JPH093100A - Hair surface layer recognizing antibody, hair treating agent and hair damage diagnostic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new hair surface layer recognizing antibody by immunizing a peptide fraction as an antigen prepared by treating a hair surface layer part with an (immobilized) protease, capable of modifying handle of hair, useful as a hair treating agent, a hair dyeing agent, a hair damage diagnostic, etc. SOLUTION: This new hair surface layer recognizing antibody is obtained by immunizing a peptide fraction shown by formula I to formula IV as an antigen obtained by treating a hair surface layer part with a protease or an immobilized protease. The hair surface layer recognizing antibody has strong affinity for the surface layer part of hair, specific bonding properties and is useful as a component for a hair treating agent, a hair dyeing agent, a hair damage diagnostic, etc., having characteristics of improving strength, softness and handle of hair. The antibody is obtained by treating cut healthy hair with agarose papain in a buffer solution containing dithiothreitol, percutaneously injecting a prepared peptide shown by any of formulae I to IV together with an adjuvant to a rabbit, additionally immunizing, collecting a blood, centrifuging the blood, collecting an antiserum and separating the antibody from the antiserum.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a surface layer of a hair which has a property of specifically binding to the surface layer of the hair with a strong affinity and, as a result, improving the strength, suppleness, and texture of the hair. The present invention relates to a hair treatment agent, a hair dye, and a hair damage diagnostic agent containing a recognition antibody and a hair surface layer recognition antibody.

[0002]

2. Description of the Related Art Conventionally, it has been shown that an antibody against human hair keratin enhances the breaking strength of hair and has a hair-protecting effect (Journal of the Japanese Dermatological Society 104, 855,
1994; Japan Society of Animal Science, Vol. 65, p. 580, 1994).
In addition, a hair modifier using an antibody against a constituent component of hair (Japanese Patent Application Laid-Open No. 4-29912, Japanese Patent Application Laid-Open No. 4-4141).
No. 3) and a hair dye in which a coloring agent is bound to this antibody (JP-A-6-227955). However, although the disclosed antigens produce antibodies against the components that make up the interior of the hair, they do not produce antibodies that recognize the surface layer, or have a very low content even when they are produced. When used in applications such as hair agents that need to be bound to the hair surface, the treatment or hair dyeing effect is not sufficiently exhibited.

On the other hand, an antibody against the S100 protein, which is known to exist in the brain and nerves, is widely used for tissue staining, but there is no report that it is expressed in the surface layer of the hair, and recognition of the surface layer of the hair. There is no report of using it as an antibody. In addition, gene cloning has also been carried out for S100A3 protein, a kind of S100 protein (Pr
oc. Natl. Acad. Sci. USA 90, 6547, 1993), but there is no report that it is expressed in the surface layer of hair and that it is used as an antibody for recognizing the surface layer of hair.

[0004]

Therefore, the object of the present invention is to specifically bind to the surface layer of the hair with a strong affinity, and as a result, the strength, suppleness, and texture of the hair can be improved. Hair treatment agent with properties that make it good,
It is an object of the present invention to provide a hair dye capable of specifically dyeing hair without dyeing the skin and a hair surface recognizing antibody useful as a hair damage diagnosing agent capable of easily diagnosing hair damage by visual observation.

[0005]

That is, the present invention provides a peptide fraction obtained by treating the surface layer of hair with a proteolytic enzyme such as papain, a kind of thiol protease, or an immobilized proteolytic enzyme, and a multi-antigenic peptide thereof. Or a hair surface recognizing antibody obtained by immunizing with a carrier conjugate thereof as an antigen, a hair treatment agent containing the hair surface recognizing antibody, a hair dye containing a colorant bound to the hair surface recognizing antibody, and the hair The present invention relates to a hair damage diagnostic agent containing a surface-recognizing antibody.

H-Ala-Gln-Tyr-Asp-Asp-Ile-Ala-Ser-Arg-OH ... (I) H-Ser-Lys-Ala-Glu-Ala-Glu-Ala-Trp-Tyr- Gln-OH ... (II) H-Ser-Val-Leu-Asp-Thr-Asn-Lys-Asp-Cys-Glu-Val-Asp-Phe-Val-Glu-OH ... (III) H- Leu-Tyr-Cys-His-Glu-Tyr-Phe-Lys-Asp-Cys-Pro-Ser-Glu-Pro-Pro-Cys-Ser- Gln-OH ... (IV)

The structure of the present invention will be described below in detail.

In the present specification, amino acids, amino acid residues, amino acid derivatives, peptides and protecting groups are IUPAC-
Symbols recommended by the IUB Biochemistry Nomenclature Committee (Biochemistr
y, vol. 11, p. 1726, 1972, IUPAC Appl., vol. 40, p. 317, 1
974) or a symbol conventionally used in the art, and the L symbol for L-amino acid and its residue is omitted. Such symbols are as follows, for example.

Ala: Alanine, Asn: Asparagine, Asp: Aspartic acid, Arg: Arginine, Cys: Cysteine, Gln:
Glutamine, Glu: Glutamic acid, Gly: Glycine, His: Histidine, Ile: Isoleucine, Leu: Leucine, Lys: Lysine Met: Methionine, Phe: Phenylalanine, Pro: Proline, Ser: Serine, Thr: Threonine, Trp: Tryptophan, Tyr. : Tyrosine, Val: Valine, Fmoc: 9-fluorenylmethyloxycarbonyl, Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl, tBu: t-butyl, OtBu: t-butyl ester , Trt: trityl, Boc: t-butyloxycarbonyl

The antibody for recognizing the hair surface layer of the present invention is obtained by treating a component derived from the hair surface layer with a proteolytic enzyme to immunize a reaction solution, that is, a peptide fraction as it is, or by analyzing the amino acid sequence of the obtained component. It can be obtained by chemically synthesizing based on this primary structure as an antigen.

