JPH0776600A - Monoclonal antibody and hybridoma capable of producing the same - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody, useful for analyzing a high-order structural change of hair proteins occurring in a process of keratinization or damage or as a damage sensor, etc., for the hair and applicable to a hair damage restoring agent or a hair cosmetic by combination thereof with other existing substances. CONSTITUTION:This monoclonal antibody is against a keratinized hair shaft part. The antibody is obtained by fusing a cell capable of producing antibody of a mammal immunized with hair powder obtained from the keratinized hair shaft part to a cell of a myeloma in a mammal, constructing a hybridoma, culturing the resultant hybridoma in a culture medium and separating the prepared monoclonal antibody from the culture supernatant. The monoclonal antibody is capable of recognizing a protein high-order structure present in the keratinized hair shaft part and the presence of the recognition thereof entirely varies with a difference in chemical structure or damage degree of the hair used in preparing the monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a monoclonal antibody,
More specifically, for recognizing the hair shaft after keratinization, for analysis of higher-order structural changes in hair proteins that occur during keratinization or damage,
The present invention relates to a monoclonal antibody useful as a hair damage sensor and the like, and a hybridoma producing the monoclonal antibody.

[0002]

Hair is a structure derived from the epidermis. Hair is formed in an organ called a hair follicle, which is derived from epithelial cells. The basal part of the hair follicle is in contact with the dermal papilla derived from mesenchymal cells so that it is called the hair bulb part. In addition, undifferentiated cells that rapidly divide in the lower half of the bulb are called hair matrix cells. Divided and proliferated hair mother cells continuously move to the upper half of the hair bulb, and are morphologically (elongated in the longitudinal direction) and physiologically (accumulate a large amount of a hard protein called keratin). The differentiated cells further move to an area called the keratinized zone above the hair follicle. There are many cysteine residues in the keratin molecule, and in the keratinized zone, a cystine bond within the keratin molecule or between the molecules (SS bond between cysteine residues).
Are produced in large amounts, and keratin fibers are formed. Keratin fibers fill the cells, resulting in the collapse and elimination of intracellular organelles and the hair becoming a very strong structure.

It is called keratinization of hair that the keratinous zone is filled with keratin fibers, disappears of intracellular organelles, and the cells die and dry to form a strong structure.
In the keratinized region, intracellular proteins (mainly keratin proteins) form disulfide bonds or γ-glutamyllysine bonds intramolecularly or intermolecularly, and by crosslinking, the hair has a very strong structure, When various chemical treatments such as perming and bleaching are carried out along with the extension of the hair shaft, partial cleavage of the above-mentioned cross-linking structure occurs and the higher order structure of the protein is further changed. Such higher-order structure of hair proteins and structural change due to chemical treatment are deeply related to the substance, quality and shape of hair, and in some cases may lead to hair damage.

Control of hair substances, improvement of hair quality of damaged hair,
Furthermore, in order to elucidate the mechanism of keratinization of hair, it is necessary to analyze in detail the subtle changes in the higher-order structure of hair proteins that occur during the process of damage due to keratinization and chemical treatment. However, hair is an aggregate of various proteins, and there is currently no effective method for analyzing the protein structure.

On the other hand, an antibody recognizes a certain structural site (epitope) corresponding to several to several dozen amino acid residues and specifically binds thereto. The affinity of the antibody for the antigen is changed by changing or modifying this epitope. Therefore, in order to carry out the structural analysis of the hair protein by utilizing the molecular recognition ability of the antibody, a large number of antibodies having specificity for a certain higher order structure of the hair protein are required.

[0006] Polyclonal antibodies have a high affinity for antigens and can be prepared easily and in large quantities from the blood or egg yolk of immunized animals, but many types of non-isolated antibodies with different epitopes and affinities are available. It is a heterogeneous set of molecules. On the other hand, a monoclonal antibody is a set of one kind of antibody molecule and recognizes a certain epitope in an antigen.
The latter is more useful than the former for the analysis of the structure and changes of certain sites in hair, which is an aggregate of various proteins.

