JP5900923B2 - Evaluation method of allergen reduction function - Google Patents

Description

The present invention relates to a test method for evaluating an allergen-reducing function of a material such as a flooring or a wallpaper that has been processed with an allergen-reducing agent.

Allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis are plagued by many people, and in recent years, the tendency has been increasing. The causes of these allergic diseases are various allergens present in the environment. Among them, mites, pet animal dander, pollen, and mold that inhabit indoors are typical inhalable allergens. Well known. In particular, leopard mites, which are dust mites that live in the house, are a major problem as a source of allergens. Leopard mites are a hotbed for growing textile products in the house such as tatami mats, carpets, bedding, curtains, etc., or seat sheet fabrics of moving vehicles such as trains and cars outdoors.

Among the leopard mites, Dermatophagoides farinae and Dermatophagoides pteronysinsinus are typical species, and the dead bodies and feces of these mites are strong allergen substances. In addition, pollen of various plants including cedar ( Cryptomeria japonica ) pollen scattered in early spring is also an allergen, and causes allergic rhinitis in particular. The pollen that scatters is not only cedar pollen in the early spring, but also many kinds of cypress, mugwort, ragweed, camo, etc. There is a state that some kind of pollen is scattered throughout the year, and there is a risk of causing pollen allergies at any time It is thought that there is.

In order to remove such allergens such as mites and pollen, various allergen reducing agents have been developed. Tannic acid has been known for more than 20 years to have an allergen-reducing function, and polyphenols, which are analogs of tannic acid, have been proposed. In addition to polyphenols, many allergen reducing agents have been developed.

These allergen reducing agents can be considered as a method of removing or reducing allergen substances present in the environment by spraying as a spray, but in many cases, air cleaners, air conditioner filters, clothing, etc. Fiber products, wooden materials such as flooring, which are building materials, and resin products such as wallpaper.

It is necessary to evaluate the performance of fiber products, wood materials, wallpaper, and the like processed with the allergen reducing agent in order to prove the allergen reducing effect. In this method, an allergen-containing aqueous solution is immersed or brought into contact with a test substrate processed with an allergen-reducing agent for a certain period of time, and the amount of allergen in the aqueous solution thereafter is measured (Patent Documents 1 to 4). .

In addition, as a method of bringing the allergen aqueous solution into contact with the test substrate, a method of placing the allergen aqueous solution on the surface of the test substrate and covering the film has been proposed, but the amount of the allergen aqueous solution to be placed is limited, and the contact area is limited. There was a scene where accurate evaluation was difficult because it was not sufficient (Patent Document 5).

JP 2001-212806 A JP 2010-116450 A JP 2010-235701 A JP 2008-239721 A JP 2011-47778 A

It is an object of the present invention to provide a highly reproducible test method for accurately evaluating the effect of reducing allergens such as flooring, wallpaper, etc. processed with an allergen reducing agent on the entire surface or the entire surface.

As a result of earnest research to solve such problems, the present inventor made contact by sandwiching an allergen aqueous solution between two test substrates, and measuring the amount of allergen in the allergen aqueous solution after contact. It was found that it is possible to evaluate allergen reduction performance with high accuracy and high reproducibility.

That is, the present invention is (1) a test method for evaluating the allergen reducing performance of a test substrate processed with an allergen reducing agent, wherein the test substrate processed with the allergen reducing agent has a known concentration on the surface. The allergen aqueous solution is placed on the surface of the test substrate by placing the allergen aqueous solution on top of the other test substrate processed with the allergen reducing agent. It is a test method for allergen reduction performance by analyzing the amount of allergen. (2) In the test method, it is a test method for allergen reduction performance in which an aqueous solution of allergen and a gap holder are sandwiched between two test substrates. (3) The method for analyzing allergen reduction performance, wherein the method for analyzing the allergen amount in the allergen aqueous solution is an enzyme immunoassay. (4) The test base material in which the allergen reducing agent is processed is any flat plate or sheet selected from wood materials, plastic products, rubber, glass, stones and ceramics, or these flat plates This is a test method for allergen reduction performance in which a coating film is formed on the surface of a test substrate in the form of a sheet or sheet.

By using the test method of the present invention, accurate and high reproducibility can be achieved for flat or sheet-like test substrates such as wood materials, plastic products, rubber, glass, stones and ceramics processed with allergen reducing agents. It is possible to evaluate the allergen-reducing performance possessed.

The test substrate to be evaluated is not particularly limited, but may be a wood material used for flooring, resin or plastic products used for wallpaper, rubber or plastic sheets, glass or ceramic or stone tile plates, etc. Examples include building materials, which are usually flat. The allergen reducing agent can be added by kneading when manufacturing these building materials. Moreover, it is also possible to process the surface of these building materials by adding an allergen reducing agent to the coating agent and paint which are processed on the surface of these building materials, and applying them.