The method for obtaining the antigen used in the present invention can be obtained, for example, by treating the hair with a proteolytic enzyme or an immobilized proteolytic enzyme capable of efficiently obtaining the surface layer component of the hair. Alternatively, the reaction solution obtained by centrifuging the reaction solution to remove hair, immobilized proteolytic enzyme and the like can be used as an antigen.

As the proteolytic enzyme used in the present invention, a well-known and publicly used proteolytic enzyme is used. However, the immobilized proteolytic enzyme hardly penetrates into the hair and undesired proteolytic products inside the hair are removed. Since it is hardly eluted, it is preferable in that an antigen that easily obtains an antibody that specifically recognizes the hair surface layer can be obtained.

The proteolytic enzyme used in the present invention includes trypsin, chymotrypsin, pepsin, papain, cathepsin B1 and the like, and it is preferable to use thiol protease such as papain and cathepsin B1 which are activated in the presence of a reducing agent. , Especially papain is preferred.

As the reducing agent used when the reducing agent and the thiol protease such as papain or cathepsin B1 are used in the enzymatic treatment, dithiothreitol, 2-mercaptoethanol, cysteine, sodium sulfite and the like can be mentioned.

The carrier for the immobilized proteolytic enzyme used in the present invention includes agarose gel, high molecular polymers such as polystyrene, latex such as carboxy-modified polystyrene latex, insoluble carriers such as liposomes, and amylose, dextran, gelatin, collagen, Examples thereof include water-soluble polymers such as polylysine.

The treatment of the surface layer of the hair is carried out, for example, by cutting the hair cut into pieces of several cm with a proteolytic enzyme or an immobilized proteolytic enzyme in the presence of a reducing agent. When dithiothreitol is used as the reducing agent, it is usually preferably 0.05 to 1% (W / V), for example, 10 to 50 units of agarose-papain per 1 g of hair is added to a buffer containing 0.2% (W / V). Add and incubate at 25 ℃ for 24 hours.

The method for obtaining the antigen used in the present invention includes a method in which hair is treated with a proteolytic enzyme, and individual peptide components are purified from the resulting reaction solution by a known method and isolated. However, it is also possible to determine the amino acid sequence of the isolated peptide and synthesize the peptide based on the sequence to provide an antigen. Furthermore, in order to enhance the antigenicity of the synthesized peptide, a carrier conjugate in which the peptide is bound to a carrier is used, or a plurality of the peptides are bound to synthesize a peptide such as a multi-antigenic peptide in which the antigenicity of the peptide is enhanced. Moreover, it can be provided as an antigen.

Examples of the method for producing the carrier conjugate used in the present invention include a method in which the peptide obtained by isolation or synthesis is bound to a carrier protein such as bovine serum albumin or hemocyanin using a crosslinking agent. To be The cross-linking agent used is m-maleimidobenzoyl-N-hydroxysuccinimide ester, dimethyl adipimidate,
Examples include dimethyl suberimidate.

As the multi-antigenic peptide used in the present invention, specifically, for example, the following formulas (V), (VI) and (VI) using the peptides represented by the above (I) to (IV) are used.
I) and the multi-antigenic peptide represented by (VIII),
Examples include multi-antigenic peptides using two or more of the peptides represented by (I) to (IV).

[0020]

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[0021]

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[0022]

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[0023]

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As the method for producing the multi-antigenic peptide used in the present invention, an amino acid having two or more amino groups or carboxyl groups in one molecule such as lysine is used in one molecule. A method of connecting a plurality of peptides in parallel (see J. Biol. Chem. 263, 1719, 1988).
Etc., but can also be synthesized based on the amino acid sequence.

As a method for synthesizing a multi-antigenic peptide, for example, a solid phase method (basics and experiments of peptide synthesis, Nobuo Izumiya et al., Maruzen Co., Ltd., 1985, p. 194) can be used.
Examples include a method in which a protected amino acid is condensed and synthesized.
Examples of the solid phase carrier include 4- (hydroxymethyl) phenylacetamidomethyl poly (styrene-cordyvinylbenzene) (see J. Amer. Chem. Soc. Vol. 98, page 7357, 1976).
Is preferred.

For the protection of the α-amino group of amino acids, a protecting group which is easily cleaved with piperidine, for example, Fmoc group is used. A protecting group that is difficult to be cleaved by piperidine is used as a side chain functional group of amino acids. That is, the guanidyl group of Arg is Pm
c group, hydroxyl groups of Ser, Thr and Tyr are tBu groups, carboxyl groups of Asp and Glu are OtBu groups, Asn and Gln
It is preferred that the carbamide group of, the thiol group of Cys, the imidazole group of His be Trt group, and the ε-amino group of Lys be protected by Boc group.

Β-Ala (Fmoc) is introduced into the hydroxyl group of the solid-phase carrier, and then α-, ε- (Fmoc) Lys (Fmoc) is bonded to the α-, α- (Fmoc) Lys (Fmoc).
Α-, ε- (Fmoc) Lys (Fmoc) on both amino groups of ε and ε
Is generally repeated to synthesize a multi-antigenic peptide skeleton having 4, 8 or 16 amino groups per β-Ala molecule. The extension of the peptide chain on the carrier is
It can be performed according to a usual solid phase method. That is, the deprotection of the N-terminal amino group and the condensation reaction with the protected amino acid are repeated to sequentially extend the peptide chain from the C-terminal on the carrier to obtain a protected peptide-resin.

