylsulfonyl structure A divalent organic group having a direct bond or a direct bond.)

Here, in equation (15) (16), R 15 ~R 26 is hydrogen or a hydrocarbon group. Examples of the hydrocarbon group include alkyl groups such as methyl, ethyl, propyl, and butyl groups, cycloalkyl groups such as cyclobutyl, cyclopentyl, and cyclohexyl groups, alkenyl groups such as vinyl groups, and cycloalkenyl groups such as cyclobutenyl groups, An aryl group such as a phenyl group and an ethylphenyl group, an aralkyl group and the like can be mentioned, and it is preferable that all of R ^{15 to} R ²⁶ are hydrogen.

  Further, in the formula (15), X is a divalent organic group selected from the group consisting of a methylene group, an ethylene group, a propylidene group, a butylidene group and a sulfonyl group, or a direct bond, and a methylene group, a propylidene group, a sulfonyl group Groups are preferred.

In the formulas (15) and (16), Z ¹ and Z ² are a divalent organic group selected from the group consisting of a methylene group, an ethylene group, a propylene group, a hydroxytrimethylene group and a butylene group, and a phenylsulfonyl structure A divalent organic group or a direct bond having a methylene group, a hydroxytrimethylene group, or a divalent organic group having a phenylsulfonyl structure or a direct bond, and Z ¹ represents a hydroxytrimethylene group or a phenylsulfonyl structure. A divalent organic group or a direct bond is more preferable, and Z ² is more preferably a divalent organic group having a phenylsulfonyl structure.

  And although it does not specifically limit as a bivalent organic group which has the said phenylsulfonyl structure, For example, the organic group which has a structure shown to following formula (17)-(19) is mentioned.

  When the aromatic polyether compound is a compound containing the structural unit represented by the formula (15) and / or the formula (16) as a main repeating unit, the structural unit of the formula (15) or the formula (16) The main repeating unit may be used, or the constituent units of the formula (15) and the formula (16) may be used as the main repeating unit, and other constituent units may be contained.

  Specific examples of the compound containing the structural unit represented by the above formula (15) as a main repeating unit include polysulfone obtained by polycondensation of bisphenol A and bis (4-chlorophenyl) sulfone; Epoxy resins obtained by polycondensation of bisphenol F and epichlorohydrin are preferred.

  As the compound containing the structural unit represented by the above formula (16) as a main repeating unit, specifically, by a solution polycondensation method using a potassium salt of 4-chloro-4′-hydroxydiphenylsulfone. Polyethersulfone obtained, polyethersulfone obtained by solution polycondensation method using potassium salt of 4-fluoro-4'-hydroxydiphenylsulfone; polycondensation of 4,4'-biphenyldisulfonyl chloride and diphenyl ether The resulting polyallylsulfone is preferred.

  When the weight average molecular weight of the aromatic polyether compound is small, the allergen suppressing effect may not be exhibited. Therefore, 1500 or more is preferable, and 2500 or more is more preferable. Is preferably 500,000 or less.

  And as a method of making the base material contain the said allergen inhibitor, the method of making an allergen inhibitor chemically couple | bond or physically adhere | attach is mentioned.

  Examples of the method for chemically binding the allergen inhibitor to the substrate include a method of chemically binding the allergen inhibitor to the substrate by a grafting reaction. The grafting reaction is not particularly limited.For example, (1) a graft polymerization method in which a polymerization initiation point is formed on a substrate and an allergen inhibitor is polymerized as a branch polymer, and (2) the allergen inhibitor is based on a polymer reaction. Examples include a polymer reaction method that chemically bonds to a material.

  The method for physically fixing the allergen inhibitor to the substrate is not particularly limited.For example, (1) the substrate is immersed in the allergen inhibitor dispersion obtained by dispersing the allergen inhibitor in, A method of containing an allergen inhibitor dispersion in a substrate and fixing the allergen inhibitor to the substrate, (2) a method of applying the allergen inhibitor dispersion on the substrate surface by means such as spraying, ( 3) Dissolve or disperse the binder resin in the allergen inhibitor dispersion, immerse the base material in the allergen inhibitor dispersion, contain the allergen inhibitor dispersion in the base material, and use the allergen inhibitor as the binder resin. (4) The binder resin is dissolved or dispersed in the allergen inhibitor dispersion, and the allergen inhibitor dispersion is sprayed on the substrate surface. There was applied, and a method of fixing the allergen inhibitor to the substrate by the binder resin.

