JPH08143431A - Pretreating agent for dyeing hair - Google Patents

Abstract

PURPOSE: To obtain a pretreating agent for dyeing hair capable of expecting hair dyeing effects on selective dyeing of the hair in using an avidin- or a biotin-binding pigment as a dyeing agent. CONSTITUTION: This pretreating agent for dyeing hair contains an antihair antibody containing biotin or avidin bound thereto. The antihair antibody containing the biotin or avidin bound thereto as the pretreating agent is applied to the hair and an avidin- (or a biotin)-binding pigment is then applied to sufficiently manifest the hair binding power of the antihair antibody. Furthermore, the hair can be dyed with higher hair dyeing effects than those of hair dyeing simply with the avidin or biotin without using the antibody. The biotin-binding antibody is obtained by previously activating carboxyl group of the biotin and then introducing the activated carboxyl group into the free amino group in the antibody. The avidin-binding antibody is obtained by previously introducing N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) into the free amino group in avidin or the antibody, subsequently treating the resultant antibody with a reducing agent, producing free sulfhydryl group and reacting the prepared compound with the antibody or avidin into which the SPDP is introduced.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is used in advance when using an avidin- or biotin-bonded pigment as a hair dye, and is a dye capable of selectively dyeing hair without binding to the skin. The present invention relates to a pretreatment agent for hair dye, which is expected to have a hair effect.

[0002]

2. Description of the Related Art Heretofore, as a hair dye capable of selectively dyeing hair, a hair dye using an anti-hair antibody which is an antibody against a constituent component of hair has been developed (JP-A-6-2).
No. 27955).

However, although a hair dye using this anti-hair antibody has a high dyeing rate, when a pigment is used as a dyeing component, the antibody may be bound to the pigment at the antibody active site. It has the drawback that it is difficult to exert a binding force commensurate with the amount of antibody used and that it tends to be poor in color retention as a hair dye.

On the other hand, avidin and biotin have been used as research reagents and clinical test agents by utilizing their specific binding. For example, a highly sensitive method for detecting an antigen-antibody reaction by binding an enzyme-labeled avidin to a biotinylated antibody and measuring the enzyme activity has been reported (J. Histochem.
See Cytochem., 27, 1131, 1979). However, there are no reports of using avidin / biotin as a hair dye.

[0005]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As a result of intensive studies to eliminate the above-mentioned drawbacks, the present inventors have found that after applying a biotinylated (or avidinized) antibody to hair as a pretreatment agent, By applying an avidinized (or biotinylated) pigment, the hair-binding strength of the anti-hair antibody can be sufficiently exerted, and a higher hair dyeing effect than that obtained by dyeing with avidin / biotin alone without using an antibody is obtained. The present invention has been found to be obtained, and the present invention has been completed. An object of the invention is to provide a pretreatment agent for hair dyeing, which can achieve a high hair dyeing rate and excellent antibody binding properties. .

[0006]

The above-mentioned object is achieved by a pretreatment agent for hair dyeing, characterized by containing an anti-hair antibody conjugated with biotin or avidin.

The avidin used in the present invention is a glycoprotein having a molecular weight of about 68,000, which is present mainly in avian egg whites such as chicken egg whites and has a very high affinity for biotin (vitamin H).

The biotin used in the present invention is a known water-soluble vitamin also called vitamin H and has a property of binding to avidin.

The anti-hair antibody used in the present invention is an antibody having an immunological activity on human hair. Examples of such an antibody include various kinds of hair constituting the hair to be colored. Antibodies to the components are included. Such an antibody can be obtained, for example, by immunizing an animal with a hair keratin protein, a hair cuticle protein, 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 the antibody from the above-mentioned antibody raw material may be carried out by a known method. For example, after removing the lipid by an appropriate method, the antibody is purified by the ammonium sulfate fractionation method, the alcohol precipitation method or the membrane separation method. A crudely purified antibody can be obtained. 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 a 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. Also, 2-
An H chain or L chain obtained by reducing the antibody with mercaptoethanol may be used.

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

The antibody having an immunological activity against the anti-hair antibody used in the present invention is heterologous with an antibody derived from an animal of the same species as the animal immunized to obtain the anti-hair antibody or a fragment thereof as an antigen. Obtained by immunizing an animal. For example, when the anti-hair antibody is obtained from milk, an animal other than cow, such as rabbits and chickens, may be immunized with the bovine-derived antibody. Further, a monoclonal antibody having an immunological activity against the anti-hair antibody can also be used. Furthermore, 2-
An H chain or L chain obtained by reducing the antibody with mercaptoethanol may be used. Antibodies having immunological activity against these anti-hair antibodies can be purified in the same manner as the above-mentioned anti-hair antibodies. 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.