The condensation reaction is carried out by the active ester method, especially the protected amino acid 2- (1H-benzotriazol-1-yl) -1,1,3,3-
Tetramethylronium hexafluorophosphate (HBT
The active ester method using U) ester is desirable because of its high reactivity. Cleavage of the peptide from the carrier and removal of the protective group can be carried out with trifluoroacetic acid, but if necessary, it can also be carried out in the coexistence of ethanedithiol, thioanisole, phenol and the like. The crude peptide or a salt thereof obtained by cleaving and deprotecting the peptide from the carrier can be purified by column chromatography, high performance liquid chromatography or the like.

Various analyzes of peptide and protein fractions used as antigens in the present invention were carried out by the following methods.

Amino acid sequence analysis: Gas phase protein sequencer (477A manufactured by Applied Biosystems) was used. Amino acid analysis of acid hydrolysates: hydrolysis with 6N hydrochloric acid (110
The resulting hydrolyzate was analyzed by an amino acid analyzer.

Mass spectrometry value: Time-of-flight mass spectrometer (Las
ermat 2000, manufactured by Finnigan) using α-cyano-4-hydroxycinnamic acid as a matrix.

Further, as an antigen used in the present invention, S
The S100 protein is an acidic soluble protein existing in nerve tissue, adipose tissue and the like, and is a general term for many protein families having a calcium ion binding site.

As the S100 protein, 10 cysteines were used.
% S100A3 protein containing 9% amino acid residues
Examples of α and β of 1 and 93 include S100 for producing an antibody having stronger selectivity for hair.
A3 protein is preferred.

The S100 protein can be obtained by extraction from bovine brain or the like by a known method, but in order to produce an antibody having higher selectivity for hair, it is necessary to extract it from hair, preferably cuticle. Is preferred.

Next, a method for immunizing the antigen thus obtained to produce a hair surface recognizing antibody will be described.

The hair surface recognizing antibody of the present invention can be obtained by immunizing mammals, birds and the like by a known method. Animals used for the production of the antibody include
You can choose suitable livestock such as cow, horse, sheep, goat, rabbit, chicken.

As the immunization method, a usual method such as subcutaneous injection, intraperitoneal injection, intramuscular injection, intravenous injection, nasal drop,
It can be performed by a method such as eye drops. If necessary, an adjuvant such as Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA) may be used in combination with the antigen.

The antibody can be obtained from animal serum, normal milk or colostrum, or egg yolk.

As the dosage form of the hair treatment agent of the present invention, there are various treatments, rinses, shampoos, hair creams, hair lotions, hair packs, permanent agents and the like, which are used for the hair.

The content of the hair surface recognizing antibody in the hair treatment composition of the present invention is 0.01% by weight to the total amount of the composition.
5% by weight is preferred, and 0.1% to 1% by weight is particularly preferred.

The dosage form of the hair dye of the present invention includes treatments, rinses, shampoos, hair creams and hair lotions.

The content of the coloring agent bound to the hair surface recognizing antibody in the hair dye of the present invention is based on the total amount of the composition.
0.01% to 5% by weight is preferable, and 0.1% to 1% by weight is particularly preferable.

Examples of the colorant that binds to the hair surface layer-recognizing antibody of the present invention include dyes, pigments, and dyes included in a polymer compound. Examples of the polymer compound include synthetic polymer polymers such as polystyrene, Examples thereof include liposomes.

The weight ratio of the binding amount of the hair surface recognizing antibody to the coloring agent is preferably 1: 100 to 10: 1, and particularly preferably 1:10 to 1: 1.

Examples of the hair damage diagnosing agent of the present invention include physiologically isotonic buffer (PBS) and the like in which an antibody for recognizing the surface of the hair or an antiserum containing the antibody for recognizing the surface of the hair is dissolved.

The content of the hair surface recognizing antibody in the hair damage diagnostic agent of the present invention is preferably 0.0001% by weight to 1% by weight, particularly preferably 0.001% by weight to 0.1% by weight.

[0047]

The present invention will be described in more detail with reference to the following examples.

Example 1 (A hair surface recognizing antibody obtained by using a peptide fraction obtained by treating hair with agarose-papain as an antigen)

1. Preparation of antigen After washing 10 g of chopped healthy hair, dithiothreitol
To 200 ml of 0.1 N phosphate buffer (pH 6.7) containing 0.2%, 200 units of agarose-papain (8.5 units / mg, manufactured by Elastin Products) were previously dispersed in the buffer and then centrifuged to discard the supernatant. The precipitated agarose-papain was added, and the mixture was rotated at 25 ° C. for 24 hours for reaction.

The reaction solution obtained was centrifuged to remove hair and agarose-papain, and a part of the supernatant was freeze-dried. The remaining supernatant was poured into Sep-Pak C ₁₈ (Waters), and the adsorbed peptide was eluted with methanol and freeze-dried to obtain a peptide fraction. The obtained peptide fraction was analyzed and the following analysis results were obtained.

Amino acid analysis value of acid hydrolyzate (mol%) Peptide fraction: Asx, 5.42; Thr, 4.18; Ser, 9.42; Glx, 10.6
4; Pro, 10.51; Gly, 6.67; Ala, 5.20; Val, 8.55; Cys-Cys / 2,1
0.17; Met, 1.09; Ile, 7.08; Leu, 7.70; Tyr, 2.84; Phe, 3.05;
Lys, 3.86; His, 0.83; Arg, 2.77

2. Preparation of antiserum and antibody 1 mg of the obtained antigen was dissolved in 1 ml of PBS, and 1 ml of FCA and 1:
The mixture was mixed at a ratio of 1 to form a water-in-oil emulsion. This was subcutaneously injected into several places on the back of a rabbit (Japanese white breed, 9 weeks old, Kbl: JW, female). After 3, 6, and 9 weeks, the adjuvant mixed with the antigen was changed to FIA and subcutaneously injected. One week after the final booster (10 weeks), whole blood was collected from the carotid artery and centrifuged to collect serum.