  The binder resin is not particularly limited as long as it can fix the allergen inhibitor to the substrate surface. For example, urethane resins such as one-component urethane resins and two-component urethane resins, acrylic resins, and urethane acrylates. Examples thereof include resins, polyester resins, unsaturated polyester resins, alkyd resins, vinyl acetate resins, vinyl chloride resins, epoxy resins, epoxy acrylate resins, silicone resins, and the like, and urethane resins and silicone resins are preferable.

Next, the sample obtained as described above and containing the allergen inhibitor in the base material is brought into contact with the allergen, and the allergen inhibitor and the allergen in the sample are brought into contact with each other. The method for bringing the sample into contact with the allergen is not particularly limited. For example, a method of immersing the sample in a liquid containing the allergen, an atmosphere having an absolute humidity of 50 g / m ³ or less, and being placed on the sample. Examples include a method of sprinkling allergens and a method of exposing a sample to living space.

  The liquid used in the method of immersing the sample in the liquid containing the allergen is not particularly limited, and examples thereof include phosphate buffers such as water and phosphate buffered saline. Acid buffered saline is preferred.

Of the methods of bringing the sample into contact with the allergen, the method of immersing the sample in a liquid containing the allergen is preferable because the contact time between the sample and the allergen can be shortened, while the absolute humidity is 50 g / The method of sprinkling the allergen on the sample under an atmosphere of m ³ or less is preferable in that the contact time between the sample and the allergen can be shortened and the sample and the allergen can be brought into contact under conditions close to the actual living space.

  The time for contacting the sample and the allergen as described above is not particularly limited. However, depending on the type of allergen inhibitor, it may take 24 to 500 hours to inactivate the allergen. Is preferable, and 72 to 350 hours are more preferable.

  Subsequently, among allergens contained in the sample, allergens that maintain reactivity with the specific antibody are desorbed using a desorbing solution and recovered in the desorbing solution. There is no particular limitation on the method for desorbing the allergen maintaining the reactivity with the specific antibody from the sample to the desorbing solution. For example, the sample and the desorbing solution are placed in a sealable container, and the container is sealed. In addition to the method of desorbing the allergen contained in the sample, which maintains the reactivity to the specific antibody, to the desorption solution by shaking well, the sample is immersed in a liquid containing allergen. When the sample and the allergen are brought into contact with each other, the surfactant or the liquid containing the surfactant is added to the liquid containing the allergen to remove the liquid containing the allergen. For example, a method of desorbing the allergen contained in the sample and maintaining the reactivity to the specific antibody into the desorbing solution may be mentioned.

  Here, if the allergen that maintains the reactivity to the specific antibody cannot be sufficiently desorbed from the sample, the allergen that maintains the reactivity to the specific antibody remains in the sample. In the antigenicity test of allergens, the amount of allergens whose reactivity to specific antibodies was not suppressed by the allergen inhibitor was evaluated to be less than the actual amount, making it impossible to accurately evaluate the suppressive ability of the allergen inhibitor Therefore, it is necessary to use a desorbing solution that can reliably desorb allergens that maintain reactivity with the specific antibody from the sample.

  Accordingly, as the desorbing solution used in the present invention, a buffer solution containing a surfactant or a buffer solution having a pH of 4 to 6 or 8.5 to 11 is used. These desorbing liquids may be used alone or in combination of two or more.

  The desorbing liquid containing the surfactant is not particularly limited as long as it contains a surfactant. For example, a buffer such as a phosphate buffer, a phosphate buffered saline, or a Tris-hydrochloric acid buffer. A liquid, physiological saline, purified water, or the like containing a surfactant is used.

