JP2000063207A - Control of allergen and formulation therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To suppress adverse effects and simply remove a wide range of allergens such as a mite, a Japanese cedar pollen or a cockroach at a low cost without leaving toxic substances in an environment in order to suppress the onset of allergy by denaturing the allergens utilizing an organic solvent such as an alcohol and inactivating the allergens. SOLUTION: An organic solvent such as an alcohol (preferably the alcohols such as ethanol, methanol or propanol, hydrocarbons such as hexane, toluene or xylene or halogenated hydrocarbons, etc., such as chloroform or dichlorobenzene) is brought into contact with allergens by spraying such as an aerosol to thereby denature and inactivate the allergens and remove the allergens. Ethanol at 20-100 vol./vol.% concentration is more preferably utilized as the organic solvent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preventive technique for allergy, atopy, asthma and the like, and more particularly to a method and a preparation for removing allergen that inactivates or insolubilizes allergens in the environment.

[0002]

2. Description of the Related Art The number of allergic patients is increasing year by year, and it is said that the number of patients in Japan is 7.5 million, which is currently a very serious problem. Countermeasures for allergic diseases can be divided into treatment of people with allergic diseases and methods of removing allergens themselves.

[0003] Steroids and antihistamines have been administered for the treatment of people with allergic diseases, but they are not at a satisfactory level in view of their effects and side effects. In addition, treatment methods to improve physical constitution are also being considered,
The method has not been established. On the other hand, it has been reported that removing allergens from the environment is very effective in preventing the development of allergies (Allergy 41
1405-1412, THELANCET 336
895-897). In order to reduce the allergen density in the environment, acaricides, bactericides, vacuum cleaners and futon dryers, etc., which have an acaricidal effect are commercially available. Further, JP-A-61-44821 and JP-A-6-279273 disclose a method of removing an allergen by treating the environment with tannic acid or a substance having a similar structure.

[0004]

The above-mentioned products are effective in suppressing the growth of mites and molds themselves, but since allergens are contained in large amounts in the mold body, carcasses of mites and excrement, Just killing mites and molds does not reduce the amount of allergens in the room. Therefore, in order to suppress the onset of allergies, it is necessary to remove these allergens. The removal of allergens is generally performed by suction with a vacuum cleaner, futon tapping, cleaning, or the like.
However, it is very difficult to remove the allergen by performing the above-mentioned method normally, and it takes time and cost even in the most effective cleaning (allergy 4043).
9-443). Also, when the environment is treated with a substance such as tannic acid, the polyhydric phenol group forms a chelate with the metal ion and a specific color reaction occurs, causing the walls and rugs to be colored and before actual use. There are many problems to be solved. From the above, an inexpensive and simple allergen removal method is desired.

The present invention improves the above-mentioned problems of the prior art.
The present invention provides a novel technique which is not only effective in preventing the development of allergies, but which suppresses side effects, does not leave harmful substances in the environment, and can be used for a wide range of allergens.

[0006]

Means for Solving the Problems As a result of intensive studies for solving the above-mentioned problems, the present inventors have found that an allergen composed of a protein is brought into contact with an organic solvent by spraying with an aerosol or the like. It was discovered that there is a change in physiological activity. It was also clarified that the same effect can be obtained with solvent vapor.

The organic solvent includes alcohols such as ethanol, methanol and propanol, hydrocarbons such as hexane, toluene and xylene, halogenated hydrocarbons such as chloroform and dichlorobenzene, phenol,
Phenols such as cresol, ethers such as diethyl ether and tetrahydrofuran, carboxylic acids such as acetic acid and oleic acid, nitrogen compounds such as acetonitrile and aniline, esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, dimethyl sulfoxide and the like A very good effect appears when one or a mixture of two or more selected from sulfur compounds is used.

Further, the above-mentioned allergen has a remarkable effect when targeting allergens such as mites, cedar pollen, cockroaches and molds existing in the environment.

Further, by using the organic solvent, it is possible to prevent a specific color-matching reaction from occurring and to prevent contamination such as coloring.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below with reference to Examples. However, it goes without saying that the present invention is not limited to these examples.