The obtained anti-hair keratin antibody does not react with epidermal keratin but specifically reacts with hair-derived keratin (see Japanese Dermatological Association Magazine, 104, 855, 1994) and is therefore selective for hair. Can be expected to be combined with.

The biotin-conjugated anti-hair antibody used in the present invention is manufactured as follows. In order to efficiently bind biotin to hair, it is desirable to introduce biotinamide capryate N-hydroxysuccinimide ester or the like in which the carboxyl group of biotin is previously activated into the free amino group of the anti-hair antibody. These activated biotins easily react by adding 1/100 to 10 times the amount of anti-hair antibody and stirring, and unreacted substances can be easily removed by a method such as gel filtration or dialysis.

Next, a method for producing the avidin-conjugated anti-hair antibody used in the present invention will be described. Anti-hair antibody and avidin can also be performed by using a cross-linking agent such as glutaraldehyde that is usually used for preparing a protein complex, but anti-hair antibody can be reduced by reducing the binding between anti-hair antibodies or avidin. In order to efficiently bind avidin, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) was introduced into the free amino group of anti-hair antibody or avidin in advance, and then free sulfhydryl was prepared by treating with a reducing agent. N-succinimidyl generating group
It is desirable to react -3- (2-pyridyldithio) propionate (SPDP) or succinimidyl-4- (p-maleimidophenyl) butyrate (SMPB) with avidin or an anti-hair antibody introduced into the free amino group.

The hair treated with the pretreatment agent for hair dyeing of the present invention obtained as described above can be specifically colored with the following avidin- or biotin-bonded pigment. When the pretreatment agent for hair dye is an avidin-conjugated anti-hair antibody, a biotin-conjugated pigment is used, and when the pretreatment agent for hair dye is a biotin-conjugated anti-hair antibody, an avidin-conjugated pigment is used.

The avidin-bonded colorant is obtained by binding avidin to the colorant by physical adsorption or chemical bonding, and then washing the colorant to remove excess avidin.

The biotin-bonded colorant can be prepared as follows. Although it is possible to directly introduce free functional groups on the colorant with activated biotin, it is also possible to biotinate bovine serum albumin in advance and adsorb it to the colorant. This is then washed and the excess biotinylated bovine serum albumin is removed to give the biotin-conjugated colorant. When bovine serum albumin is used after being biotinylated, a sufficient amount of biotin can be introduced onto the pigment surface in a state in which the pigment such as titanium black has good dispersibility, and thus the pigment is strongly bonded to hair, which is preferable.

Examples of the colorant include dyes, pigments, and dyes included in a polymer compound, and examples of the polymer compound include synthetic polymer polymers such as polystyrene and liposomes.

[0024]

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

Prior to the examples, a method for producing an anti-hair antibody and a hair dye test method used for evaluating a hair dye will be described.

1. Method for Producing Anti-Hair Antibody Anti-keratin antibody was produced as follows according to the method described in Japanese Society of Animal Science, Volume 62, 580, 1994.

After washing 10 g of healthy hair, 8 M urea and 0.2
The extract obtained by repeating the extraction operation at 50 ° C. for 1 hour in 2.5 l of 0.2 M Tris-HCl buffer (pH 9.2) containing M 2-mercaptoethanol was centrifuged. 400 g Tris solution 76 in the supernatant
200 g of monoiodoacetic acid solution was added to 0 ml, and the mixture was reacted with stirring for 1 hour under room temperature light shielding. Stop the reaction by adding 7 ml of 2-mercaptoethanol, dialyze against a sufficient amount of water and then add 2μ.
and a permeate was obtained. To 4 parts by volume of this permeate, 1 part by volume of 0.5 M sodium acetate buffer (pH 4.2) was added, and the pH was adjusted to 4.2 with acetic acid to precipitate the hair keratin isoelectrically. The precipitate obtained by centrifugation is dissolved in physiological saline and 2 μm
After sterilization through a filter of No. 1 and concentration with an ultrafiltration membrane, a hair keratin antigen was obtained.

The hair keratin antigen was dissolved in physiological saline (protein concentration: 20 mg / ml) and mixed with Freund's complete adjuvant in an equal volume to prepare a water-in-oil emulsion. 5.0 ml of this emulsion was subcutaneously administered to the neck of a pregnant Holstein cow two months before birth. After 10 days, an emulsion containing the same amount of antigen as the initial immunization prepared with Freund's incomplete adjuvant was administered subcutaneously for the 1st to 3rd boosters and intramuscularly for the 4th and 5th boosters. did.