60 ml of PBS was added to 60 ml of the obtained antiserum,
To this, 80 ml of saturated sodium ammonium sulfate solution (pH 7.0) was gradually added with stirring, and the mixture was stirred for 1 hour under ice cooling. 10,000g
After centrifugation for 10 minutes, the precipitate was collected and dissolved in 60 ml PBS, and the ammonium sulfate fractionation was repeated. About 12 of the resulting precipitate
It was dissolved in 0 ml of PBS and filtered through a 0.2 μm filter to obtain an antibody.

Example 2 (H-Ala-Gln-Tyr-Asp-Asp-Ile-Al
Antibody for recognizing hair surface layer that recognizes a-Ser-Arg-OH) 1. Preparation of Antigen (1) Purification of H-Ala-Gln-Tyr-Asp-Asp-Ile-Ala-Ser-Arg-OH Sep-Pak C ₁₈ (manufactured by Waters) obtained by the same method as in Example 1.
One-fifth each of the adsorbed peptide components under reduced pressure was subjected to anion exchange liquid chromatography under the following conditions, and fractions with a retention time of about 15.1 minutes were collected.

Column: TSKgel DEAE-5PW (8.0 × 75 mm, manufactured by Tosoh Corporation) Elution: 10 mM Tris-HCl buffer (pH 9.6) to 0.7 M NaC
Linear concentration gradient of 10mM Tris-HCl buffer (pH 9.6) containing l (concentration gradient applied over 70 minutes) Flow rate: 0.5ml / min Detection: Absorbance at 220nm

The obtained fraction was dried under reduced pressure, and 1/2 of the fraction was subjected to reverse-phase liquid chromatography under the following conditions to collect a fraction having a retention time of about 4.4 minutes.

Column: A-312 ODS (6.0 × 150mm, YMC
Elution: Water-acetonitrile-trifluoroacetic acid (90:10:
0.1) to water-acetonitrile-trifluoroacetic acid (30: 7
(0: 0.1) linear concentration gradient (concentration gradient applied over 60 minutes) Flow rate: 2.0 ml / min Detection: Absorbance at 220 nm

The obtained fraction was dried under reduced pressure and further subjected to gel filtration liquid chromatography under the following conditions to collect a fraction having a retention time of about 3.5 minutes.

Column: G2000SWXL (7.8 × 300 mm, manufactured by Tosoh Corporation) Elution: Water-acetonitrile-trifluoroacetic acid (55:45:
0.1) Flow rate: 0.5 ml / min Detection: Absorbance at 220 nm

The obtained fraction was dried under reduced pressure and the following analysis results were obtained. Results of amino acid sequence analysis: H-Ala-Gln-Tyr-Asp-Asp-Ile-Al
Amino acid analysis value (molar ratio) of a-Ser-Arg-OH acid hydrolyzate: Asx, 1.71; S
er, 1.15; Glx, 1.49; Ala, 2.26; Ile, 0.91; Tyr, 0.80; Arg, 0.
67 Mass Spec: 1039.1 (calculated 1038.02)

(2) (H-Ala-Gln-Tyr-Asp-Asp-Ile-Ala-S
er-Arg) ₈ -Lys ₄ -Lys ₂ -Lys-βAla-OH was synthesized by a solid phase method at room temperature using a model 431A peptide synthesizer manufactured by Applied Biosystems.

Fmoc ₈ -Lys ₄ -Lys ₂ -Lys-βAla-resin (manufactured by Applied Biosystems) 0.125 mmol (0.30 g) was used as a starting material, and Fmoc-Arg (Pmc), Fmoc-Ser (tBu), Fmoc-Ala,
Fmoc-Ile, Fmoc-Asp (OtBu), Fmoc-Tyr (tBu), Fmoc-Gln
Each protected amino acid of (Trt) was used, and each step of deprotection washing of N-terminal Fmoc and condensation reaction washing was repeated to extend the peptide chain from the C-terminal to the resin.

Deprotection of N-terminal Fmoc was carried out using 20% piperidine-
It was carried out by treating with N-methylpyrrolidone for 1 minute and then for 7.6 minutes. Wash with N-methylpyrrolidone 4
I went there. Condensation reaction is carried out with 1 mmol of protected amino acid and 0.9
The solution was prepared by adding 1 ml of 2.0 M diisopropylethylamine / N-methylpyrrolidone to an N-methylpyrrolidone solution of mmol HBTU. Washing was performed with N-methylpyrrolidone.

After completion of the final condensation reaction of Fmoc-Ala, N-terminal
After deprotection of Fmoc, washing and operation, the residue was dried to obtain 0.42 g of protected peptide-resin.

Crystallized phenol / ethanedithiol / thioanisole / water / trifluoroacetic acid (3: 1: 2: 2: 40 w / v / v / v / v)
6 ml of the mixed solution was added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was filtered through a Polyflon filter PF060 (manufactured by Advantech), washed with 1 ml of trifluoroacetic acid and then with 10 ml of dichloromethane, and the filtrate was concentrated under reduced pressure at 40 ° C. or lower to about 1 ml. 50 ml of cold ether was added to this to precipitate the peptide. This is 3
It was filtered with a μm-pore PTFE membrane (manufactured by Advantech) and dissolved in acetic acid: water (1: 1).

The obtained crude peptide was subjected to reverse phase liquid chromatography under the following conditions, and fractions eluting at a retention time of about 9 to 20 minutes were collected.

Column: RESOURCE RPC 3 ml (6.4 × 100 m
m, Pharmacia Biotech) Elution: Water-acetonitrile-trifluoroacetic acid (90:10:
0.1) to water-acetonitrile-trifluoroacetic acid (50: 5
(0: 0.1) linear concentration gradient (concentration gradient applied over 40 minutes) Flow rate: 2.0 ml / min Detection: Absorbance at 220 nm

The obtained fraction was freeze-dried to obtain 18.0 mg of the title peptide. The following analysis results were obtained for this peptide.

