JPH06265544A - Medicine and method for diagnosing hair damage - Google Patents

Abstract

PURPOSE:To decide the damage of hair easily, quickly with high sensitivity by causing antigen-antibody reaction between a diagnostic medicine prepared by fixing an insoluble carrier and a hair or a protein derived therefrom for an antibody exhibiting immune activity to the protein of human hair. CONSTITUTION:An antibody exhibiting immune activity to the protein for human hair can be obtained through immune response of animal using the component of hair, e.g. hair keratein protein or hair cuticle protein, as an antigen. The insoluble carrier to be fixed to the antibody preferably includes a synthetic polymer, especially polystyrene having or not having a functional group. When the insoluble carrier and the antibody are fixed by physical adsorption or chemical bonding, an antibody exhibiting immune activity to human hair protein decorated by insoluble carrier can be prepared in the form of various tablets. For example, hair is immersed into a diagnostic medical solution and the degree of adhesion of diagnostic medicine produced through antibody-antigen reaction to the surface of hair is measured thus deciding the extent of damage of hair.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hair damage diagnostic agent and a method for diagnosing hair damage, and more specifically, a modification in which an insolubilized carrier is immobilized on an antibody having an immunological activity against human hair protein. The present invention relates to a hair damage degree diagnostic agent comprising an antibody and a method for diagnosing a hair damage degree using the diagnostic agent.

[0002]

2. Description of the Related Art Hair is constantly exposed to damage due to external environment, physical factors, chemical factors, and the like.
However, because hair is an assembly of already dead cells, once damaged, it cannot be repaired. Therefore, it is required to correctly evaluate the degree of damage to hair and take some measures (hair treatment etc.) to minimize the damage as early as possible. Further, in order to perform appropriate treatments such as permanent wave, hair coloring and treatment, it is important to perform accurate hair diagnosis and then perform those treatments.

The conventional measurement of the degree of hair damage is carried out by the following method. First of all, "hair is dry" and "matte" as if touched with fingers or combed.
There is a method for sensuously evaluating "a lot of split ends", "the hair is not stiff", "combing is poor", and the like.

In addition, as a method using various devices,
<Observation with a microscope><measuring frictional resistance><measuring tensile strength><measuring swelling ratio><measuring alkali solubility><measuring compositional changes of amino acids>
And the like. Specifically, the condition of the hair surface can be observed with an optical microscope or an electron microscope, the increase in frictional resistance due to damage to the hair surface can be measured, the elongation of the hair can be measured, and the water absorption of hair. Is obtained by increasing the weight of the hair to obtain the expansion coefficient, the change in the hair weight due to the alkali treatment, and the amount of cystine in the hair is measured to obtain the decrease rate. For these measurements, an optical microscope, an electron microscope, a microscope, a hair tester (tension meter), a pH meter, a pH test paper, a sump method set, a friction resistance measuring device, a hair gauge, etc. are used.

Although it is desirable to comprehensively determine the degree of hair damage by performing the above-mentioned test, in actual cases, the degree of hair damage may be determined by the sense of touch with the hand or the sense of weight due to shampooing. Many. It is because the general people do not have the above-mentioned measuring device,
This is because not all of them can be prepared in general beauty salons. Further, the measuring method thereof cannot be said to be simple, nor is it possible to measure many hairs at once in a short time. Therefore, the judgment of the degree of hair damage depends on sensory things, and individuals cannot always judge easily and accurately.

[0006] By the way, the hair is covered with a cuticle (hair epidermis) on the outside, a cortex which is a hair fiber, and a central medulla (hair medulla) on the inside. Hair is constantly damaged by various factors, and exfoliation of cuticles or degeneration or decomposition of internal keratin protein occurs. Therefore, it can be said that washing the hair with shampoo or the like causes outflow of the intercalation substance or the like that binds the keratin protein and the like, and the greater the elution amount, the more severe the hair is damaged.

[0007]

The present inventors have completed the present invention by discovering that the above-mentioned eluted substances can be quantified and the damaged portion of hair can be detected by an antibody having an immunological activity against human hair protein. An object of the present invention is to provide a method for easily, quickly, accurately and sensitively determining the degree of hair damage and a diagnostic agent therefor.