  The surfactant is not particularly limited as long as it does not denature allergens or affects antigenicity tests. For example, polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene Polyoxyethylene alkyl ether sulfonates such as sodium lauryl ether sulfonate, alkyl benzene sulfonates such as linear sodium alkyl benzene sulfonate, alkyl diphenyl ether disulfonates such as sodium alkyl diphenyl ether disulfonate, polyoxyethylene oleate diester, poly Oxyethylene oleic acid monoester, polyoxyethylene stearic acid monoester, polyoxyethylene polyoxypropylene block polymer, lauryl And polyoxyethylene alkyl ether, polyoxyethylene alkyl ether sulfonate, and alkyl diphenyl ether disulfonate are preferable. Polyoxyethylene alkyl ether, sodium polyoxyethylene alkyl ether sulfonate, alkyl diphenyl ether disulfone Sodium acid is more preferred. Commercially available detergents can also be used. These surfactants may be used alone or in combination of two or more. In addition, lauryl benzene sulfonate cannot be used because it easily modifies allergens.

  When the surfactant content in the desorption solution containing the surfactant is small, the allergen that maintains the reactivity to the specific antibody may not be sufficiently desorbed from the sample. In many cases, the allergen that maintains the reactivity to the specific antibody desorbed from the sample is denatured by the surfactant, and the inhibitory ability of the allergen inhibitor may not be accurately evaluated. 30 mg / mL is preferable and 0.005-8 mg / mL is more preferable.

  In addition, the pH of the leaching solution containing the surfactant is too low or too high, and the allergen that maintains the reactivity to the specific antibody is denatured, and the allergen inhibitor can be suppressed. 4 to 11 is preferable.

  When a buffer solution is used as the desorbing solution, if the pH of the buffer solution is higher than 6 and lower than 8.5, the allergen that maintains the reactivity to the specific antibody is sufficiently removed from the sample. It was not possible to desorb, and the inhibitory ability of the allergen inhibitor could not be accurately evaluated. On the other hand, when the pH of the buffer solution was lower than 4 and higher than 11, it was desorbed from the sample. Since the allergen that maintains the reactivity with the specific antibody is denatured and the inhibitory ability of the allergen inhibitor cannot be accurately evaluated, the pH of the buffer solution is limited to 4-6 or 8.5-11. .5 to 5.5 or 9.5 to 11 is preferable. When the desorption solution is a mixture of two or more types of buffer solutions, the pH of the desorption solution is the apparent pH of the entire desorption solution.

  However, when a buffer containing a surfactant is used as the desorption solution, an allergen that maintains the reactivity to the specific antibody is sampled even if the pH of the desorption solution is 6 to 8.5. The pH of the desorbed solution is preferably 4 to 11 because it can be sufficiently desorbed from the inside.

  The buffer solution having a pH of 4 to 6 is not particularly limited, and examples thereof include a citric acid-sodium citrate buffer solution, an acetic acid-sodium acetate buffer solution, a succinic acid-sodium hydroxide buffer solution, and hydrogen phthalate. Examples include potassium-sodium hydroxide buffer.

  The buffer having a pH of 8.5 to 11 is not particularly limited, and examples thereof include boric acid-potassium chloride-sodium hydroxide buffer, glycine-sodium hydroxide buffer, and diethanolamine-hydrochloric acid buffer. Is mentioned.

  In addition, some allergen inhibitors suppress the reactivity of allergens to specific antibodies by adsorbing allergens. In such a case, when the sample is treated with the detachment solution, the allergen inhibitors The allergen adsorbed on the allergen may be detached from the allergen inhibitor, and the reactivity of the allergen to the specific antibody may be restored. In this way, the allergen amount at which the reactivity to the specific antibody is restored is the allergen inhibitor. This is a small percentage of the allergen adsorbed on the allergen and does not affect the measurement of the allergen inhibitory ability of the allergen inhibitor.

  Then, the antigenicity of the allergen that has been detached from the sample and recovered in the detachment solution and that maintains the reactivity with the specific antibody is measured by antigenicity test. To improve the sensitivity of the antigenicity test. In addition, it is preferable to measure the antigenicity of the allergen that maintains the reactivity with the specific antibody in the concentrated solution obtained by concentrating the desorbed solution.

  The method for concentrating the desorbed solution is not particularly limited, but it is preferable to carry out the method in which the allergen contained in the desorbed solution and maintaining the reactivity against the specific antibody is difficult to denature. Examples of such a method include a method of filtering with an ultrafiltration membrane and lyophilization.