<Example 1> Example 1 is a case of immersion in a solvent, and the effects are as follows. Freeze-dried 1 mg of mite bodies isolated from mite cultures gave 0, 50, 10
Immerse in 1 ml of 0 v / v% ethanol solution for 10 minutes, and
The binding ability with IgE (immunoglobulin E) of allergic patients was investigated by the LISA method. This ELISA method is as follows.

Bicarbonate buffer
The antigen solution diluted to 10 μg / ml with a buffer was added to a microtiter plate (Falcon 3912, B).
ecton Dickinson and Co. 50 μl was added to the well of (1) and allowed to stand at 25 ° C. for 2 hours to immobilize the antigen. PBST (PBS container
ng 0.05% Tween20), after washing with PBS,
200 μl of blocking buffer
er) (PBST, maintaining 5% ski)
(n milk) was added and the mixture was allowed to stand at 25 ° C. for 2 hours. Wash again with PBST and PBS, then with PBS 4, 8, 16
50 μm each of the mite asthma patient pool serum diluted twice
1 of each was added, and the mixture was allowed to stand at 25 ° C for 12 hours. PBST, P
Affinity-separated goat antibody anti- (Affinity-isolated) diluted 1000 times after washing with BS
Goat Antibodies anti- (Hu
man IgE epsilon chain)) biotin conjugate (Biotinconjugate (BIO)
SOURCE international)) was applied to the plate in an amount of 50 μl, and the plate was allowed to stand at 25 ° C. for 2 hours. After washing with PBST and PBS, 1000-fold diluted HPR-streptavidin (HPR-st
50 μl of reptavidin) (Zymed) was applied, and the mixture was allowed to stand at 25 ° C. for 1 hour. After washing with PBST and PBS, add 100 μl of coloring solution to develop color, and
Absorbance was measured in nm.

The results are shown in Table 1 below, which shows the effect when immersed in a solvent, and IgE in the ethanol immersion treatment.
The binding ability (IgE binding ability of the water treatment area (water immersion area)) is 100.
The binding capacity (%) is shown in FIG.

[0014]

[Table 1]

As is clear from Table 1 and FIG. 1, 50%
Water treatment area (water immersion area) shows binding ability with ethanol solution
Of 40%. This result indicates that ethanol acts on the allergen to reduce the valency of lgE for allergic patients or the binding of the allergen to the microplate, which changes the conventional physiological activity. That is clear.

<Embodiment 2> Embodiment 2 is a case of contact with a solvent vapor, and the effects are as follows. Put 0,8,22,100 v / v% ethanol solution in a closed container, fully saturate the inside of the container, put a glass petri dish containing 1 mg of mite body, and contact with saturated ethanol vapor at 25 ° C for 10 hours. It was

The results are shown in Table 2 below, which shows the histamine release rate based on the water treatment group at each antigen concentration, and FIG. 2 which shows the histamine release rate of the allergen that was in spatial contact with ethanol. Water treatment at each antigen concentration The histamine release ratio based on the plot is shown in FIG. 3, and the antigen concentration at 50% histamine release is shown in FIG. 4, respectively.

[0018]

[Table 2]

As is clear from FIG. 2, the activity was most reduced in the 22% ethanol solution (about 70% ethanol concentration in the air).

Further, as is clear from Table 2 and FIG.
The lower the antigen concentration, the more remarkable the decrease in activity, and the most effective effect was when the antigen concentration was 0.05 μg / ml.
The activity was reduced by a factor of 5.

Further, as is clear from FIG. 4, the antigen concentration required when the histamine release rate (the method for measuring the histamine release rate will be described later) is 50% is 0.9 μg / ml in the water treatment area. However, it was 2.1 μg / ml in the 22% ethanol treated group. In other words, the activity as an antigen was the same although the amount of the antigen taken into the living body was 2.3 times.

In this way, the allergen can be deactivated extremely effectively when the concentration is 20 v / v% to 100 v / v% in the ethanol solution.

Next, a method for measuring the histamine release rate will be described. Allergy is caused by IgE binding to basophils
It develops when antibodies bind to antigens and are crosslinked, and basophils release chemical mediators such as histamine. Therefore, an antigen with reduced allergen activity has reduced binding to IgE, and thus the amount of histamine released from blood cells is reduced. Therefore, the antigen was treated with ethanol and the amount of histamine released from the blood cells of allergic patients was measured by high performance liquid chromatography.