[0008]

Means for Solving the Problems The above-mentioned object is to provide a hair damage diagnostic agent comprising a modified antibody in which an insoluble carrier is immobilized on an antibody having immunological activity against human hair protein, and the hair damage diagnostic agent. It is achieved by a method for diagnosing the degree of hair damage, which comprises inducing an antigen-antibody reaction between the hair and the hair or a protein derived from the hair.

The present invention will be described in detail below.

Examples of the antibody having an immunological activity against the human hair protein used in the present invention include antibodies against various components constituting hair. Such an antibody can be obtained, for example, by immunizing an animal with a hair keratin protein, a hair cuticle protein, an intercellular substance, a hair matrix protein, or a fragment thereof as an antigen. Further, it can be obtained by immunizing animal nails, body hair, feathers, etc., or extracts or fragments thereof, etc., but considering the difference in species, human hair, body hair is preferable, and particularly hair is constituted. The various ingredients mentioned are most preferred.

Animals used for immunization include cattle, horses,
Appropriate livestock can be selected from sheep, goats, rabbits, chickens and the like.

The antibody can be obtained from the normal milk or colostrum of these animals, serum, egg yolk, etc., but the antibody obtained from bovine normal milk or colostrum or egg yolk is preferable because a large amount can be obtained. Purification of antibodies from these antibody raw materials may be carried out by a known method. For example, after removing lipids by a suitable method, purification is carried out by ammonium sulfate fractionation method, alcohol precipitation method, membrane separation method, etc. Obtainable. If necessary, further purification can be performed by ion exchange chromatography, gel filtration chromatography or the like to obtain a purified antibody. Further, if necessary, high-purity purified antibody can be obtained by performing affinity chromatography using the antigen used for immunization as a ligand.

Further, an antibody can be obtained as a monoclonal antibody from a fused cell of an antibody-producing cell producing the desired antibody and a myeloma cell.

The antibody thus obtained may be an antibody fragment obtained by treating with an enzyme such as papain or pepsin to remove the Fc portion of immunoglobulin.

Further, it may be a product of a microorganism or a cultured cell into which an immunoglobulin gene fragment cloned from spleen cells or lymphocytes of an immunized animal has been introduced.

The insoluble carrier used in the present invention includes
Polymer particles or the like are used. As the polymer particles, any material can be used as long as it is insoluble in an aqueous system. For example, synthetic high molecular polymers, metal particles, pigments, insoluble proteins and the like can be mentioned, but the point that they can be molecularly designed and can meet various conditions, and that dyes and pigments can be easily retained,
In addition, synthetic high molecular polymers are preferable, and polystyrene and polystyrene having a functional group are particularly preferable, because the size is uniform and the quantitativeness as a diagnostic agent is high.

Examples of the synthetic high molecular polymer include the following. Polystyrene, poly (α-methylstyrene), polyvinyltoluene, polychloromethylstyrene, polychlorostyrene, polyvinyl chloride, polyvinyl bromide, polyacrylonitrile, polymethacrylonitrile, ethyl polyacrylate, polyoctyl acrylate, poly Hydroxypropyl acrylate, butyl polyacrylate, methoxyethyl polyacrylate, hydroxyethyl polyacrylate, lauryl polyacrylate, ammonium polyacrylate, polymethylmethacrylate, polyethylmethacrylate, polypropylmethacrylate, polymethacrylic acid Butylaminoethyl, polyhydroxymethacrylic acid, polyvinyl acetate, polyacrylic acid, polymethacrylic acid, polymaleic acid, polystyrenesulfonic acid, poly (2-acrylamido-2-methyl) Lopansulfonic acid), polyacrylamide, polymethacrylamide, poly {N- (2-hydroxypropyl) methacrylamide}, poly (2-hydroxyethyl methacrylate), poly (glycerol monomethacrylate), poly (2-oxyethyl acrylate), Examples thereof include poly (2-oxyethyl methacrylate), polyethylene glycol methacrylate, polyethylene, polypropylene, polybutene, polyisobutene and copolymers thereof, polyurethane, copolymers of polyurethane and silicone, nylon beads and the like.

