JPH08151397A - Hair gel carrier and purification of antibody - Google Patents

Abstract

PURPOSE: To provide a hair gel carrier composed of a gel produced by extracting hair in the presence of a reducing agent and treating the obtained hair-extracted protein with an oxidizing agent, useful e.g. for the easy purification of an anti-hair antibody to be used in cosmetic and pharmaceutical fields and producible at a low cost. CONSTITUTION: The objective hair gel carrier capable of easily purifying an anti-hair antibody to be used in cosmetic and pharmaceutical fields can be produced at a low cost by washing hair with 2% aqueous solution of sodium polylaurylsulfate, cutting the washed hair with scissors into small pieces, adding a reducing agent consisting of 0.2M tris-HCl buffer solution/8M urea/0.2M β- mercaptoethanol (pH9.2), leaving the hair at rest in the presence of nitrogen gas at 50 deg.C for 1hr, grinding the hair, centrifuging the ground product, recovering the supernatant liquid to obtain a hair-extracted protein, concentrating the solution of the hair-extracted protein, adding ethyl acetate, a surfactant and sodium brominate and subjecting the mixture to gelation by oxidization.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention is useful for purifying anti-hair antibodies used in the fields of cosmetics and pharmaceuticals, is a cost-effective hair gel carrier, and is convenient for purifying the anti-hair antibodies. And a method for purifying an antibody.

[0002]

2. Description of the Related Art Conventionally, as a technique for purifying an antibody from a crude antibody purified product, a method in which an antigen for the antibody to be purified is bound to a synthetic gel carrier having an existing functional group by a chemical method is used. It was taken.

However, since the synthetic gel carriers used in these methods are extremely expensive, the purification cost is high, and moreover, it is necessary to combine the antigens by a chemical method. Has been pointed out.

[0004]

DISCLOSURE OF THE INVENTION The inventors of the present invention have made extensive studies in view of the above circumstances, and as a result, succeeded in obtaining a hair gel carrier by adding an oxidizing agent to the hair extract protein, and used the hair gel carrier. It was found that the anti-hair antibody can be easily purified by the above, and the present invention has been completed. The purpose of the present invention is to provide an inexpensive hair gel carrier and an anti-hair that can be easily purified. A method of purifying an antibody is provided.

[0005]

[Means for Solving the Problems] The above-mentioned object is characterized by comprising a gel obtained by treating a hair extract protein with an oxidizing agent, and using the hair gel carrier. It is achieved by a method for purifying an anti-hair antibody.

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

The hair extract protein used in the present invention is
It is a protein obtained by treating an antigen protein derived from hair, to which an antibody to be purified can specifically bind, with a reducing agent to cleave the S—S bond.

As the antigen protein, whole hair extract protein,
Hair protein components obtained by further purifying them, for example, hair keratin protein, hair cuticle protein, hair matrix protein and the like can be mentioned.

As a reducing agent for cleaving the S--S bond, 2-mercaptoethanol, thioglycolic acid, 2
-Mercaptopropionic acid, thioglycerol, mercaptosuccinic acid, cysteine and the like.

Examples of the oxidizing agent used in the present invention for gelling the hair extract protein include sodium bromate, aqueous hydrogen peroxide and the like.

When the hair extract protein is used as a gel carrier,
It is preferable to add an organic solvent and a surfactant in addition to the oxidizing agent and stir.

The organic solvent used when the hair extract protein is made into a gel carrier is preferably an organic solvent which is insoluble in water and forms a two-layer system. Specific examples thereof include ethyl acetate, hexane and chloroform. .

Examples of the surfactant used when the hair extract protein is used as a gel carrier include Tween 20, sodium polyoxylauryl sulfate, coconut oil, protein hydrolyzate derivatives (Promois ECP, etc.) and the like.

The size of the hair gel carrier of the present invention is
It is preferably 1 μm to 1 cm.

In preparing the hair gel carrier of the present invention,
When no organic solvent is added, a gel mass is formed, but it may be cut into a suitable size with a nylon mesh or the like.

The anti-hair antibody referred to in the present invention refers to an antibody that specifically binds to a hair protein component among the antibodies obtained by immunizing the above-mentioned hair-derived antigen protein.

Examples of the buffer used for purifying the anti-hair antibody using the hair gel carrier of the present invention include physiological phosphate buffer (hereinafter abbreviated as PBS), physiological Tris buffer and the like.

Examples of the organic solvent used for purifying the anti-hair antibody using the hair gel carrier of the present invention include methanol and ethanol.

When an anti-hair antibody is purified using the hair gel carrier of the present invention, the extract used to release the adsorbed antibody from the hair gel carrier is a strongly acidic solution such as glycine hydrochloride or ethylene glycol. Examples thereof include a strong alkaline solution such as silane.

The anti-hair antibody used in the present invention includes
It is an antibody having an immunological activity on human hair, and examples of such an antibody include an antibody having an immunological activity on each component constituting the hair.

Such an antibody may be, for example, a whole hair extract,
It can be obtained by immunizing an animal with hair keratin protein, hair cuticle protein, hair matrix protein or fragments thereof as an antigen.

Animals used for immunization include cattle, horses,
Appropriate livestock can be selected from sheep, rabbits, chickens, etc.

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.

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 may be roughly purified from the above-mentioned antibody raw material by a known method. For example, the lipid is removed by an appropriate method and then purified by an alcohol precipitation method or a membrane fractionation method. Is preferably used, but the crude purification does not necessarily have to be performed.

The crude antibody purified product can be used as it is for the purification method of the present invention, but it can also be used for the purification method of the present invention by processing for long-term storage by freeze-drying or addition of glycerin. good.