Amino acid analysis value (molar ratio) of acid hydrolyzate: Asx, 18.7; Ser, 10.1; Glx, 9.9; Ala, 20.9; Ile, 10.9;
Tyr, 11.6; Arg, 7.7; Lys, 8.2; β-Ala, 1.0

2. Preparation of antiserum and antibody Antibodies and antibodies were obtained by immunizing rabbits with 250 µg of the obtained antigen in the same manner as in Example 1. The obtained antiserum was used for the following tests.

The antibody titer against the multi-antigenic peptide was measured by the ELISA method. That is, 100 μm of a 50 mM carbonate buffer solution (pH 9.6) containing 1 μg of the multi-antigenic peptide in each well of a 96-well plate (Immron 2, manufactured by Dynatech)
Each of them was placed in an amount of 1 and left overnight at 4 ° C. to be adsorbed on the plate. The plate was washed 6 times with physiological saline containing 0.05% Tween 20, and then 1% bovine serum albumin and 0.05% Tween 20 were added.
100 μl of antiserum diluted with PBS containing was added to each well. After reacting at 37 ° C. for 1 hour, the plate was washed 6 times.

As a secondary antibody, peroxidase-conjugated goat anti-rabbit IgG Fc fragment antibody (manufactured by Kapel) was used.
Solution diluted 7,000 times with PBS containing 0.05% Tween 20 1
Add 00 μl to each well and incubate at 25 ℃ for 30 minutes.
It was washed 6 times. Next, 0.2 M disodium phosphate-0.1 M citrate buffer (pH 5.0) was added to 50 ml of ortho-phenylenediamine.
Prepare a substrate solution in which 20 mg and 10 μl of 31% hydrogen peroxide solution are dissolved, add 100 μl to each well, and protect from light at 25 ° C for 2
Color was developed for 0 minutes. The reaction was stopped by adding 100 μl of 3N sulfuric acid. After the completion of color development, the absorbance of each well at 492 nm was measured. The antibody titer was the dilution ratio at which the absorbance was 0.2. Table 1 shows changes in antibody titer during the immunization process.

[0073]

[Table 1]

Example 3 (H-Ser-Lys-Ala-Glu-Ala-Glu-Al
Antibody for recognizing hair surface layer that recognizes a-Trp-Tyr-Gln-OH) 1. Preparation of antigen (1) H-Ser-Lys-Ala-Glu-Ala-Glu-Ala-Trp-Tyr-Gln-OH
Purification of Sep-Pak C ₁₈ (manufactured by Waters) obtained in the same manner as in Example 1.
1/5 each of the adsorbed peptide components under reduced pressure was subjected to anion exchange liquid chromatography under the following conditions, and fractions with a retention time of about 24.3 minutes were collected.

Column: TSKgel DEAE-5PW (8.0 × 75 mm, manufactured by Tosoh Corporation) Elution: 10 mM Tris-HCl buffer (pH 9.6) to 0.7 M NaC
Linear concentration gradient of 10mM Tris-HCl buffer (pH 9.6) containing l (concentration gradient applied over 70 minutes) Flow rate: 0.5ml / min Detection: Absorbance at 220nm

The obtained fraction was dried to dryness under reduced pressure, and 1/2 of the fraction was subjected to reverse phase liquid chromatography under the following conditions to collect a fraction having a retention time of about 9.3 minutes.

Column: A-312 ODS (6.0 × 150mm, YMC
Elution: Water-acetonitrile-trifluoroacetic acid (90:10:
0.1) to water-acetonitrile-trifluoroacetic acid (30: 7
(0: 0.1) linear concentration gradient (concentration gradient applied over 60 minutes) Flow rate: 2.0 ml / min Detection: Absorbance at 220 nm

The obtained fraction was dried to dryness under reduced pressure and then subjected to gel filtration liquid chromatography under the following conditions to collect a fraction having a retention time of about 3.4 minutes.

Column: G2000SWXL (7.8 × 300 mm, manufactured by Tosoh Corporation) Elution: Water-acetonitrile-trifluoroacetic acid (55:45:
0.1) Flow rate: 0.5 ml / min Detection: Absorbance at 220 nm

The obtained fraction was dried under reduced pressure and the following analysis results were obtained. Results of amino acid sequence analysis: H-Ser-Lys-Ala-Glu-Ala-Glu-Al
a-Trp-Tyr-Gln-OH mass spec: 1183.3 (calculated 1182.19)

(2) (H-Ser-Lys-Ala-Glu-Ala-Glu-Ala-T
rp-Tyr-Gln) ₈ -Lys ₄ -Lys ₂ -Lys-βAla-OH synthesis Fmoc ₈ -Lys ₄ -Lys ₂ -Lys-βAla-resin (Applied Biosystems) 0.125 mmol (0.30 g) As a starting material, Fmoc
-Gln (Trt), Fmoc-Tyr (tBu), Fmoc-Trp, Fmoc-Ala, Fm
oc-Glu (OtBu), Fmoc-Lys (Boc), and Fmoc-Ser (tBu) were used in the solid phase method in the same manner as in Example 2 to prepare protected peptide-resin 0.39 g. Got

0.1 g of the obtained protected peptide-resin was cooled under ice-cooling with ethanedithiol / water / trifluoroacetic acid (1: 1:
38 v / v / v) mixed solution (5 ml) was added, and the mixture was stirred at room temperature for 3 hours.
The reaction solution was treated in the same manner as in Example 2 to remove the peptide-cleaving protecting group from the carrier to obtain a crude peptide.