Examples of the metal particles include titanium oxide and magnetic particles.

Examples of the pigment include inorganic pigments such as titanium dioxide, zinc oxide, chromium oxide and carbon black, and organic pigments such as fast yellow, lake red R4, brilliant fast scarlet and phthalocyanine blue.

As the insoluble protein, a neutral insoluble protein or a water-soluble protein crosslinked to be insolubilized / polymerized may be used. Specific examples include fibroin, gelatin, collagen and the like.

The form of these insoluble carriers is not particularly limited and may be spherical or plate-like, but spherical ones having a uniform particle size are preferred.
The size is not particularly limited, but preferably 0.001 to 100 μm.

The amount of antibody to the insoluble carrier is not generally specified, but the higher the amount of antibody, the higher the sensitivity as a diagnostic agent. Therefore, it is preferable to immobilize the antibody until it is saturated.

The insoluble carrier and the above antibody are immobilized by physical adsorption or chemical bonding. An amino group, a carboxyl group, a thiol group, a sugar chain portion and the like can be considered as the functional group of the above-mentioned antibody used for chemical bonding, and they can be bonded in the same manner as in the case of bonding the dye and the carrier. For example, when the sugar chain portion is used for binding, the sugar chain portion can be oxidized with metaperiodic acid or the like to generate an aldehyde group, and can be bound with the amino group of the carrier by Schiff base formation. When a thiol group is used, it can be bound using an SPDP reagent or the like.

It is preferable that the insoluble carrier according to the present invention is further colored by holding a dye, a pigment or the like, because the color makes the determination easier.

The dyes and pigments used for coloring include various synthetic dyes such as tar dyes, reactive dyes,
Examples thereof include fluorescent dyes, synthetic melanin dyes, natural dyes derived from animals, plants and microorganisms, organic or inorganic pigments and the like.

As a method for holding the dye, pigment and the like on the insoluble carrier, a physical method and a chemical method can be considered. As a physical method, a dye, an adsorption method of adsorbing a pigment to an insoluble carrier, a dye at the time of making an insoluble carrier, an internal addition method of adding a pigment, a dye in an insoluble carrier, an encapsulation method of wrapping a pigment, a dye in an insoluble carrier. There is a polymerization method in which a precursor is polymerized.

When the insoluble carrier has a functional group, it can be bound by a chemical method. As the functional group, an amino group, a carboxyl group, an aldehyde group, a hydroxyl group, a thiol group or the like is used.

The amount of the dye, pigment or the like added is not particularly limited, but when adsorbed on the surface of latex or the like, the range of 0.1 to 10% by weight is preferable with latex as 100, and it is visually observed. The amount that can be determined is sufficient. For example, when Lake Red R4, which is an organic pigment, is used, sufficient coloring can be obtained with 1% by weight. When internally adding to a synthetic high molecular polymer, 1
The range of 0.1 to 50% by weight is preferable as 00, and it is sufficient if the amount can be visually determined. For example, when phthalocyanine blue, which is an organic pigment, is used, sufficient coloring can be obtained at 15% by weight.

The antibody having immunological activity against the human hair protein modified with the insoluble carrier obtained as described above can be prepared in various dosage forms, for example, a solution using an appropriate solvent such as an aqueous solution. Or as a dispersion.

The method for diagnosing the degree of hair damage of the present invention can be carried out, for example, as follows.

Human hair is immersed in the solution (or dispersion) of the diagnostic agent for the degree of hair damage of the present invention, and the degree of adhesion of the diagnostic agent to the hair surface caused by the antibody-antigen reaction is measured. The degree of hair damage is determined based on the degree.

In addition, the hair is treated with physiological phosphate buffer (hereinafter referred to as PBS).
Is abbreviated. ) Etc. and extracted with a solution of proteins (these are called hair-derived proteins) and the hair damage diagnostic agent of the present invention are mixed, and the degree of aggregation caused by the antibody-antigen reaction is measured. It is also possible to determine the degree of hair damage.