Colostrum of the immunized cow was collected for 3 days after delivery. The fat layer was removed from the colostrum using a cream separator to obtain skim milk. Add 0.1N hydrochloric acid to this skim milk to adjust the pH.
It was adjusted to 4.5 and casein was precipitated. After removing the precipitate with a filter cloth, a supernatant was obtained by continuous centrifugation at 2,500 × g. After neutralization, ammonium sulfate was added to reach 33% saturation, and the antibody was salted out. The precipitate was collected by continuous centrifugation at 2,500 xg and dissolved in physiological phosphate buffer. The obtained solution was dialyzed against 10 mM phosphate buffer (pH 7.5) and equilibrated with the same buffer, 2 l of DEAE cellulose column (DE-52,
Whatman) was applied 5 times. After washing off non-adsorbed protein with the same buffer, the antibody was eluted with the same buffer containing 50 mM sodium chloride to obtain 200 g of the antibody. The purity of this antibody was 90% or more. This antibody was applied to 400 ml of an affinity carrier (Affigel 15, Bio-Rad Co.) to which hair keratin was bound by a conventional method, in 5 batches. The anti-hair antibody bound to the affinity carrier was eluted with 0.2 M glycine-hydrochloric acid buffer (pH 2.5), and the pH was immediately adjusted to about 8 with 3 M Tris solution, and the anti-hair antibody that specifically bound to the hair keratin antigen was bound. Hair keratin antibody was obtained. Less than,
It is simply described as an anti-keratin antibody.

2. Hair dyeing test method (1) Preparation of avidin-bonded pigment 20 ml of distilled water was added to 1 g of titanium black 10S (manufactured by Mitsubishi Metals Co., Ltd.) and dispersed by ultrasonic waves. To this dispersion was added 15 μl of a zircoaluminate coupling agent having a carboxyl group (Type C, manufactured by Manchem), and the mixture was stirred at room temperature for 2 hours. The dispersion was dried under reduced pressure with an evaporator while heating at 40 ° C, and then dried at 110 ° C for 10 minutes. 10 mg of titanium black treated with a zircoaluminate coupling agent was dispersed in 1 ml of purified water, and the pH was adjusted to 5 with hydrochloric acid.
After adjusting to 0, 1 ml of 0.002M EDC aqueous solution was added and stirred at room temperature for 2 hours. After centrifugation at 12000 rpm for 20 minutes at 20 ° C., the supernatant was discarded and redispersed in 1 ml of purified water. Avidin (3 mg) was dissolved in physiological phosphate buffer (1 ml), and the mixture was stirred at 4 ° C. for 24 hours. After centrifugation, unreacted avidin was removed and the mixture was dispersed in a physiological phosphate buffer containing 0.1% bovine serum albumin. Centrifuge again for 0.
After redispersion in physiological phosphate buffer containing 1% bovine serum albumin and leaving it at room temperature for 1 hour, 0.1% bovine serum albumin and
0.1% polyoxyethylene sorbitan monolaurate (Tw
een 20) and dispersed in a physiological phosphate buffer solution to obtain an avidin-bonded pigment.

(2) Preparation of biotinylated bovine serum albumin binding pigment 1 mg bovine serum albumin was added to physiological phosphate buffer (pH 7.2)
After dissolving in 1 ml, pH was adjusted to 9.0 by adding 0.2 ml of 1M sodium hydrogen carbonate. 40 μl of biotinamide capryate N-hydroxysuccinimide ester (50 mg / ml) previously dissolved in dimethyl sulfoxide was added, and the mixture was kept overnight at 4 ° C.
I shook it up. This reaction solution was added to physiological phosphate buffer (pH 7.
It was applied to Sephadex G-25 (manufactured by Pharmacia) equilibrated in 2) and biotinylated bovine serum albumin that was eluted in the flow-through fraction was collected. In addition, the bound biotin was treated with Metohds Enzymo
As a result of quantification by the method described in L. 18A, page 418, 1970, 1.8 molecules of biotin were bound per molecule of bovine serum albumin.

10 mg of titanium black treated with a zircoaluminate coupling agent was dispersed in 1 ml of purified water, and the pH was adjusted with hydrochloric acid.
After adjusting to 5.0, 1 ml of 0.002M EDC aqueous solution was added and stirred at room temperature for 2 hours. After centrifugation at 12000 rpm for 20 minutes at 20 ° C., the supernatant was discarded and redispersed in 1 ml of purified water. To this, 1 mg of biotinylated bovine serum albumin was dissolved in 1 ml of physiological phosphate buffer, and the mixture was stirred at 4 ° C. for 24 hours. After centrifugation, unreacted avidin was removed and the mixture was dispersed in a physiological phosphate buffer containing 0.1% bovine serum albumin. Centrifuge again, redisperse in physiological phosphate buffer containing 0.1% bovine serum albumin, leave at room temperature for 1 hour, then
The pigment was dispersed in a physiological phosphate buffer containing 1% bovine serum albumin and 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) to obtain a biotinylated bovine serum albumin-bound pigment.