The obtained crude peptide was subjected to reverse phase liquid chromatography under the following conditions, and fractions eluting at a retention time of about 15 to 25 minutes were collected.

Column: RESOURCE RPC 3 ml (6.4 × 100 m
m, Pharmacia Biotech) Elution: Water-acetonitrile-trifluoroacetic acid (90:10:
0.1) to water-acetonitrile-trifluoroacetic acid (50: 5
(0: 0.1) linear concentration gradient (concentration gradient applied over 40 minutes) Flow rate: 2.0 ml / min Detection: Absorbance at 220 nm

The obtained fraction was freeze-dried to obtain 17.5 mg of the title peptide. The following analysis results were obtained for this peptide.

Amino acid analysis value (molar ratio) of the acid hydrolyzate: Ser, 7.6; Glx, 29.4; Ala, 30.6; Tyr, 11.4; Lys, 13.1;
β-Ala, 1.0

2. Preparation of Antisera and Antibodies Recognizing Hair Surface Layer 250 μg of the obtained antigen was immunized to rabbits in the same manner as in Example 1 to obtain antisera and antibodies. The obtained antiserum was used for the following tests.

The antibody titer against the multi-antigenic peptide was measured by the ELISA method in the same manner as in Example 2.
Table 2 shows changes in antibody titer during the immunization process.

[0089]

[Table 2]

Example 4 (H-Ser-Val-Leu-Asp-Thr-Asn-Ly
(S-Asp-Cys-Glu-Val-Asp-Phe-Val-Glu-OH recognizing hair surface layer)

1. Preparation of antigen (1) H-Ser-Val-Leu-Asp-Thr-Asn-Lys-Asp-Cys-Glu-Va
Purification of l-Asp-Phe-Val-Glu-OH Sep-Pak C ₁₈ (manufactured by Waters) obtained in the same manner as in Example 1.
One-fifth each of the adsorbed peptide components under reduced pressure was subjected to anion exchange liquid chromatography under the following conditions, and fractions with a retention time of about 27.6 minutes were collected.

Column: TSKgel DEAE-5PW (8.0 × 75 mm, manufactured by Tosoh Corporation) Elution: 10 mM Tris-HCl buffer (pH 9.6) to 0.7 M NaC
Linear concentration gradient of 10mM Tris-HCl buffer (pH 9.6) containing l (concentration gradient applied over 70 minutes) Flow rate: 0.5ml / min Detection: Absorbance at 220nm

The obtained fraction was dried under reduced pressure, and 1/2 of the fraction was subjected to reverse-phase liquid chromatography under the following conditions to fractionate a retention time of about 14.5 minutes. Column: A-312 ODS (6.0 × 150 mm, YMC) Elution: Water-acetonitrile-trifluoroacetic acid (90:10:
0.1) to water-acetonitrile-trifluoroacetic acid (30: 7
(0: 0.1) linear concentration gradient (concentration gradient applied over 60 minutes) Flow rate: 2.0 ml / min Detection: Absorbance at 220 nm

The obtained fraction was evaporated to dryness under reduced pressure and then subjected to gel filtration liquid chromatography under the following conditions to collect a fraction having a retention time of about 2.8 minutes.

Column: G2000SWXL (7.8 × 300 mm, manufactured by Tosoh Corporation) Elution: Water-acetonitrile-trifluoroacetic acid (55:45:
0.1) Flow rate: 0.5 ml / min Detection: Absorbance at 220 nm

The obtained fractions were dried under reduced pressure and the following analysis results were obtained. Amino acid sequence analysis results: H-Ser-Val-Leu-Asp-Thr-Asn-Ly
s-Asp-Cys-Glu-Val-Asp-Phe-Val-Glu-OH

(2) (H-Ser-Val-Leu-Asp-Thr-Asn-Lys-A
sp-Cys-Glu-Val-Asp-Phe-Val-Glu-OH) ₈ -Lys ₄ -Lys ₂ -Lys-
Synthesis of βAla-OH Fmoc ₈ -Lys ₄ -Lys ₂ -Lys-βAla-resin (manufactured by Applied Biosystems) 0.125 mmol (0.30 g) was used as a starting material, and Fmoc
-Glu (OtBu), Fmoc-Val, Fmoc-Phe, Fmoc-Asp (OtBu), Fm
oc-Cys, Fmoc-Lys (Boc), Fmoc-Asn (OtBu), Fmoc-Thr,
Using protected amino acids of Fmoc-Leu and Fmoc-Ser (tBu), synthesis was carried out by the solid phase method in the same manner as in Example 2 to obtain 0.40 g of protected peptide-resin.

0.1 g of the obtained protected peptide-resin was cooled under ice-cooling with ethanedithiol / water / trifluoroacetic acid (1: 1:
38 v / v / v) mixed solution (5 ml) was added, and the mixture was stirred at room temperature for 3 hours.
The reaction solution was treated in the same manner as in Example 2 to remove the peptide-cleaving protecting group from the carrier to obtain a crude peptide.

The obtained crude peptide was subjected to reverse phase liquid chromatography under the following conditions, and fractions eluting at a retention time of about 10 to 26 minutes were collected.

Column: RESOURCE RPC 3 ml (6.4 × 100 m
m, Pharmacia Biotech) Elution: Water-acetonitrile-trifluoroacetic acid (90:10:
0.1) to water-acetonitrile-trifluoroacetic acid (30: 7
(0: 0.1) linear concentration gradient (concentration gradient applied over 40 minutes) Flow rate: 2.0 ml / min Detection: Absorbance at 220 nm

The obtained fraction was freeze-dried to obtain 37.8 mg of the title peptide. The following analysis results were obtained for this peptide.