[0033]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 (Preparation of antigen) 5 g of normal male hair and 5 normal female hair
g, mixed with 2% sodium polyoxyethylene lauryl sulfate (3EO) aqueous solution, and then washed with 8M urea,
It was added to 2.5 L of 0.2 M tris-hydrochloric acid buffer solution (pH 9.2) containing 0.2 M 2-mercaptoethanol, stirred for 1 hour under nitrogen bubbling at 50 ° C., and mashed using a Teflon homogenizer. Further, this extraction operation was repeated, centrifugation was performed at 10,000 × g for 30 minutes to remove insoluble matter, and hair protein was extracted and used as an antigen.

(Immunization of Rabbit) The protein concentration of the antigen solution of Example 1 was adjusted to 2 mg / ml with PBS, and the solution was mixed with Freund's complete adjuvant at a volume ratio of 1: 1 to prepare oil. An aqueous emulsion was prepared by subcutaneously administering 2 ml per animal to several sites on the backs of 3 rabbits, and after 2 weeks and 4 weeks, the same amount was further administered subcutaneously. At the 7th week, the same amount of the antigen solution was gently administered from the ear vein slowly over 30 minutes for immunization.

(Collection of antiserum) One week after the final administration (8th week), blood was collected from the test animal, centrifuged, and serum was collected.

(Purification of antibody) Next, the obtained antiserum 60
60 ml of PBS was added to ml, 80 ml of a saturated ammonium sulfate solution (pH 7.0) was gradually added to this with stirring, and the mixture was stirred for 1 hour under ice cooling. Centrifuge (10,000
After 0 × g), the precipitate was collected, dissolved in 60 ml of PBS, and the ammonium sulfate salting out fractionation was repeated. About 1 of the obtained precipitate
Dissolve in 20 ml PBS, dialyze against PBS,
The resulting precipitate was removed by centrifugation to obtain an antibody.

An antibody was purified from rabbit serum that was not immunized in the same manner as this method, and this was used as a control antibody.

(Preparation of Hair Damage Diagnostic Agent) Carboxy-modified polystyrene latex (0.19 μm, white) was dispersed in distilled water at a solid concentration of 1% and then adjusted to pH 5. with hydrochloric acid.
Adjusted to 0. 1 volume part of 0.01M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is added to 1 volume part of a latex dispersion solution having a concentration of 1%, and the mixture is stirred and left at room temperature for 2 hours. After centrifugation, the supernatant is removed, and 1 part by volume of physiological phosphate buffer solution (hereinafter abbreviated as PBS) is added to carry out a redispersion operation. To this, 1 part by volume of the above antibody diluted to 1 mg / ml with PBS is added, stirred, and left to stand at room temperature for 2 hours. After centrifugation, the supernatant was removed, and bovine serum albumin (hereinafter abbreviated as BSA) was added at a concentration of 0.1% to P.
After adding 1 part by volume of BS and carrying out a redispersion operation, the mixture is left at room temperature for 1 hour. After further centrifugation, BSA and polyoxyethylene sorbitan monolaurate (20 EO /
Hereinafter referred to as Tween 20. ) Was added at a concentration of 0.1% and redispersed by adding 1 part by volume of PBS to obtain a hair damage diagnostic agent.

In the present hair damage diagnostic agent, 0.5 mg of the above antibody was bound per 10 mg of latex weight.

Example 2 As the insoluble carrier, a carboxy-modified polystyrene latex (0.19 μm) adsorbed with a blue dye (Oil Blue B, manufactured by Shirado Chemical Co., Ltd.) was used, and a hair damage diagnostic agent was prepared in the same manner as in Example 1. Was prepared. In the present hair damage diagnostic agent, 0.5 mg of the antibody of Example 1 was bound per 10 mg of latex.

Comparative Example 1 A control agent was used to prepare a hair damage diagnostic agent in the same manner as in Example 1. In this hair damage diagnostic agent, 0.5 mg of the control antibody was bound per 10 mg of the latex weight.

Comparative Example 2 A blue dye was directly bound to the antibody having an immunological activity against the human hair protein prepared in Example 1 to prepare a hair damage diagnostic agent.