[0007] So far, a monoclonal antibody was prepared using a solubilized protein of hair as an antigen, and it was reported that this reacts with the keratinized part of hair follicle and root sheath in immunohistochemical analysis of hair. Has been done. It is known that an egg yolk antibody, which is a polyclonal antibody produced from chicken egg yolk using hair powder as an antigen, reacts with the hair shaft after keratinization. However, at present, there is no report on a monoclonal antibody that reacts with the keratinized hair shaft and a method for producing the same.

[0008]

Therefore, it has reactivity to the hair shaft of hair and is used for analysis of higher-order structure of hair proteins and changes thereof, and as a hair damage sensor or hair cosmetics. There was a desire for a monoclonal antibody that can.

[0009]

Under such circumstances, the inventors of the present invention have conducted extensive studies and as a result, as a result, a monoclonal antibody to the keratinized hair shaft was used as an immunogen using hair powder obtained from the keratinized hair shaft. We succeeded in obtaining the antibody and completed the present invention.

That is, the present invention provides a monoclonal antibody against the keratinized hair shaft and a hybridoma producing the same.

The monoclonal antibody of the present invention is produced by a hybridoma obtained by fusing mammalian antibody-producing cells immunized with hair powder obtained from the keratinized hair shaft and mammalian myeloma cells. To be done.

As the hair powder used as an immunogen in the present invention, whole hair of healthy hair or damaged hair caused by perms or the like, or a hair constituting tissue such as hair pellicle or fur, has a length of 1
It is possible to use a powder (hair powder) pulverized to a size of 00 μm or less, or a powder obtained by chemically treating hair powder such as perm.

As a method for producing hair powder, as described in JP-A-57-163392, hair is swollen with water and then freeze-pulverized, or lithium bromide, urea, guanidine hydrochloride or the like is used. There is a method of treating with a protein denaturing agent, swelling, then freeze-pulverizing, and pulverizing with an existing pulverizer such as a mortar and a sand mill under liquid nitrogen. However, the method is not limited to these and other methods may be used. Further, as the structural structure of hair, there are hair pellicle, fur texture, and hair pulp. The hair pellicle powder is cut into pieces of 1 cm or less, and the hair is mechanically peeled by shaking it with sterile Teflon balls in sterile water. Can be obtained by Further, fur-like powder can be obtained by removing the hair pellicle by Vantian treatment (JP-A-55-39702) or the like according to the above-mentioned hair crushing method.

Then, the obtained hair powder is used as an immunogen to prepare antibody-producing cells according to a conventional method. That is, first, a mammal is immunized with hair powder as an immunogen. The mammal to be immunized here is preferably selected in consideration of compatibility with myeloma cells used for cell fusion in a subsequent operation, and specific examples thereof include mouse and rat. In particular, it is preferable to use an autoimmune disease animal because human hair proteins have high homology with proteins of body hair of other mammals, and hair powder used as an immunogen in the present invention has extremely low immunogenicity. . NZB, NZW, NZBW as autoimmune disease animals that can be used
Mention may be made of F ₁ , MRL / 1, male BXSB, and mice with autoimmune disease such as SL / Ni.

Balb / c and the like, which have enhanced autoantibody-producing ability by administering a Gram-negative bacterial lipid polysaccharide (LPS), a polyclonal B cell activator (PBA) such as dextran sulfate, as an immunizing mammal. The mouse can be used in an autoimmune disease state.

As a method of immunizing a mammal with the above-mentioned immunogen, for example, hair powder alone or in combination of two or more kinds, and subcutaneously, intraperitoneally, intravascularly, intramuscularly or intraspleenally injecting this into a mammal. A usual method such as a method by injection or a method of orally administering or immunizing with feed or water can be adopted. Moreover, when immunizing, it can be used in combination with an adjuvant if necessary.