  In addition, if the average pore size of the ultrafiltration membrane is large, allergens that maintain the reactivity to specific antibodies may pass through the ultrafiltration membrane and may not be concentrated, so it is preferably 5 nm or less. If it is too small, it may take too much time to concentrate, so 1 to 5 nm is more preferable.

  The method for measuring the antigenicity of the allergen contained in the desorbed solution or concentrated solution and maintaining the reactivity to the specific antibody is not particularly limited. For example, an ELISA kit (Indoor) is used. ELISA method to be used, and a method using an allergen measuring instrument (trade name “Mighty Checker” manufactured by Shinto Fine Co., Ltd.).

  The method for evaluating the inhibitory ability of the allergen inhibitor of the present invention is the treatment of the above sample with a desorbing solution containing a surfactant after contacting the sample containing the allergen inhibitor with the substrate and the allergen. By desorbing allergens that maintain reactivity to specific antibodies from the sample and collecting them in the desorbed solution, and measuring the antigenicity of the allergen in the desorbed solution. Therefore, the allergen that maintains the reactivity to the specific antibody contained in the sample is surely desorbed and recovered in the desorbed solution, hardly remains in the sample, and is desorbed from the sample. In addition, since the allergen that maintains the reactivity to the specific antibody is hardly denatured, the inhibitory ability of the allergen inhibitor can be accurately evaluated.

  Moreover, since the sensitivity of the antigenicity test is improved by performing the antigenicity test using the concentrated solution obtained by concentrating the above detachment solution, the ability to suppress the allergen inhibitor should be evaluated with higher accuracy. Can do.

Hereinafter, embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples. In the following description, the term "untreated cotton" refers to cotton cloth 5 cm ² of 135 g / m ² to treatment with allergen inhibitor has not been performed, the "untreated hollow cotton" is the allergen inhibitor This refers to 0.2 g of hollow cotton that has not been subjected to the treatment. In addition, as phosphate buffered saline (PBS buffer), NaH ₂ PO ₄ .multidot.NaH ₂ PO ₄ .12H was dissolved in A solution prepared by dissolving 3.58 g of Na ₂ HPO ₄ .12H ₂ O and 5.84 g of NaCl in 1 L of ion exchange water. A solution prepared by dissolving 0.78 g of 2H ₂ O and 2.92 g of NaCl in 500 mL of ion-exchanged water while adding pH while measuring the pH with a pH meter, and adjusting the pH to 7.60 was used.

(Example 1)
A mite extract freeze-dried powder (trade name “Mite Extract-Df” manufactured by Cosmo Bio Inc.) was added to phosphate buffered saline to prepare an allergen solution having a protein amount of 10 μg / mL.

  On the other hand, polyethersulfone (trade name “A-300A” manufactured by Solvay Advanced Polymers), which is an allergen inhibitor, 10% by weight, special silicon emulsion (trade name “TF-3500”, manufactured by Kitahiro Chemical Co., Ltd.) 2.5% by weight and An untreated cotton cloth was immersed in an allergen inhibitor dispersion composed of 87.5% by weight of ion-exchanged water, stirred for 5 minutes with a stirrer, and then dried to obtain an allergen inhibitor-treated cotton cloth.

  Next, the allergen inhibitor treated cotton cloth and the untreated cotton cloth are separately immersed in 3.5 mL of the allergen solution in a sealed container and shaken at 100 rpm for 7 days at a temperature of 37 ° C. to suppress allergen. The agent-treated cotton cloth and the untreated cotton cloth were each taken out from the container.

  Subsequently, as a desorption operation, the above-mentioned allergen inhibitor-treated cotton fabric and untreated cotton fabric were separately sealed in a sealed container (trade name “Emar 20C” manufactured by Kao Corporation, surfactant component: polyoxyethylene) (Sodium lauryl ether sulfonate, surfactant component content: 25% by weight) Mixing 0.5 mL of a surfactant solution prepared by dissolving 0.33 mg in 0.5 mL of ion-exchanged water and 3.5 mL of phosphate buffered saline Soaked in 4.0 mL of the desorbed liquid (pH: 7.6), shaken at 100 rpm for 30 minutes at 37 ° C., stirred for 3 minutes with a touch mixer, and then treated with an allergen inhibitor Cotton cloth and untreated cotton cloth were removed from the container.