The test substrate is usually flat, and the test is performed by sandwiching the allergen aqueous solution using two flat test substrates. However, if the allergen aqueous solution can be sandwiched, the test substrate is a curved surface having a certain curvature. There is no problem.

Although it does not specifically limit to the magnitude | size of a test base material, the surface area of a test base material is 4-225 cm < ² >, Preferably 9-100 cm < ² > is suitable. Although it does not specifically limit about a shape, Usually, it is a square whose side is 2-15 cm, Preferably it is a square whose side is 3-10 cm. However, it may be round or rectangular, and may be distorted if there is no obstacle to contact between the allergen aqueous solution and the test substrate.

The allergen aqueous solution sandwiched between the test substrates can be arbitrarily selected as long as the liquid amount necessary for measuring the allergen amount can be recovered. This liquid volume correlates with the surface area of the test substrate, and the larger the surface area, the larger the liquid volume. In many cases, about 100 μL is required for measurement of the amount of allergen, and it is preferable to use 100 μL or more as a sufficient amount of liquid for measurement in the test. When the test substrate has a sufficient area and there are moderate irregularities on the surface of the test substrate, the test can be performed by sandwiching the allergen aqueous solution between the two test substrates. If the amount of allergen aqueous solution is large relative to the surface area of the test substrate and the allergen aqueous solution may spill, the test substrate may be deformed or partially deformed with a certain curvature to form a dish-like allergen. It is possible to prevent the aqueous solution from spilling. Furthermore, if the test substrate has high flatness and has a certain amount of weight, there is a risk of spilling when the allergen aqueous solution is sandwiched, and in order to prevent this, secure a gap to hold the allergen aqueous solution. Therefore, a minute object (gap holder) having a certain thickness can be used.

This gap holder is for holding the allergen aqueous solution between the two test substrates. By adjusting the thickness, the amount of the allergen aqueous solution provided to the test substrate of a certain area can be adjusted. If the test substrate has high flatness, a small amount of allergen aqueous solution can be spread on the surface of a wide test substrate by using a thin gap holder. The material of the gap holder is not particularly limited as long as it does not cause allergen adsorption or allergen reaction, but stainless steel or glass can be used. As the stainless steel gap holder, for example, a stainless steel wire having an appropriate thickness cut to a length of about 5 mm can be used, and as the glass gap holder, a thin glass plate can be made small. Divided ones can be used. In order to obtain a constant gap with the gap holder, at least three gap holders are required for one test substrate, and usually 3 to 4 gap holders can be used.

The allergen reducing agent used for the test substrate is not particularly limited as long as it has an allergen reducing action, and the test method of the present invention is applied to a test substrate processed for general use. Can be applied. Examples of such allergen reducing agents include tannic acid, gallotannin, epicatechin gallate, epicatechin, epigallocatechin, epigallocatechin gallate, and natural polyphenols such as persimmon astringent, polymers of paravinylphenol as synthetic polyphenols, Examples thereof also include hydroxybenzoic acid compounds such as dihydroxybenzoic acid and 2,4,6-trihydroxybenzoic acid or salts thereof, polystyrene compounds such as polystyrene sulfonate, and extracts such as olive leaves and ginkgo leaves. Furthermore, zinc oxide which is a metal oxide, titanium oxide and tungsten oxides having a photocatalytic function, and inorganic salt compounds such as calcium, strontium and rare earth salts which are water-soluble inorganic salts. Among these various allergen reducing agents, those that are processed into building materials such as flooring and wallpaper are preferably water-insoluble, and water-insoluble organic or inorganic allergen reducing agents are applied. be able to. The dosage form of the allergen reducing agent is not particularly limited, but usually a liquid or solid form is used. In the case of liquid, the allergen-reducing agent active ingredient can be dissolved in water or a solvent, and if not dissolved, it can be made into a flowable agent dispersed together with a dispersant. In the case of a solid form, any form such as a powder form or a granule form can be used, but a powder form is preferred. Two or more of these allergen reducing agents may be used in combination.

The test method of the present invention will be described in detail. First, an allergen aqueous solution is placed on the surface of a test substrate. The allergen amount contained in this allergen aqueous solution is 0.1 to 1000 ng / mL, preferably 1 to 50 ng / mL, more preferably 2 to 25 ng / mL as a purified allergen.