(3) Hair dyeing test Avidin-conjugated pigment or biotinylated bovine serum albumin-conjugated pigment was added to physiological phosphate buffer containing 0.1% bovine serum albumin and 0.1% polyoxyethylene sorbitan monolaurate (Tween 20). Disperse to 0.1% (pigment weight) with a liquid, and add 1 ml of the human hair bundle (50 mg) to 1% mercaptoethanol at 30 ° C for 10 minutes, then add to the reduced hair washed with purified water and rotate for 1 hour. It was Then 0.05% Tween 20
It was washed with a physiological saline solution containing and dried.

After the hair dyeing operation, the degree of coloring was visually evaluated according to the following criteria. 4 ... It was dyed in a remarkably dark black color. 3 ... It was dyed in a dark black color. 2 ... It was dyed black. 1 ... It was dyed in a light black color. 0 ... It didn't stain at all.

Example 1 (Preparation of pretreatment agent for hair dye containing biotinylated anti-keratin antibody) (1) Preparation of biotinylated anti-keratin antibody 1 mg of anti-keratin antibody was added to 1 ml of physiological phosphate buffer (pH 7.2)
Then, add 0.2 ml of 1M sodium hydrogen carbonate to the pH.
Was set to 9.0. 40 μl of biotinamide capryate N-hydroxysuccinimide ester (50 mg / ml) previously dissolved in dimethyl sulfoxide was added, and the mixture was shaken overnight at 4 ° C. This reaction solution was applied to Sephadex G-25 (manufactured by Pharmacia) equilibrated with physiological phosphate buffer (pH 7.2) to collect biotinylated antibodies that were eluted in the flow-through fraction. The bound biotin was bound to Metohds Enzymol. 18A, 418, 1970.
Anti-keratin antibody 1 as a result of quantification by the method described in
8.0 molecules of biotin were bound per molecule.

(2) Preparation of pretreatment agent for hair dye Biotinylated anti-keratin antibody was dissolved in physiological phosphate buffer containing 0.1% bovine serum albumin and 0.1% polyoxyethylene sorbitan monolaurate (Tween 20). Then, a pretreatment agent for hair dyeing was prepared.

Comparative Example 1 (Preparation of biotin-containing pretreatment agent for hair dye) The same amount of biotin as the biotinylated anti-keratin antibody was added to 0.1% bovine serum albumin and 0.1% polyoxyethylene sorbitan monolaurate (Tween 20). A pretreatment agent for hair dye was prepared by dissolving it in a physiological phosphate buffer solution containing.

Test Example 1 (Hair dyeing test of human gray hair treated with a biotinylated antibody-containing pretreatment agent for hair dye using an avidin-bonded pigment) The biotinylated anti-keratin antibody prepared by the method of Example 1 (10 or 100 μg / ml) ) Pretreatment agent for hair dye,
Alternatively, 1 ml of the biotin-containing pretreatment agent for hair dye prepared by the method of Comparative Example 1 was added to a human white hair bundle (50 mg) and the mixture was rotated for 1 hour. Then, after washing with a physiological saline containing 0.05% Tween 20, a hair dyeing test was carried out using an avidin-bonded pigment. The test results are shown in Table 1.

[0039]

[Table 1]

Human hair treated with the pretreatment agent for hair dye containing the biotinylated anti-keratin antibody of Example 1 was sufficiently colored with the avidin-bonded pigment. In contrast, human hair treated with the biotin-containing pretreatment agent for hair dye of Comparative Example 1 was not colored with the avidin-bonded pigment.

Example 2 (Preparation of pretreatment agent for hair dye containing avidin-anti-keratin antibody conjugate) (1) Preparation of avidin-anti-keratin antibody conjugate Avidin 10 mg containing 0.3M NaCl and 0.5% n-butanol N-succinimidyl-3-dissolved in 1 ml of 0.05M carbonate buffer (pH 9.0) and previously dissolved in dimethylformamide
(2-Pyridyldithio) propionate (SPDP) (6mg / ml)
Was added at 25 μl and shaken for 30 minutes at room temperature. This reaction solution was equilibrated with physiological phosphate buffer (pH 7.2) to give CW-35 (1.5
X 5.5 cm, manufactured by Tosoh Corporation) and elute in the flow-through fraction P
DP-Avidin (3 ml) was collected.