  Then, by preparing a new detachment liquid and performing the detachment operation twice more, the allergen that has been maintained in the reactivity to the specific antibody contained in the allergen inhibitor-treated cotton cloth and the untreated cotton cloth. Was desorbed in the desorbing solution. The desorbed liquid obtained by the above three desorption operations is mixed, and the mixed desorbed liquid is ultrafiltered using an ultrafiltration membrane (trade name “Centriprep YM-10” manufactured by Millipore). As a result, 1 mL of concentrated liquid was obtained.

  On the other hand, tick extract freeze-dried powder (trade name “Mite Extract-Df” manufactured by Cosmo Bio Inc.) was added to phosphate buffered saline, and the total amount of protein was the same as the amount impregnated on the allergen inhibitor-treated cotton fabric. A 3.5 mL allergen solution was prepared.

  Thereafter, the same amount of polyethersulfone as impregnated in the allergen inhibitor-treated cotton cloth was supplied into 3.5 mL of the allergen solution, and the mixture was shaken at 100 rpm for 7 days at a temperature of 37 ° C. Next, 0.5 mL of the surfactant solution is supplied into the allergen solution, shaken at 100 rpm for 30 minutes at a temperature of 37 ° C., then stirred for 3 minutes with a touch mixer and concentrated in the same manner. 1 mL of liquid was produced.

(Example 2)
A mite extract freeze-dried powder (trade name “Mite Extract-Df” manufactured by Cosmo Bio Inc.) was added to phosphate buffered saline to prepare an allergen solution having a protein amount of 20 μg / mL.

  On the other hand, an untreated cotton cloth is immersed in an allergen inhibitor dispersion composed of 5% by weight of poly-4-vinylphenol (Aldrich, weight average molecular weight: 8000) and 95% by weight of ethanol, which is an allergen inhibitor, and stirred for 5 minutes with a stirrer. After drying, an allergen inhibitor-treated cotton fabric was obtained.

  Next, the allergen inhibitor-treated cotton cloth and the untreated cotton cloth are separately immersed in 3.5 mL of the allergen solution in a sealed container and shaken at 100 rpm for 7 days at a temperature of 37 ° C. Surfactant obtained by diluting the agent (trade name “Sandet AL” manufactured by Sanyo Kasei Co., Ltd., surfactant component: sodium alkyldiphenyl ether disulfonate, surfactant component content: 25% by weight) 100 times with ion-exchanged water Agent solution (surfactant component concentration: 2.5 mg / mL) 0.1 mL each to the allergen solution in each container to form a detachment solution (pH: 7.6), each container is sealed again, touch mixer For 5 minutes, the allergen maintaining the reactivity to the specific antibody was desorbed from the allergen inhibitor-treated cotton fabric and untreated cotton fabric. The desorbed solution was ultrafiltered using an ultrafiltration membrane (trade name “Centriprep YM-10” manufactured by Millipore) to obtain 1 mL of a concentrated solution.

  On the other hand, an allergen solution was prepared in the same manner as described above. Thereafter, the same amount of poly-4-vinylphenol as the amount impregnated in the allergen inhibitor-treated cotton cloth was supplied into the allergen solution, and the mixture was shaken at 100 rpm for 7 days at a temperature of 37 ° C. Next, 0.1 mL of the surfactant solution was supplied into the allergen solution, stirred for 3 minutes with a touch mixer, and concentrated in the same manner to prepare 1 mL of a reference solution.

(Example 3)
First, an allergen solution having a protein amount of 10 μg / mL was prepared in the same manner as in Example 1. On the other hand, an allergen inhibitor-treated cotton fabric was obtained in the same manner as in Example 2.

  Next, the allergen inhibitor-treated cotton cloth and the untreated cotton cloth are separately immersed in 3.5 mL of the allergen solution in a sealed container and shaken at 100 rpm for 5 days at a temperature of 37 ° C. The inhibitor-treated cotton cloth and the untreated cotton cloth were taken out from the container.