-Method of treating ethanol and preparation of antigen solution 1 mg of mite bodies separated and freeze-dried from mite culture were separately placed in a glass petri dish having a diameter of 6 cm and placed in a closed container (510 ml) at 0, 8, 22, 100 v / 50 ml of v% ethanol solution was poured and the inside of the container was sufficiently saturated with ethanol of each concentration. A petri dish containing the antigen was placed in each container and left at 25 ° C. for 10 hours to bring it into contact with ethanol in the air. After the contact, Hanks' buffer was added and suspended to recover the antigen.

Preparation of blood cells of allergic patients Blood of allergic patients was centrifuged at 1400 rpm for 15 minutes to separate into blood cells and plasma. Blood cells were added with 5 times the amount of Hanks' buffer and added to 1400
After centrifugation at rpm for 15 minutes, the supernatant was removed and the plate was washed 3 times. The separated plasma was added to the washed blood cells and used as a blood cell fluid for antigen stimulation.

Preparation of a sample for measuring the amount of free histamine The prepared antigen was added to Hanks 'buffer (Hanks' buff).
er) 0.0005, 0.001, 0.005, 0.
01, 0.05, 0.1, 0.5, 1, 5, 10, 5
Dilute to a concentration of 0,100 μg / ml and add 12 to the tube.
0 μl was dispensed. To each diluted solution, 120 μl of the blood cell solution prepared in the preparation of the blood cell solution for allergy patients was added, and the mixture was incubated at 37 ° C. for 3
Incubated for 0 minutes. After centrifugation at 1400 rpm for 10 minutes, 12 μl of 60% HClO ₄ was added to 120 μl of the supernatant, and the mixture was stirred to precipitate a protein component, and then 12000 rp
100 μl of the supernatant after centrifugation for 10 minutes was used as a quantitative sample. At the same time, after adding an equal volume of Hanks' buffer to 120 μl of blood cell solution, 60%
After adding 24 μl of HClO ₄ to destroy blood cells, 120
Centrifugation was performed at 00 rpm for 10 minutes, and 100 μl of the supernatant was used as a sample for quantifying total histamine. As a blank, the amount of histamine released in the case of no antigen stimulation was measured.

Measurement of free histamine amount by high performance liquid chromatography As a histamine measurement system, a high performance liquid chromatograph analysis system manufactured by Hitachi Ltd. was used. WH as a pretreatment column
-C18 (4.6 mm ID x 50 mm L), CM-825 (8.0 mm ID x 75 mm) as an analytical column
L), and quantified using F-1050 as a fluorescence detector.

The ratio of the amount of histamine released by each concentration of antigen stimulation (the amount of histamine in each sample) to the total amount of histamine in 100 μl of blood cell fluid was calculated as the histamine release rate by the following formula. Histamine release rate (%) = (Histamine amount of each sample-
Blank) / (total histamine amount-blank) x 100

<Example 3> Example 3 is a case where the allergen is insolubilized, and the effects thereof are as follows. 2 mg of mite bodies that had been separated from the mite culture and lyophilized were placed in a plastic Petri dish with a diameter of 3.5 cm. The water in the petri dish was evaporated along with the time treatment. The antigen was recovered with 1 ml of PBS, centrifuged at 12000 rpm for 10 minutes, and the amount of protein in the supernatant soluble antigen solution was measured. The measurement of this protein amount is as follows.

Using a protein assay reagent (Bio-Rad), the amount of soluble protein in each ethanol-treated group was measured by the Bradford method according to its protocol. That is, 200 μl of the staining solution was added to 10 μl of the sample and stirred. 15
After standing at room temperature for a minute, the absorption at OD ₆₀₀ nm was measured.

The antigen insolubilization rate of each ethanol-treated group with respect to the water-treated group was calculated by the following formula. Antigen insolubilization rate (%) = {1- (measured value of each treated section-blank) / (measured value of water treated section-blank)} × 100

The results are shown in FIG. 5 which shows the antigen insolubilization rate of each ethanol treatment group with respect to the water treatment group.

As is clear from FIG. 5, when treated with 75 v / v% ethanol, about 45% insolubilization was observed. It is speculated that the insolubilization of the allergen reduces the permeability of the mucosa and facilitates the removal of the allergen from the environment by the effect of aggregation and precipitation. In fact, the insolubilized allergen was recovered in a short time even at low G during centrifugation, and could be easily recovered using a membrane of constant pore size. Insolubilizing the allergen in this manner facilitates removal of the allergen.