Test Example 1 Aggregation test The hair extract protein, which is the antigen prepared in Example 1, was diluted to 100 μg / ml with PBS. 1 part by volume of this antigen solution and 1 part by volume of each of the hair damage diagnostic agents of Examples 1 and 2 or Comparative Examples 1 and 2 were mixed, and an agglutination reaction was observed.
The results of the agglutination test are shown in Table 1. If no agglutination is observed at all-, if it is difficult to determine the presence or absence of agglutination ±, and if agglutination is clearly observed, the order of strong agglutination is ++, ++,
It was judged as +.

[0045]

[Table 1]

As can be seen from Table 1, the hair damage diagnostic agents of Examples 1 and 2 specifically reacted with the hair extract protein, and the agglutination could be visually judged by the insoluble carrier. Further, the hair damage degree diagnostic agent of Example 2 also contained a pigment, so that the determination was easy.
On the other hand, the hair damage diagnostic agent of Comparative Example 1 using the control antibody did not react with the hair extract protein. Further, the hair damage degree diagnostic agent of Comparative Example 2 in which only the dye was bound without using an insoluble carrier had poor color development, and it was difficult to visually determine the presence or absence of aggregation.

Test Example 2 Diagnosis of Damage (1) Extraction of Hair Protein A hair bundle (0.1 g) was immersed in 10 ml of PBS containing 0.1% Tween 20, and the mixture was stirred at room temperature for 30 minutes to obtain a normal hair extract. Obtained. The hair was then treated several times with a commercially available bleaching agent to give damaged hair. A hair bundle (0.1 g) of damaged hair was immersed in 10 ml of PBS containing 0.1% Tween 20, and the mixture was stirred at room temperature for 30 minutes to obtain a damaged hair extract.

(2) Agglutination reaction The extracts of normal hair and damaged hair were treated with 0.1% Tween.
Diluted 10-fold and 100-fold with n20-containing PBS.
Next, the hair damage diagnostic agent of Example 1 was diluted with PBS containing 0.1% Tween 20 by the multiple dilution method. 1 part by volume of each diagnostic diluent was mixed with 1 part by volume of a hair extract, and the agglutination reaction was observed. The results of the agglutination test are shown in Table 2. In addition, when aggregation was not observed at all, − was determined if presence or absence of aggregation was difficult to determine, and when aggregation was clearly observed, it was determined as ++, ++, + in order of strong aggregation.

[0049]

[Table 2]

As can be seen from Table 2, the hair damage diagnostic agent of Example 1 showed strong aggregation with the extract of damaged hair.

Further, the same test was carried out for the hair damage diagnostic agent of Comparative Example 1, but no aggregation was observed at all.

Test Example 3 Damage Diagnosis Hair was treated with a commercially available bleaching agent to prepare damaged hair treated differently. A hair bundle (0.1 g) of each damaged hair was dipped in 10 ml of PBS containing 0.1% Tween 20, and stirred at room temperature for 30 minutes to obtain a damaged hair extract. Then, the same operation as in Test Example 2 is performed,
The aggregation reaction of the hair damage degree diagnostic agent and the damaged hair extract of Example 2 was observed. The results of the aggregation test are shown in Table 3. In addition, when aggregation was not observed at all, − was determined if presence or absence of aggregation was difficult to determine, and when aggregation was clearly observed, it was determined as ++, ++, + in order of strong aggregation.

[0053]

[Table 3]

From Table 3, it was found that the coagulation became stronger according to the number of treatments with the bleaching agent, and the present invention is useful as a hair damage diagnostic agent.

Test Example 4 Damage Diagnosis The same operation as in Test Example 3 was performed using a commercially available permanent agent, and the aggregation reaction of the hair damage diagnostic agent and the damaged hair extract was observed.

Also in this test example, it was shown that the coagulation becomes stronger in accordance with the number of treatments with the permanent agent.

Example 3 A polystyrene latex (0.19 μm) having a blue dye (Oil Blue B, manufactured by Shirado Chemical Co., Ltd.) adsorbed thereon was dispersed in distilled water at a solid content concentration of 1%. PB in 1 volume part of the dispersion liquid
1 part by volume of an antibody having an immunological activity against the human hair protein prepared in Example 1 diluted with S to 1 mg / ml was added, and the mixture was stirred and allowed to stand at room temperature for 2 hours. After centrifugation, the supernatant is removed, and 1 part by volume of PBS containing 0.1% BSA is added to carry out a redispersion operation. After further repeating centrifugation and redispersion operations, 0.1% BSA and 0.1% Tween 20 were added.
1 part by volume of PBS containing was added and redispersed to obtain a hair damage degree diagnostic agent.