  Then, as the detachment operation, the above-mentioned allergen inhibitor-treated cotton cloth and untreated cotton cloth are separately sealed in a sealed container (trade name “Emulgen 108” manufactured by Kao Corporation, surfactant component: polyoxyethylene lauryl). Surfactant solution (surfactant component concentration: 33 mg / mL) obtained by diluting ether, surfactant component content: 100% by weight with ion-exchanged water 30 times and phosphate buffered saline Included in the allergen-inhibitor-treated cotton cloth and untreated cotton cloth by immersing in 1.1 mL of a detachment liquid (pH: 7.6) mixed with 1.0 mL and stirring for 5 minutes with a touch mixer. The allergen maintaining the reactivity with the specific antibody was desorbed in the desorption solution.

  On the other hand, tick extract freeze-dried powder (trade name “Mite Extract-Df” manufactured by Cosmo Bio Inc.) was added to phosphate buffered saline, and the amount of protein was the same as the amount impregnated in the allergen inhibitor-treated cotton cloth. An allergen solution was prepared.

  Thereafter, the same amount of poly-4-vinylphenol as the amount impregnated in the allergen-inhibitor-treated cotton cloth was supplied into the allergen solution, and the mixture was shaken at 37 ° C. and 100 rpm for 5 days. Next, a release solution composed of 0.1 mL of the surfactant solution and phosphate buffered saline was supplied into the allergen solution and stirred for 5 minutes with a touch mixer to prepare a reference solution. The above-mentioned reference solution is a specific antibody contained in the allergen inhibitor-treated cotton cloth by adjusting the amount of phosphate buffered saline in the allergen solution and the amount of phosphate buffered saline in the detachment solution. It was prepared so as to be the same amount as the desorbed solution after desorbing the allergen maintaining the reactivity with respect to.

Example 4
A mite extract freeze-dried powder (trade name “Mite Extract-Df” manufactured by Cosmo Bio Inc.) was added to phosphate buffered saline to prepare an allergen solution having a protein amount of 4 μg / mL.

  On the other hand, poly 4-vinylphenol which is an allergen inhibitor (trade name “Marcalinker M” manufactured by Maruzen Petrochemical Co., Ltd., weight average molecular weight: 5500) 5% by weight and an allergen inhibitor dispersion composed of 95% by weight of ethanol is untreated Hollow cotton was immersed for 5 minutes and dried to obtain allergen inhibitor-treated hollow cotton.

  Next, the allergen inhibitor-treated hollow cotton and untreated hollow cotton are each separately immersed in 3.5 mL of the allergen solution in a sealed container and shaken at 100 rpm for 3 days under a temperature condition of 37 ° C. In each of the above containers, a surfactant (trade name “Emulgen 108” manufactured by Kao Corporation, surfactant component: polyoxyethylene lauryl ether, surfactant component content: 100 wt%) is diluted 30 times with ion-exchanged water. The surfactant solution (surfactant component concentration: 33 mg / mL) thus obtained was added in an amount of 0.1 mL each to form a desorbing solution (pH: 7.6), and each container was sealed again. By stirring for a minute, the allergen maintaining the reactivity with respect to the specific antibody contained in the allergen inhibitor-treated hollow cotton and the untreated hollow cotton was desorbed in the desorbing solution.

  On the other hand, an allergen solution was prepared in the same manner as described above. Thereafter, the same amount of poly-4-vinylphenol as the amount impregnated in the allergen inhibitor-treated hollow cotton was supplied into the allergen solution, and the mixture was shaken at 100 rpm for 3 days at a temperature of 37 ° C. Next, 0.1 mL of the surfactant solution was supplied into the allergen solution and stirred for 3 minutes with a touch mixer to prepare a reference solution.

(Comparative Example 1)
Except that the surfactant solution was not added to the allergen solution, a 1 mL concentrated solution and a reference solution were obtained in the same manner as in Example 2.

(Comparative Example 2)
The reactivity with respect to the specific antibody contained in the allergen inhibitor treated hollow cotton and untreated hollow cotton in the same manner as in Example 4 except that 0.1 mL of the surfactant solution was not added to the allergen solution. A desorbed solution (pH: 7.6) from which allergens maintaining the pH were desorbed and a reference solution were obtained.

  In Examples 1 to 4 and Comparative Examples 1 and 2, the untreated allergen solution was prepared by stirring the allergen solution before contacting with the sample at 100 rpm under a temperature condition of 37 ° C. In addition, the stirring days of allergen liquid were made into the same number of days as the days which contacted the allergen liquid and the sample in each of Examples 1-4 and Comparative Examples 1 and 2.