Next, spleen cells collected from the immunized animal are fused with mouse myeloma cells. When an autoimmune mouse is used as an immunized animal, it is preferable to collect spleen cells at 4 to 8 months, preferably 6 months or older, in order to efficiently obtain IgG type antibodies. As myeloma cells, various cells already known, for example, NS-1, SP-
2, X63.6.5.3, P3-U1 and the like can be used. The fusion method can be performed according to a known method. Moreover, polyethylene glycol (PEG), Sendai virus (HVJ), or the like can be used as the fusion promoter. The mixing ratio of spleen cells and myeloma cells is 1: 1.
10: 1 is preferable. Depending on the case, cell fusion may be performed by an electrofusion method or the like.

After cell fusion, the hybridoma can be selectively obtained by culturing in a usual selection medium. For example, since the above-mentioned myeloma cells cannot grow in HAT medium (including hypoxanthine, aminopterin and thymidine), HAT medium may be used and cells that grow in the medium may be selected. When the hybridoma colony becomes sufficiently large, a strain producing the desired antibody is searched and cloned. The search for an antibody specific to an antigen can be performed by a method generally used for detecting an antibody, such as an ELISA method or a RIA method. In addition, cloning can be performed by the limiting dilution method or the soft agar method. At this time, it is preferable to use mouse thymocytes, peritoneal macrophages, or known additives having the same effect as these as a feeder.

The hybridoma of the present invention thus obtained can be stored in liquid nitrogen for a long period of time.

To obtain the monoclonal antibody of the present invention using the hybridoma of the present invention, the hybridoma is cultured in a medium and separated from the culture supernatant, or the hybridoma is intraperitoneally administered to a mouse and the ascites fluid is collected. do it. Further, the obtained monoclonal antibody can also be purified by a conventional method, for example, using ammonium sulfate precipitation, gel filtration, ion exchange column chromatography and the like.

The hair powder used as an antigen in the present invention does not undergo the solubilization treatment of the hair protein by a chemical reaction, so that the higher order structure of the hair protein is preserved. Therefore, the monoclonal antibody of the present invention can recognize the protein higher-order structure present in the hair shaft portion after keratinization.
In addition, if hair powder prepared from hair damaged by various chemical treatments such as perm and bleach is used as an antigen, the protein higher-order structure present in the undamaged hair shaft is not recognized and damage is caused. It is also possible to produce a monoclonal antibody that recognizes the higher-order structure of the protein present in the received hair shaft and firmly and specifically binds thereto. Therefore, the monoclonal antibody of the present invention is useful for analyzing the conformational change of the hair protein that occurs during the process of keratinization or damage. Further, a monoclonal antibody specific to the protein structure of damaged hair can be used as a hair damage sensor. Furthermore,
By combining with other existing substances, it can be applied as a hair damage repairing agent or a hair cosmetic.

[0022]

INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention recognizes the higher-order structure of the protein present in the hair shaft portion after keratinization, and furthermore, the difference in the chemical structure of the hair used in the preparation of the monoclonal antibody and the degree of damage Whether or not they are recognized depends on the difference. Therefore, this monoclonal antibody is useful for analysis of changes in the higher-order structure of hair proteins that occur during the process of keratinization or damage, as a hair damage sensor, etc., and in combination with other existing substances, it repairs hair damage. It can also be applied as an agent or a hair cosmetic.

[0023]