Comparative Example 3 A control agent was used to prepare a hair damage diagnostic agent in the same manner as in Example 3.

The diagnostic agent of Example 3 was also shown to react specifically with the hair extract protein as in Examples 1 and 2, and also showed an agglutination reaction with damaged hair.

Further, the same test was conducted for the hair damage diagnostic agent of Comparative Example 3, but no aggregation was observed.

Example 4 A hair damage diagnostic agent was prepared in the same manner as in Example 3, using polystyrene latex dyed with a fluorescent dye or a commercially available fluorescent latex for a biochemical test as the insoluble carrier. . This hair damage diagnostic agent
The antibody of Example 1 was 0.5 per 10 mg of latex.
mg bound.

Comparative Example 4 A control agent was used to prepare a hair damage diagnostic agent in the same manner as in Example 4.

Comparative Example 5 A fluorescent dye was directly bound to the antibody of Example 1 to prepare a hair damage diagnostic agent.

Test Example 5 Hair was treated with a commercially available perm agent to prepare damaged hair treated differently. A bundle of damaged hair (0.1 g) is immersed in 1 ml of the diagnostic agent of Example 4 or Comparative Examples 4 to 5,
Rotate for 10 minutes at room temperature. Then 0.05% Twee
After washing with a physiological saline solution containing n20, it was dried. Table 4 shows the results obtained by irradiating a light beam having an excitation wavelength with a light source having an excitation wavelength of a fluorescent dye (such as a commercially available ultraviolet lamp) in a dark place. When fluorescence is not observed, −, when it is difficult to determine the presence or absence of aggregation ±, when fluorescence is observed, + in order of strong fluorescence
It was judged as + and +.

[0065]

[Table 4]

From Table 4, it was found that the diagnostic agent for hair damage of Example 4 had an increased fluorescence intensity corresponding to the number of times of perm treatment, and the present invention is useful as a diagnostic agent for hair damage. On the other hand, the hair damage diagnostic agent of Comparative Example 4 using the control antibody did not show fluorescence. Further, the hair damage degree diagnostic agent of Comparative Example 5 to which only the fluorescent dye was bound had poor color development, and it was difficult to visually determine the presence or absence of fluorescence.

[0067]

EFFECT OF THE INVENTION With the hair damage diagnostic agent of the present invention,
The degree of hair damage can be determined accurately, quickly and easily.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Umeji Murakami 5-328, Kotobuki-cho, Odawara-shi, Kanagawa Inside Kanebo Co., Ltd. Biochemical Research Laboratory (72) Inventor Junichiro Hiraoka 5-chome, Kotobuki-cho, Odawara, Kanagawa No. 3-28 Kanebo Co., Ltd. Biochemistry Research Institute (72) Inventor Kenichi Sugimoto 5-3 28, Kotobukicho, Odawara-shi, Kanagawa Kankobo Co., Ltd. Cosmetic Research Institute (72) Inventor Hiromi Minamino Hisashi Odawara-shi, Kanagawa Prefecture 5-3-28 Machi Kanebo Co., Ltd. in the Cosmetic Research Laboratory (72) Inventor Masashi Yakumaru 5-3 28 Kotobukicho, Odawara-shi, Kanagawa Kanbo Spinning Co., Ltd. (72) Inventor Toru Matsuo Odawara, Kanagawa Prefecture 5-3-28, Kotobuki-cho, Kanebo Co., Ltd., Cosmetic Research Laboratory

Claims (2)

[Claims] 1. A diagnostic agent for a degree of hair damage, which comprises a modified antibody obtained by immobilizing an insoluble carrier on an antibody having an immunological activity against human hair protein. 2. A hair damage characterized by mixing the diagnostic agent for a degree of hair damage according to claim 1 and a hair or a hair-derived protein to induce an antibody-antigen reaction between a modified antibody and the hair or the hair-derived protein. Degree diagnostic method.