  And the untreated allergen solution of Examples 1 to 4 and Comparative Examples 1 and 2, the allergen solution obtained in Examples 1 to 4 and Comparative Examples 1 and 2 after completion of contact with the sample, Examples 1 and 2 And the concentrated liquid obtained in Comparative Example 1, the desorbed liquid obtained in Examples 3, 4 and Comparative Example 2, and the reference liquid obtained in Examples 1 to 4 and Comparative Examples 1 and 2, The antigenicity of the allergen that maintains the reactivity to the antibody was evaluated by the Mighty Checker and by the ELISA method in the following manner, and the results are shown in Tables 1 and 2. In Tables 1 and 2, allergen inhibitor-treated cotton cloth and allergen inhibitor-treated hollow cotton are represented as “treated sample”, and untreated cotton cloth and untreated hollow cotton are represented as “untreated sample”. In Tables 1 and 2, the evaluations of the concentrates obtained in Examples 1 and 2 and Comparative Example 1 are described in the column “Desorbed liquid”.

(Evaluation by Mighty Checker)
Untreated allergen solutions of Examples 1 to 4 and Comparative Examples 1 and 2, allergen solutions obtained in Examples 1 to 4 and Comparative Examples 1 and 2 after completion of contact with samples, Examples 1 and 2 and comparison The concentrated liquid obtained in Example 1, the desorbed liquid obtained in Examples 3 and 4 and Comparative Example 2, and the standard liquid obtained in Examples 1 to 4 and Comparative Examples 1 and 2, After immersing a tick simple inspection kit (trade name “Mighty Checker” manufactured by Shinto Fine Co., Ltd.), the degree of color development on the test line (T line) of the Mighty Checker was visually observed and evaluated according to the following criteria.
A Color development of the T line was not recognized. B The T line was slightly colored. C The T line was colored. D The T line was clearly colored. The ET line was thick and clearly colored.

(Quantification of Der f2 by ELISA method)
Untreated allergen solutions of Examples 1 to 4 and Comparative Examples 1 and 2, allergen solutions obtained in Examples 1 to 4 and Comparative Examples 1 and 2, immediately after contact with the sample, Examples 1 and 2 and comparison The concentrated solution obtained in Example 1, the detachment solution obtained in Examples 3 and 4 and Comparative Example 2, and the Der f2 in the reference solutions obtained in Examples 1 to 4 and Comparative Examples 1 and 2 The worm body-derived allergen) was quantified using an ELISA kit (Indoor).

Claims (6)

After contacting the allergen with a sample containing an allergen inhibitor in the base material, the sample is treated with a desorbing solution containing a surfactant to react with the specific antibody from the sample. The allergen that maintains the above is desorbed and recovered in the desorbed solution, and the antigenicity of the allergen in the desorbed solution is measured, and the method for evaluating the inhibitory ability of the allergen inhibitor is characterized by the following. By contacting the sample containing the allergen inhibitor in the substrate with the allergen, and then treating the sample with a desorption solution comprising a buffer solution having a pH of 4 to 6 or 8.5 to 11, An allergen inhibitor characterized by desorbing allergens that maintain reactivity to a specific antibody from the sample and recovering them in the desorbed solution, and measuring the antigenicity of the allergen in the desorbed solution To evaluate the ability to inhibit The method for evaluating the inhibitory ability of an allergen inhibitor according to claim 1 or 2, wherein the sample is immersed in a liquid containing the allergen and the sample and the allergen are brought into contact with each other. The method for evaluating the inhibitory ability of an allergen inhibitor according to claim 1 or 2, wherein the allergen is sprinkled on the sample in an atmosphere having an absolute humidity of 50 g / m ³ or less. 5. The antigenicity of an allergen that maintains the reactivity to a specific antibody in a concentrate obtained by concentrating the detachment solution, is measured according to any one of claims 1 to 4. Evaluation method of inhibitory ability of allergen inhibitor. The method for evaluating the inhibitory ability of an allergen inhibitor according to any one of claims 1 to 5, wherein the sample is made of a substrate made of cellulose or a substrate made of a porous material.