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

Example 1 (1) Preparation of Hair Powder: Healthy hair or perm-treated hair was washed with hexane, immersed in an 11 M aqueous solution of lithium bromide, and treated with hot water at 90 ° C. for 90 minutes to swell and to give a rubbery form. Denatured. Excess liquid was removed from the treated hair with a nylon net, transferred to a mortar that had been cooled beforehand, and frozen with liquid nitrogen. Then, while being appropriately supplemented with liquid nitrogen, it was ground in a frozen state with a pestle for 3 hours, the obtained ground hair was transferred to a centrifuge tube, and lithium bromide was washed out from the hair with ion-exchanged water. The washed hair powder was collected by centrifugation and this was repeated 3 times to completely wash away the lithium bromide from the hair. By this treatment, the hair was crushed to 100 μm or less. Next, finer powder was selected from this pulverized product. That is, the above pulverized product was vigorously shaken and dispersed in ion-exchanged water in a centrifuge tube, and then allowed to stand for 10 minutes.
At this time, a large powder precipitates, but a fine powder remains dispersed in the supernatant, so this supernatant was collected. The same procedure was repeated from the remaining precipitate to obtain a supernatant containing fine powder. Then, the fine powder was collected from the supernatant by centrifugation. To make it even finer, the fine powder was dispersed at 1% in distilled water and crushed three times using a French press at a pressure of 2000 psi. The fine powder was collected by freeze-drying. By this treatment, the hair was crushed to 10 μm or less. The powders obtained from healthy hair and permed hair were designated as healthy hair powder and permed hair powder, respectively.

(2) Immunization method: Healthy hair powder prepared by the above method was suspended in physiological saline at a concentration of 0.4% (w / v), and the same amount of Freund's complete adjuvant (FC was used).
An emulsion for immunization was prepared by mixing with A) for 10 minutes. 500 μl of this adjuvant for immunization (hair powder 1 mg / mouse) was intraperitoneally injected into Balb / c (female, 8 weeks old). After 21, 27 and 38 days, booster immunization was performed in the same manner. However, FCA was not used in the final immunization,
250 μl of 0.4% (w / v) healthy hair powder suspension was injected intraperitoneally.

(3) Cell fusion: Three days after the final immunization, spleen cells were extracted from the immunized Balb / c, and RPMI was used.
It was washed with 1640 medium. On the other hand, mouse myeloma cells P3 / NSI / 1-Ag4-1 in the logarithmic growth phase were collected and R
It was washed with PMI1640 medium. Spleen cells 1-2 x 10
The suspension of ^{8 and} the suspension of mouse myeloma 2 × 10 ⁷ were mixed, and the medium was removed by centrifugation. 3 for mixed cells
50% polyethylene glycol 4000 heated to 7 ℃
-1 ml of RPMI1640 medium was gradually added over 1 minute to perform fusion by gently stirring for 1 minute. RPMI1
10 ml of 640 medium was added over 5 minutes with gentle stirring. Remove the medium by centrifugation and add 5 x 10 ⁶ to the cells.
HAT medium containing mouse thymocytes / ml (RPMI1 containing 15% fetal bovine serum, 1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine)
640 medium) was added so as to have a spleen cell concentration of 5 × 10 ⁶ / ml, and 0.1 ml per well was distributed to a 96-well plate. After 4 days, 0.1 ml of HAT medium was added to each well. After 2 weeks of culture, hybridoma growth was observed in 60% or more of the wells.

(4) Selection of hybridoma: The antibody in the hybridoma culture supernatant was searched for by the modified ELISA method using the healthy hair powder obtained by the above-mentioned preparation method as the antigen. 0.2% (w / v) of healthy hair powder in 1.5% normal rabbit serum-containing phosphate buffered saline (PBS-NRS)
96-well multiplate (Plate with a low protein adsorption membrane filter sealed at the bottom, Multiscreen GV filtration plate, Millipore)
50 μl of the solution was dispensed into each well, and the solution was aspirated and discarded.
100 μl of the culture supernatant after 2 weeks of culture was reacted with the antigen in each well. Further, a peroxidase-labeled goat anti-mouse IgG antibody was reacted, and 2,2′-azino-bis- (3-ethylbenchazoline-6-sulphonic acid) diammonium (ABTS) was used as a coloring agent.
The antibody titer was measured by measuring the absorbance at 5 nm. As a result, an antibody that reacts with healthy hair powder was detected in four wells. The obtained hybridoma is HA
After transferring from T medium to HT medium without aminopterin and culturing, it was cloned by the limiting dilution method. That is, thymocytes 1 × 10 ⁷ / ml, R containing 15% fetal calf serum
Using PMI1640 medium, cells were diluted and cultured in a 96-well multiplate at a density of 1 per well. After the appearance of colonies, antibody-producing cells were selected by a modified ELISA method. The cloning was further repeated to obtain 4 stable hybridomas NH1 to NH4. With the mouse monoclonal antibody subtyping kit (manufactured by Amersham), the classes of the produced monoclonal antibodies were all determined to be IgM and κ type.