Amino acid analysis value (molar ratio) of acid hydrolyzate: Ser, 5.7; Val, 22.1; Leu, 5.9; Asx, 22.8; Thr, 5.2; Gl
x, 16.6; Lys, 11.5; β-Ala, 1.0

2. Preparation of antiserum and antibody Antibodies and antibodies were obtained by immunizing rabbits with 250 µg of the obtained antigen in the same manner as in Example 1. The obtained antiserum was used for the following tests.

The antibody titer against the multi-antigenic peptide was measured by the ELISA method in the same manner as in Example 2.
Table 3 shows changes in the antibody titer during the immunization process.

[0105]

[Table 3]

Example 5 (H-Leu-Tyr-Cys-His-Glu-Tyr-Ph
e-Lys-Asp-Cys-Pro-Ser-Glu-Pro-Pro-Cys-Ser-Gln-OH that recognizes the surface layer of hair) 1. Preparation of antigen (H-Leu-Tyr-Cys-His-Glu-Tyr-Phe-Lys-A
sp-Cys-Pro-Ser-Glu-Pro-Pro-Cys-Ser-Gln-OH) ₄ -Lys ₂ -L
Synthesis of ys-βAla-OH

Fmoc ₄ -Lys ₂ -Lys-βAla-resin (manufactured by Applied Biosystems) 0.125 mmol (0.29 g) was used as a starting material, and Fmoc-Leu, Fmoc-Tyr (tBu), Fmoc-Cys (Trt). , Fmoc
-His (Trt), Fmoc-Glu (OtBu), Fmoc-Phe, Fmoc-Lys (Bo
c), Fmoc-Asp (OtBu), Fmoc-Pro, Fmoc-Ser (tBu), Fmo
c-Gln (Trt), each protected amino acid was used and synthesized by the solid phase method in the same manner as in Example 2 to give the protected peptide-resin 0.4.
I got 9g.

0.2 g of the obtained protected peptide-resin was cooled under ice-cooling with ethanedithiol / water / trifluoroacetic acid (1: 1:
38 v / v / v) mixed solution (5 ml) was added, and the mixture was stirred at room temperature for 3 hours.
The reaction solution was treated in the same manner as in Example 2 to remove the peptide-cleaving protecting group from the carrier to obtain a crude peptide.

The resulting crude peptide was dialyzed against 0.2M acetic acid and freeze-dried to obtain 57.2 mg of the title peptide.

[0110] 2. Preparation of antiserum and antibody Antibodies and antibodies were obtained by immunizing rabbits with 250 µg of the obtained antigen in the same manner as in Example 1. The obtained antiserum was used for the following tests.

The antibody titer against the multi-antigenic peptide was measured by the ELISA method in the same manner as in Example 2.
Table 4 shows changes in the antibody titer during the immunization process.

[0112]

[Table 4]

Example 6 (Preparation of anti-S100A3 protein antibody) Preparation of antigen (production of S100A3 protein fraction from hair epithelium) 800 mg of hair epithelium containing 0.2M 2-mercaptoethanol
The mixture was added to 3 ml of 0.2 M Tris (pH 9.5), incubated at 50 ° C. for 1 hour, and then ground using a glass homogenizer. After further incubation at 50 ° C. for 1 hour, centrifugation (10,000 g) was performed and the supernatant was collected. The residue was triturated with a glass homogenizer, incubated at 50 ° C for 1 hour, and then centrifuged (10,000 g) to collect the supernatant, which was repeated 6 times in total. A protein fraction (8.9 mg as protein mass) was obtained.

[0114] 2. Preparation of antiserum and antibody Using the obtained hair epithelial protein fraction, antiserum and antibody were obtained in the same manner as in Example 1. The obtained antiserum was used for the following tests.

The antibody titer of the antiserum to the hair epithelial protein fraction was measured by the ELISA method in the same manner as in Example 2. However, 10 ng of hair epithelial protein fraction was adsorbed to each well of the 96-well plate. The antibody titer of the serum obtained by collecting whole blood 10 weeks after the first immunization was about 6900.

Test Example 1 (Hair binding of antiserum) [0116] The hair binding of antiserum obtained by whole blood sampling at 10 weeks to a multi-antigenic peptide and an anti-hair extract was evaluated by ELISA.
Performed by law.

Healthy hair was added to each well of a 96-well multiscreen DV filter plate (0.65 μm, Millipore).
Or agarose obtained in Example 2 as damaged hair-
Antiserum containing 1 mg of papain-treated hair diluted with PBS containing 1% bovine serum albumin and 0.05% Tween 20
100 μl was added to each well. After reacting at 37 ° C. for 1 hour, the plate was washed 6 times. As a secondary antibody, peroxidase-conjugated goat anti-rabbit IgG Fc fragment antibody (manufactured by Capel) was used in PBS containing 0.05% Tween 20.
Add 100 μl of 4000 times diluted solution to each well and
After reacting for 30 minutes with, it was washed 6 times.

Next, 50 ml of 0.2 M disodium phosphate-0.1 M citrate buffer (pH 5.0) was added to 20 ml of ortho-phenylenediamine.
Prepare a substrate solution in which 10 μl of 31 mg hydrogen peroxide and 31% hydrogen peroxide was dissolved, add 100 μl to each well, and keep at 30
Color was developed for minutes. The reaction was stopped by adding 100 μl of 3N sulfuric acid. After the completion of color development, transfer the color development solution to a 96-well plate (Imron 2, manufactured by Dynatec) and 492 nm in each well.
Was measured for absorbance. The hair binding amounts of the antisera of Examples 1 to 6 are shown in Table 5.

[0119]

[Table 5]

It is clear that all of the antibodies contained in the antisera of Examples 1 to 6 bind to healthy hair and damaged hair treated with agarose-papain.

Examples 7 to 12 (Antibody-containing hair treatment agent) Antibody-containing hair treatment agents were prepared in which the antibodies obtained in Examples 1 to 6 having the compositions shown below were mixed.