[0034]

EFFECT OF THE INVENTION According to the method and preparation for removing allergen of the present invention, which contains the organic solvent as a main component, alone or in the form of adding a synergist, the allergen removing method does not leave side effects and does not leave harmful substances in the environment. Moreover, the allergen can be effectively denatured and deactivated without contamination such as coloring due to chelate formation of tannic acid or the like. Moreover, the present invention is very simple and versatile, and can reduce the amount of allergen in the environment at a low cost, by insolubilizing the allergen, facilitating the removal of the allergen, eventually effective in preventing the development of allergies. Is.

In addition, mites and cedar pollen existing in the environment,
Targeting allergens such as cockroaches and molds can produce significant effects.

The organic solvent includes alcohols such as ethanol, methanol and propanol, hydrocarbons such as hexane, toluene and xylene, halogenated hydrocarbons such as chloroform and dichlorobenzene, phenols such as phenol and cresol, Ethers such as diethyl ether and tetrahydrofuran, carboxylic acids such as acetic acid and oleic acid, nitrogen compounds such as acetonitrile and aniline,
By using one or a mixture of two or more selected from esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, and sulfur compounds such as dimethyl sulfoxide, a very good effect can be produced.

The organic solvent has a concentration of 20 v / v% to
By utilizing 100 v / v% ethanol, the allergen can be deactivated very effectively.

[Brief description of drawings]

FIG. 1 is a chart showing IgE binding ability in ethanol immersion treatment.

FIG. 2 is a diagram showing a histamine release rate of an allergen that is brought into spatial contact with ethanol.

FIG. 3 is a chart showing the histamine release ratio based on the water treatment group at each antigen concentration.

FIG. 4 is a chart showing the antigen concentration when 50% histamine is released.

FIG. 5 is a chart showing the antigen insolubilization rate of each ethanol treatment group with respect to the water treatment group.

Claims (10)

[Claims] 1. A method for removing allergen, which comprises deactivating the allergen by using an organic solvent such as alcohol. 2. A method for removing allergen, which comprises denaturing an allergen by using an organic solvent such as alcohol to denature the allergen. 3. The method for removing allergen according to claim 1, wherein the allergen is an allergen such as mites, cedar pollen, cockroaches, and molds existing in the environment. 4. The organic solvent includes alcohols such as ethanol, methanol and propanol, hydrocarbons such as hexane, toluene and xylene, halogenated hydrocarbons such as chloroform and dichlorobenzene, phenols such as phenol and cresol. , Ethers such as diethyl ether and tetrahydrofuran, carboxylic acids such as acetic acid and oleic acid, nitrogen compounds such as acetonitrile and aniline,
4. One or a mixture of two or more selected from esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, and sulfur compounds such as dimethylsulfoxide is used. Allergen removal method. 5. The organic solvent has a concentration of 20 v / v% to
The method for removing allergen according to claim 1, 2 or 3 or 4, wherein 100 v / v% of ethanol is used. 6. An allergen-removing preparation characterized by denaturing and deactivating an allergen using an organic solvent such as alcohol. 7. An allergen-removing preparation, characterized by denaturing an allergen by denaturing an allergen using an organic solvent such as alcohol. 8. The allergen-removing preparation according to claim 6, wherein the allergen is an allergen such as mites, cedar pollen, cockroaches, and molds existing in the environment. 9. The organic solvent includes alcohols such as ethanol, methanol and propanol, hydrocarbons such as hexane, toluene and xylene, halogenated hydrocarbons such as chloroform and dichlorobenzene, phenols such as phenol and cresol. , Ethers such as diethyl ether and tetrahydrofuran, carboxylic acids such as acetic acid and oleic acid, nitrogen compounds such as acetonitrile and aniline,
9. One or a mixture of two or more selected from esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, and sulfur compounds such as dimethyl sulfoxide is used. Allergen-removing preparation. 10. The organic solvent has a concentration of 20 v / v%.
The allergen-removing preparation according to claim 6, 7 or 8 or 9, wherein -100 to 100 v / v% of ethanol is used.