Example 2 (1) Immunization method: Healthy hair powder prepared in the same manner as in Example 1 was suspended in physiological saline at a concentration of 2% (w / v) and the same amount was applied. Freund's complete adjuvant (FC
An emulsion for immunization was prepared by mixing with A) for 10 minutes. NZBWF ₁ (female, 8 weeks old) was intraperitoneally injected with 100 μl of this immunizing adjuvant (hair powder 1 mg / mouse). After 19, 30, 74, 87 and 117 days, booster immunization was performed in the same manner. However, in the final immunization, F
Freund's Incomplete Adjuvant (FI
A) was used.

(2) Measurement of antibody titer in plasma of immunized mice:
Two days after the final immunization, the plasma prepared from the immunized mouse was serially diluted 50 to 800 times with PBS to prepare a plasma diluent. The antibody titer in the plasma of immunized mice was measured by the same method as this except that this plasma diluted solution was used instead of the culture supernatant in the selection of the hybridoma of Example 1.

(3) Cell fusion: Three days after the final immunization, spleen cells were extracted from the two mice having the highest plasma antibody titer in the above antibody titer measurement, and washed with RPMI1640 medium. On the other hand, mouse myeloma cells P3X63-Ag. RPMI collecting 8.6.5.3
It was washed with 1640 medium. A suspension of 2 × 10 ⁸ spleen cells and a suspension of 4 × 10 ⁷ mouse myeloma were mixed, and the medium was removed by centrifugation. 50% polyethylene glycol 4000-75 mM heated to 37 ° C was added to the mixed cells.
1.5 ml of HEPES buffer was gradually added over 1 minute to perform fusion by gently stirring for 1 minute. RPMI1
10 ml of 640 medium was added over 5 minutes with gentle stirring. Remove the medium by centrifugation and add 5 x 10 ⁶ to the cells.
HAT medium containing mouse thymocytes / ml (20% fetal bovine serum, Iscove's modified Dulbecco's medium (IM
The same composition as in Example 1 except that DM) was used) (75 ml) was added, and then 0.1 ml was distributed per well in a 96-well plate. After 4 days, 0.1 ml of HAT medium was added to each well. After 2 weeks of culture, growth of hybridomas was observed in 80% or more of the wells.

(4) Selection of hybridoma: The antibody in the hybridoma culture supernatant was searched for by the same method as described in Example 1. As a result, an antibody reactive with healthy hair powder was detected in 10 wells. The obtained hybridoma is transferred to HT medium and cultured, and the antibody in the culture supernatant is peroxidase-labeled goat anti-mouse IgG antibody (anti-IgG antibody) or biotin-labeled goat anti-mouse IgM.
It was detected by a modified ELISA method using an antibody (anti-IgM antibody). When an anti-IgM antibody was used, it was reacted with a (avidin-biotin-labeled peroxidase) complex and then a color reaction was performed. As a result, 1 antibody was detected by the anti-IgG antibody but hardly detected by the anti-IgM antibody.
Detected in two wells. 10% BM-Condime
d H1 (Boehringer Mannheim Yamanouchi), 10
Stable hybridoma nH was obtained by repeating cloning by the limiting dilution method using IMDM medium containing 15% fetal bovine serum.
The P14H6 strain was obtained. The monoclonal antibody nHP14H6 produced by this cell was determined to be IgG2a, κ type by the same method as described in Example 1.