Although the kind of allergen is not specifically limited, A mite allergen, a cedar pollen allergen, a cat allergen, a dog allergen, a cabia allergen, a cockroach allergen, etc. can be used. In the case of mite allergens, common allergens are commonly caused by allergens, such as Der f1, which is derived from feces of mites, and Der f2, which is derived from insect mites of allergic mites, as allergen proteins. Can be mentioned. A typical example of the pollen allergen is a cedar pollen allergen, and Cry j1 mainly present in the outer layer of the cedar pollen is mentioned as an allergen protein. In addition, cypress, mugwort, ragweed, hurghaya, camo, etc. are mentioned as plant species from which pollen causing allergens is derived. Sources other than mite allergens and pollen allergens include molds, dog and cat dander, insects such as cockroaches and chironomids. For example, these allergen proteins include Blag1 derived from cockroaches, Can f1 derived from dog dandruff, Fel d1 derived from cat dandruff, Alt a1 derived from mold, and the like.

Next, the allergen aqueous solution and the test substrate are brought into contact for a certain period of time. Although any time can be set for the contact time, it can usually be set in the range of 5 minutes to 72 hours, preferably in the range of 30 minutes to 24 hours, and more preferably in the range of 1 hour to 3 hours. When the contact time is set to be long, the allergen aqueous solution may decrease due to volatilization or evaporation, and the allergen concentration may change. Therefore, it is preferable to store the test substrate in a sealed container during contact. . Such a closed container is not particularly limited, and examples thereof include a glass chamber, a glass desiccator, and a polyethylene bag with a chuck. Furthermore, if the volume of the sealed container is relatively large and the amount of allergen aqueous solution is small, the decrease due to volatilization of the allergen aqueous solution in the sealed container may affect the test. It is preferable to enclose an absorbent cotton containing water or a container containing water.

The temperature at which the allergen aqueous solution is contacted with the test substrate is not particularly limited, but is preferably around room temperature in consideration of suppressing the volatilization of the allergen aqueous solution, usually 5 to 37 ° C, preferably 15 to 25 ° C. It can be arbitrarily set within the range. In order to keep the temperature constant, it is preferable to store in a thermostat, but it may be stored in a room where the temperature changes.

After contacting the test substrate with the allergen aqueous solution, the allergen aqueous solution is recovered. As a method of recovery, it can be recovered using a micropipette. If a part of the allergen aqueous solution is absorbed by the test substrate and recovery is difficult, the test substrate can be compressed and the allergen aqueous solution can be squeezed out. Is possible. The method for compressing the test substrate is not particularly limited, but it can be squeezed by hand, and an instrument such as a vise can be used.

Next, after collecting the allergen aqueous solution, the amount of allergen in the allergen aqueous solution is measured. Although it does not specifically limit regarding the measuring method of allergen, The method of using the method using the enzyme immunity method (ELISA method) and the commercially available allergen measuring simple kit is considered. The ELISA method is a method for measuring an antigen using an antigen-antibody reaction, and corresponds to an allergen measured by the antigen in the present invention.

Regarding the allergen measurement simple kit, there are some commercially available products. For example, Mighty checker (manufactured by Sumika Enviro Science Co., Ltd.), tick scan (manufactured by Asahi Food and Healthcare Co., Ltd.), etc. can be used.

The present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited thereto. In addition, all% shown below are weight%.

(Mite allergen aqueous solution)
Allergens extracted from reared mushroom mites were diluted with a phosphate buffer solution of pH 7.2 so that Der f2 was about 900 ng / mL (total protein amount), and used for the following tests.

(Sugi pollen allergen aqueous solution)
Using a phosphate buffer of pH 7.2, cedar pollen allergen Cry j1 (manufactured by Asahi Food and Healthcare Co., Ltd.) was diluted to a concentration of about 12.5 ng / mL and used for the following tests. .

(Measurement method of mite allergen concentration)
Enzyme immunoassay (Der f2 sandwich ELISA)
First, Der f2 monoclonal antibody 15E11 diluted to 2 μg / mL with phosphate buffer (pH 7.4, containing 0.1 wt% NaN ₃ ) was added per well of F16 MAXISORP NUNC-IMMUNO MODULE plate (manufactured by NUNC). 200 μL each was added and sensitized at 4 ° C. for 3 days or more. After sensitization, the solution was discarded, and a blocking reagent {1% by weight bovine serum albumin + phosphate buffer (pH 7.2, containing 0.1% by weight NaN ₃ )} was added in an amount of 200 μL per well. Reacted for 1 minute. After the reaction, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate).