[0027]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 (1) Preparation of protein extracted from hair 20 g of hair washed with a 2% sodium polylauryl sulfate aqueous solution was finely chopped with scissors, and 0.2 M Tris-HCl buffer / 8 M urea / 0.2 M β-mercaptoethano was used. -Le (p
H 9.2) in an amount of 4 liters and added in the presence of nitrogen gas
It was left to stand for 1 hour while being kept at ℃. The hair treated with an alkali and a reducing agent was ground with a dynamill in order to make the hair finer, and then centrifuged at 11500 rpm for 30 minutes to collect a supernatant. The protein concentration of the obtained supernatant is approximately 1.5 mg.
/ Ml. This hair extract protein was concentrated about 10 times by ultrafiltration.

(2) Preparation of hair gel carrier In a separatory funnel, 50 ml of the concentrated hair protein solution obtained in (1)
To the above, add an equal amount of ethyl acetate, 5 ml of Tween 20, and 0.5 g of sodium bromate and stir well,
The thiol groups in keratin are crosslinked to form a hair gel carrier and precipitate. The size of the hair gel carrier became uniform, about 2 mm. The obtained hair gel carrier is PBS
It is used for the purification of anti-hair antibody that specifically binds to the hair extract protein after adding and washing.

Example 2 50 ml of concentrated hair protein solution obtained in (1) of Example 1
To this, 5 ml of Tween 20 and 0.5 g of sodium bromate are added, and the mixture is stirred so as to be uniform to form a hair gel carrier. The hair gel carrier thus obtained becomes a large mass, so that a uniform size hair gel carrier is obtained through a nylon mesh.

Example 3 (Purification Method of Anti-Hair Antibody) (1) Preparation Method of Hair Extraction Protein 5 g of normal male hair and 5 g of normal female hair were mixed,
After washing with a 2% aqueous solution of sodium polyoxylauryl sulfate, a supernatant was obtained according to the above-described method for preparing a hair gel-supported hair extract. 200 g of monoiodoacetic acid solution was added to the supernatant (760 ml of a solution prepared by previously dissolving 400 g of Tris).
Was added), and the mixture was allowed to react with stirring for 1 hour under room temperature light shielding. The reaction was stopped by adding 7 ml of 2-mercaptoethanol, dialyzed against sufficient water, and the insoluble matter was removed through a 5 μm filter to obtain a hair keratin antigen solution. Further, 0.2 M sodium acetate buffer 1 was added to this solution 4 (prepared with acetic acid so as to have a pH of 4.2),
Hair keratin was precipitated. The precipitate was recovered by centrifugation at 1000 × g for 10 minutes. A physiological saline solution was added to the precipitate to dissolve it, and the precipitate was sterilized through a 0.2 μm filter and then aseptically concentrated with an ultrafiltration membrane to obtain a purified hair keratin antigen (2.6 g as protein).

(2) Immunization of cattle The purified hair keratin antigen solution described above was prepared with physiological saline to a protein concentration of 20 mg / ml, and the solution was mixed with Freund's adjuvant at a volume ratio of 1: 1 to obtain an oil. Use a water-in-water emulsion. The obtained emulsion was subcutaneously administered to the necks of two pregnant Holstein cows two months before delivery (one at 10 mg / head and the other at 50 mg / head). After that, at 10-day intervals, emulsions containing Freund's incomplete adjuvant, each containing the same amount of antigen as the previous time, were subcutaneously or intramuscularly administered to immunize (1 to 3 times; subcutaneous administration, 4
~ 5 times; IM).

(3) Collection and crude purification of antibody Colostrum was collected for 3 days immediately after delivery. Cream Separation
The skimmed milk was obtained with a mixer and fractionated and purified as follows.

The pH is adjusted by adding 0.1N hydrochloric acid to skim milk.
In 4.5, the casein was precipitated, the precipitate was roughly removed with a filter cloth, and the supernatant was obtained by continuous centrifugation at 2500 × g. After neutralization, ammonium sulfate was added to achieve 33% saturation and the antibody was salted out. The precipitate was recovered by continuous centrifugation at 2500 Xg, dissolved in PBS, and this salting out with ammonium sulfate was repeated. The crude antibody purified product thus obtained was further lyophilized.

(4) Method of Purifying Anti-Hair Antibody Using Hair Gel Carrier A dried crude antibody product was diluted with PBS to a concentration of 1 mg / ml.
Add. This antibody solution is added to the hair gel-supported hair extract and left to stand overnight. By allowing it to stand in this manner, only the specific anti-hair antibody is adsorbed on the hair gel carrier, and the non-specific unadsorbed antibody remains suspended in the supernatant, so that it can be easily removed by decanting.

The anti-hair antibody adsorbed on the hair gel carrier is
It can be easily released by adding 0.2 M glycine hydrochloric acid (pH 2.5). Since the antibody released from the hair gel carrier is under a strong acidic condition, 3M Tris solution is quickly added to return the pH to around neutral.

The antibody titer of the anti-hair antibody against the purified hair eluate was measured by the ELISA method. The measurement results are shown in Table 1.

[0038]

[Table 1]

[0039]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to provide a hair gel carrier which is inexpensive in cost and useful for the purification of specific antibodies derived from hair, and by using the hair gel carrier, a simple anti-hair antibody It is obvious that the purification method of

Claims (2)

[Claims] 1. A hair gel carrier comprising a gel obtained by treating a hair extract protein with an oxidizing agent. 2. A method for purifying an anti-hair antibody, which comprises using the hair gel carrier according to claim 1.