12.5 mg of anti-keratin antibody was added to 0.3 M NaCl and
0.05M carbonate buffer (pH 9.0) containing 0.5% n-butanol 1
After dissolving in 2.5 ml, 16.2 μl of N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (6 mg / ml) previously dissolved in dimethylformamide was added, and the mixture was shaken at room temperature for 30 minutes. This reaction solution was applied to CW-35 (1.5 x 5.5 cm, manufactured by Tosoh Corporation) equilibrated with 0.1 M acetate buffer (pH 4.5) containing 0.1 M NaCl, and the PDP-conjugated antibody (3 m
l) collected. To this, 46.3 mg of dithiothreitol was added and shaken for 30 minutes at room temperature. This reaction solution was equilibrated with a physiological phosphate buffer solution (pH 7.2) that had been replaced with nitrogen, and CW-35 (1.5
(× 5.5 cm, manufactured by Tosoh Corporation) was divided into two portions, and the thiopropionyl antibody (3 ml × 2) eluted in the flow-through fraction was collected.

6 ml of the thiopropionyl antibody was added to 1.5 ml of the PDP-modified avidin obtained by the above method, and the mixture was shaken at room temperature for 30 minutes. This reaction solution was applied to Sephacryl S-300 (2.8 × 19 cm, manufactured by Pharmacia) equilibrated with physiological phosphate buffer (pH 7.2), and the avidin-anti-keratin antibody conjugate (35 ml) eluted in the flow-through fraction was collected. It was SD this solution
When analyzed by S-10% polyacrylamide gel electrophoresis, a high molecular avidin-anti-keratin antibody conjugate band was confirmed as compared with avidin and anti-keratin antibody (FIG. 1).

343 nm of pyridine-2-thione released by the reaction of PDP-modified avidin and thiopropionyl antibody
As a result of estimation from the absorbance of 1., 1.1 molecule of biotin was bound to 1 molecule of the anti-keratin antibody.

(2) Preparation of pretreatment agent for hair dye Physiological phosphate buffer containing 0.1% bovine serum albumin and 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) containing avidin-antikeratin antibody conjugate To prepare a pretreatment agent for hair dye.

Comparative Example 2 (Preparation of pretreatment agent for hair dye containing avidin) Avidin was added to a physiological phosphate buffer containing 0.1% bovine serum albumin and 0.1% polyoxyethylene sorbitan monolaurate (Tween 20). A pretreatment agent for hair dye was prepared by dissolving.

Test Example 2 (Avidinization—Hair Dyeing Test of Human White Hair Treated with Pretreatment Agent for Hair Dye Containing Anti-Keratin Antibody Conjugate with Biotinylated Bovine Serum Albumin Binding Pigment) Avidin prepared by the method of Example 2 1 ml of the pretreatment agent for hair dye containing an anti-keratin antibody conjugate or the pretreatment agent for hair dye containing avidin prepared by the method of Comparative Example 2 was added to a human hair bundle (50 mg) and rotated for 1 hour. Then 0.05
After washing with physiological saline containing% Tween 20, a hair dyeing test was performed with a biotinylated bovine serum albumin binding pigment. The test results are shown in Table 2.

[0048]

[Table 2]

The human hair treated with the avidin-anti-keratin antibody conjugate-containing pretreatment agent for hair dye of Example 2 was colored better than the human hair treated with the avidin-containing pretreatment agent for hair dye of Comparative Example 2. It was

[0050]

When the pretreatment agent for hair dyeing of the present invention is used, the antibody is bound in advance and then the pigment is applied, so that the specificity of the anti-hair antibody can be sufficiently exerted, and at the same time, avidin・ Higher hair dyeing effect than hair dyeing with biotin.

[Brief description of drawings]

FIG. 1 is a drawing-substituting photograph showing a result of electrophoresis of an avidin-anti-keratin antibody conjugate prepared in Example 2. Lane 1 is phosphorylase b (94,000) bovine serum albumin (67,000), ovalbumin (43,000),
Carbonic anhydrase (30,000), α-lactalbumin (14,4
00), the second lane is avidin, the third lane is an anti-keratin antibody, and the fourth lane is an avidin-anti-keratin antibody conjugate.

Front Page Continuation (72) Inventor Junichiro Hiraoka 5-3 28, Kotobuki-cho, Odawara-shi, Kanagawa Kanebo Co., Ltd.

Claims (1)

[Claims] 1. A pretreatment agent for hair dyeing, which comprises an anti-hair antibody bound to biotin or avidin.