Next, 100 μL of mite allergen measurement sample solution was dropped into one well and reacted at 37 ° C. for 60 minutes. After the reaction, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate). The peroxidase-labeled Der f2 monoclonal antibody was dissolved in distilled water at 200 μg / mL, and was dissolved in phosphate buffer {pH 7.2, 1% by weight bovine serum albumin and 0.1% by weight polyoxyethylene (20) sorbitan monolaur. A solution diluted 1000 times with a rate containing} was added at 100 μL per well. After reacting at 37 ° C. for 60 minutes, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate) and then with distilled water. Add 100 μL per well of 13 mol of 0.1 mol / L phosphate buffer (pH 6.2) plus ortho-phenylenediamine dihydrochloride (manufactured by SIGMA CHEMICAL CO .: 26 mg Table) and 13 μL of hydrogen peroxide. And allowed to react at 37 ° C. for 5 minutes. Immediately thereafter, 50 μL of ₂ mol / L H ₂ SO ₄ was added to stop the reaction, and the absorbance (OD 490 nm) was measured with a spectrophotometer for microplate (Tecan Japan Co., Ltd.). The Der f2 concentration of the measurement sample was determined from a calibration curve similarly measured using a Der f2 aqueous solution having a known concentration.

(Sugi pollen allergen concentration measurement method)
Enzyme immunoassay (Cry j1 sandwich ELISA)
First, Cry j1 monoclonal antibody 013 diluted to 2 μg / mL with phosphate buffer (pH 7.4, containing 0.1 wt% NaN ₃ ) was added per well of F16 MAXISORP NUNC-IMMUNO MODULE plate (manufactured by NUNC). 100 μL each was added and sensitized at 4 ° C. for 1 day or longer. After sensitization, the solution was discarded, and a blocking reagent {1% by weight bovine serum albumin + phosphate buffer (pH 7.2, containing 0.1% by weight NaN ₃ )} was added in an amount of 200 μL per well. Reacted for 1 minute. After the reaction, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate).

Next, 100 μL of a cedar pollen allergen measurement sample solution was dropped into one well and reacted at 37 ° C. for 60 minutes. After the reaction, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate). Peroxidase-labeled Cry j1 monoclonal antibody 053 was dissolved in distilled water to 200 μg / mL, and then dissolved in phosphate buffer {pH 7.2, 1 wt% bovine serum albumin and 0.1 wt% polyoxyethylene (20) sorbitan mono A solution diluted 1200 times with laurate contained} was added at 100 μL per well. After reacting at 37 ° C. for 60 minutes, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate). Add 100 μL per well of 13 mol of 0.1 mol / L phosphate buffer (pH 6.2) plus ortho-phenylenediamine dihydrochloride (manufactured by SIGMA CHEMICAL CO .: 26 mg Table) and 13 μL of hydrogen peroxide. And allowed to react at 37 ° C. for 5 minutes. Immediately thereafter, 50 μL of ₂ mol / L H ₂ SO ₄ was added to stop the reaction, and the absorbance (OD 490 nm) was measured with a spectrophotometer for microplate (Tecan Japan Co., Ltd.). The Cry j1 concentration of the measurement sample was determined from a calibration curve similarly measured using a Cry j1 aqueous solution having a known concentration.

(Evaluation of allergen reducing agent processed wallpaper)
The effect of allergen reduction was measured on the wallpaper processed on the surface of the coating agent added with 20% aqueous potassium carbonate carbonate solution (manufactured by Nippon Light Metal Co., Ltd.) as the allergen reducing agent and on the wallpaper not processed with the allergen reducing agent.

Example 1
The wallpaper of the test substrate was cut into a circle having a diameter of 8 cm, and the edge portion of the wallpaper sample was bent about 5 mm so that the surface processed with the allergen reducing agent was concave. Spread 1 mL of mite allergen aqueous solution (Der f2 concentration 900 ng / mL) on this, and match the processed surface with the wallpaper sample with the edge of about 5 mm folded so that the processed surface of the allergen reducing agent is convex from above. I pinched it. In order to prevent volatilization of the allergen aqueous solution, it was put in a plastic bag with a chuck and stored at room temperature for 1 hour. 100 μL of the allergen aqueous solution was collected using a Pipetman, and the allergen concentration was measured by sandwich ELISA. The test was performed in triplicate.

Comparative Example 1
The wallpaper of the test substrate was cut into a circle with a diameter of 8 cm, placed in a plastic bag with a chuck, and 1 mL of a mite allergen aqueous solution (Der f2 concentration of 900 ng / mL) was added and swollen. After 1 hour at room temperature, recovery of the allergen aqueous solution was attempted. However, since most of the allergen aqueous solution was absorbed by the wallpaper sample, it was squeezed using a vise and 100 μL was recovered. The allergen concentration of this solution was measured by sandwich ELISA. The test was performed in triplicate.