[0122]

[Table 6]

Test Example 2 (Hair Treatment Effect of Antibody-Containing Hair Treatment Agent) Human hair bundles (5 g of black hair) were immersed in the antibody-containing hair treatment agent of each of Examples 7 to 1 for 1 hour, and then washed with running water for 10 minutes. Air dried. After the treatment, the hair bundle treated with the antibody-containing hair treatment agent was compared with the comb before treatment, as a result of a sensory evaluation on whether the hair bundle before treatment was better for combing, suppleness, and texture. The street was supple and the texture was good.

Examples 13 to 18 (Hair dye containing antibody-bonded colorant) Carboxy-modified polystyrene latex (particle size 0.19 μm, manufactured by Japan Synthetic Rubber Co., Ltd.) adsorbing a red dye was purified water with a solid content concentration of 1% by weight. Dispersed. Each of the antibodies obtained in Examples 1 to 6 was applied to a column on which an antigen was immobilized and purified, and 1 mg of the affinity-purified antibody obtained in 1 ml of physiological phosphate buffer was added to 1 cc of the latex dispersion described above.
Stir overnight at ° C. The supernatant was removed by centrifugation, and the suspension was dispersed in a physiological phosphate buffer containing 0.1% bovine serum albumin. Centrifuge again
Redispersed in physiological phosphate buffer containing 0.1% bovine serum albumin and dispersed in physiological phosphate buffer containing 0.1% bovine serum albumin and 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) for antibody binding staining An agent-containing hair dye (Examples 13 to 18) was obtained. In this case, the content of the antibody-bonded colorant was 0.01% by weight based on the total amount of the hair dye.

Test Example 3 (Hair dyeing test of hair dye containing antibody-bound colorant) The hair dye containing antibody-bound colorant of Examples 13 to 18 was added to a hair bundle (human white hair, 50 mg) and rotated for 1 hour. . Then 0.
After washing with a physiological saline containing 05% Tween 20 and drying, the degree of coloring was visually determined.

[0126]

[Table 7]

Examples 19 to 24 (Hair Diagnostic Kit Utilizing Antibody-Containing Hair Damage Diagnostic Agent) A hair diagnostic kit comprising the following four kinds of solutions was prepared and the degree of damage was diagnosed with human hair.

Solution A: The antisera obtained in Examples 1 to 6 were respectively
A hair damage diagnostic agent was prepared by diluting 1,000 times with PBS containing 0.05% Tween 20. Solution B: A peroxidase-labeled goat anti-rabbit IgG antibody (manufactured by Kappel) was diluted 7,000 times with PBS containing 0.05% Tween 20 to prepare. Solution C: Orthophenylenediamine 20 mg and hydrogen peroxide 10
0.2 M disodium phosphate-0.1 M citrate buffer containing μl, pH 5.0 (peroxidase substrate solution) D solution: physiological saline

10 mg each for damaged hair and healthy people
Solution A containing the antisera of Examples 1 to 6 was added to and the mixture was incubated at 37 ° C. for 1 hour. After washing 6 times with solution D, it was incubated with solution B for 30 minutes at room temperature. When the solution C was added after washing with the solution D 6 times, the test tube containing the hair of a damaged person was colored in any of the antisera of Examples 1 to 6.

From the above results, the hair damage diagnostic agent of the present invention containing the antibody obtained by using the hair epithelium-specific protein as an antigen,
Obviously, it is possible to know the damage or health of the hair.

[0131]

Industrial Applicability As described above, according to the present invention, the surface of the hair is specifically bound with a strong affinity, and as a result, the hair has good properties such as strength, suppleness, and texture. It is obvious that a hair treatment agent, a hair dye, a hair damage diagnostic agent and a hair surface recognizing antibody useful as these components can be provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 7/08 C07K 7/08 (72) Inventor Umeji Murakami 5-3, Shoucho, Odawara-shi, Kanagawa No. 28 Kanebo Co., Ltd. Biochemistry Research Institute

Claims (9)

[Claims] 1. A hair surface-recognizing antibody obtained by immunizing a peptide fraction obtained by treating the hair surface layer with a proteolytic enzyme or an immobilized proteolytic enzyme as an antigen. 2. The hair surface recognizing antibody according to claim 1, wherein the proteolytic enzyme is a thiol protease. 3. The hair surface recognizing antibody according to claim 1, wherein the proteolytic enzyme is papain. 4. A hair surface recognizing antibody obtained by immunizing a peptide represented by any of the following general formulas (I) to (IV), its multi-antigenic peptide or its carrier conjugate as an antigen. H-Ala-Gln-Tyr-Asp-Asp-Ile-Ala-Ser-Arg-OH ... (I) H-Ser-Lys-Ala-Glu-Ala-Glu-Ala-Trp-Tyr-Gln-OH ... (II) H-Ser-Val-Leu-Asp-Thr-Asn-Lys-Asp-Cys-Glu-Val-Asp-Phe-Val-Glu-OH ... (III) H-Leu-Tyr -Cys-His-Glu-Tyr-Phe-Lys-Asp-Cys-Pro-Ser-Glu-Pro-Pro-Cys-Ser- Gln-OH ... (IV) 5. A hair surface recognizing antibody obtained by immunizing with S100 protein as an antigen. 6. The hair surface recognizing antibody according to claim 6, wherein the S100 protein is an S100A3 protein. 7. A hair treatment agent comprising the hair surface layer recognizing antibody according to any one of claims 1 to 6. 8. A hair dye, comprising a colorant having the hair surface layer recognizing antibody according to any one of claims 1 to 6 bound thereto. 9. A hair damage diagnostic agent comprising the hair surface layer recognizing antibody according to any one of claims 1 to 6.