Comparative Example 2
The wallpaper of the test substrate was cut into a circle with a diameter of 8 cm, 1 mL of a mite allergen aqueous solution (Der f2 concentration 900 ng / mL) was spread on the processed surface, and a circular polyethylene sheet with a diameter of 7 cm was covered from above. This was stored in a plastic bag with a chuck for 1 hour at room temperature, 100 μL of the allergen aqueous solution was collected using a pipetman, and the allergen concentration was measured by sandwich ELISA. The test was performed in triplicate.

Table 1 shows the allergen concentration measurement results of Example 1 and Comparative Examples 1 and 2. In Example 1, a highly reproducible measurement result was obtained for both the unprocessed product and the processed product.

(Evaluation of allergen reducing agent processed floor materials)
The allergen-reducing effect was measured for a floor-floor plywood containing photocatalytic titanium oxide (ST-01, manufactured by Ishihara Techno Co., Ltd.) as an allergen reducing agent and a plywood with an unprocessed allergen reducing agent.

Example 2
The floor material of the test substrate was cut into a square so that the length of one side was 8 cm. Four stainless steel wires with a diameter of 0.3 mm cut into 5 mm lengths as gap holders are placed at the four corners of the flooring, and 1 mL of a cedar pollen allergen aqueous solution (Cry j1 concentration: 12.5 ng / mL) is added. I spread it and covered another floor material from the top so that the processing surface was matched. This was stored in a plastic bag with a chuck at room temperature, and after 18 hours, 100 μL of an allergen aqueous solution was recovered using a Pipetman, and the allergen concentration was measured by a sandwich ELISA method. Absorbent cotton soaked with water was placed in a plastic bag at the time of storage to suppress the volatilization of water from the allergen aqueous solution. The test was performed in triplicate.

Example 3
The floor material of the test substrate was cut into a square so that the length of one side was 8 cm. Place four pieces of 0.15 mm thick microscope cover glass as gap holders at the four corners of the floor floor sample, and spread 0.6 ml of a cedar pollen allergen aqueous solution (Cry j1 concentration 12.5 ng / mL). From the top, another floor / floor sample was placed from above so that the processed surfaces were matched. This was stored in a plastic bag with a chuck at room temperature, and after 18 hours, 100 μL of an allergen aqueous solution was recovered using a Pipetman, and the allergen concentration was measured by a sandwich ELISA method. Absorbent cotton soaked with water was placed in a plastic bag at the time of storage to suppress the volatilization of water from the allergen aqueous solution. The test was performed in triplicate.

Comparative Example 3
The floor material of the test substrate was cut into a square so that the length of one side was 8 cm. 1 mL of a cedar pollen allergen aqueous solution (Cry j1 concentration: 12.5 ng / mL) was spread on the processed surface, and a square polyethylene sheet having a side of 7 cm was covered from above. This was stored in a plastic bag with a chuck for 18 hours at room temperature, and 100 μL of an allergen aqueous solution was collected using a pipetman, and the allergen concentration was measured by a sandwich ELISA method. The test was performed in triplicate.

The allergen concentration measurement results of Examples 2 to 3 and Comparative Example 3 are shown in Table 2. In Examples 2-3, measurement results with high reproducibility were obtained for both unprocessed products and processed products.

According to the present invention, it is possible to accurately and highly reproducibly evaluate the effect of reducing allergens such as flooring and wallpaper in which an allergen-reducing agent is processed on the entire surface or wallpaper.

Claims (4)

A test method for evaluating the allergen reduction performance of a test substrate processed with an allergen reducing agent, where an allergen aqueous solution having a known concentration is placed on the surface of the test substrate processed with the allergen reducing agent, and further allergen reduction is performed. Spread the allergen aqueous solution on the surface of the test substrate by sandwiching the surface of another test substrate processed with the agent so as to match the allergen aqueous solution, collect the allergen aqueous solution after a certain period of time, and determine the amount of allergen in the aqueous solution. A method for testing allergen-reducing performance, wherein the allergen-reducing performance is evaluated by analyzing 2. The test method according to claim 1, wherein an allergen aqueous solution and a gap holder are sandwiched between two test substrates. 3. The method for testing allergen reduction performance according to claim 1, wherein the method for analyzing the amount of allergen in the allergen aqueous solution is an enzyme immunoassay. The test substrate is a flat or sheet-like material selected from wooden materials, plastic products, rubber, glass, stone and ceramics, or a coating is applied to the surface of these flat or sheet-like test substrates. The test method for reducing allergen performance according to any one of claims 1 to 3, wherein the test method is formed.