(5) Antigen specificity: EL as an antigen using the healthy hair powder or perm hair powder obtained by the above-mentioned preparation method
A modified ISA method was performed. 100 μg of each antigen blocked with PBS-NRS was placed in each well of a multiscreen-GV filtration plate (Millipore) previously blocked with PBS-NRS, and the hybridoma culture supernatant was serially diluted with PBS. Was further reacted with a peroxidase-labeled goat anti-mouse IgG antibody. The reaction mixture uses ABTS as a color former and 4
The absorbance at 05 nm was measured. The results are shown in Fig. 1. From FIG. 1, it can be seen that the monoclonal antibody of the present invention has higher reactivity to healthy hair powder than to perm hair powder. Therefore, the monoclonal antibody of the present invention is useful for analysis of higher-order structure change of hair protein by perm treatment, and can be used as a hair damage sensor by various chemical treatments.

(6) Immunohistochemistry of exfoliated hair section Human extirpated hair section, which was previously fixed with cold methanol and blocked with PBS-NRS, was diluted 10 times with PBS and reacted. It was next,
Fluorescein isothiocyanate (FITC) -labeled goat anti-mouse IgG antibody was reacted, and the reaction site of the antibody on the hair section was observed by a fluorescence microscope. As a result, it was found that the monoclonal antibody of the present invention reacts with the hair follicles after the initial stage of keratinization and the cortex of the keratinized region. Therefore, the monoclonal antibody of the present invention is not only useful for analyzing the conformational changes of hair proteins that occur during the keratinization process, but is also used as a hair damage repair agent targeting the scalp by combining with other existing substances. Alternatively, it can be applied as a hair cosmetic.

Example 3 (1) (i) Preparation of perm hair powder: (1) of Example 1
Prepared by a method similar to. (ii) Preparation of healthy fur powder: After washing healthy hair with hexane, soak it in antiformin adjusted to pH 6.5 with hydrochloric acid,
It was treated at room temperature for 50 seconds. After thoroughly washing with water, it was immersed in a 0.5% aqueous sodium pyrosulfite solution adjusted to pH 9 with aqueous ammonia, and treated at room temperature for 3 minutes. After thoroughly washing with warm water, it was air-dried, the hair dermis was peeled from the hair, and the hair dermis was removed. 6 g of hair from which the hair dermis was removed by the above method was ground to 10 μm or less by the same method as in Example 1,
A healthy fur powder was used. (iii) Preparation of healthy small leather powder: 2 g of healthy hair was washed with hexane, cut into 1 cm lengths, and placed in a 500 ml Sakaguchi flask. 1 ml with a diameter of 12.7 mm in 100 ml ion exchange distilled water
Shaking was performed at 120 rpm for 24 hours at room temperature using a reciprocal shaking incubator together with ten 6.0 mm Teflon balls each. By this treatment, the hair pellicle was mechanically peeled from the hair, the hair was removed with a nylon net, and the white turbid liquid was centrifuged. The obtained white precipitate was freeze-dried to obtain a healthy hair dermis powder.

(2) Immunization method: Perm hair powder, healthy fur powder and healthy hair dermis powder prepared by the above method are suspended in physiological saline at a concentration of 2% (w / v), An equal amount of Freund's complete adjuvant (FCA) was mixed for 10 minutes to prepare an emulsion for immunization. NZBWF
₁ (female, 9 weeks old) intraperitoneally with this adjuvant 1 for immunization
00 μl (hair powder 1 mg / mouse) was injected. After that 3
A total of 6 boosters were given every other week.

(3) Measurement of antibody titer in plasma of immunized mice:
Twenty-six days after the sixth booster immunization, the plasma prepared from the immunized mouse was serially diluted 50 to 800 times with PBS to prepare a plasma diluent. The antibody titer in the plasma of immunized mice was measured by the same method as this except that this plasma diluted solution was used instead of the culture supernatant in the selection of the hybridoma of Example 1.

(4) Cell fusion: One mouse having the highest antibody titer in plasma in the above antibody titer measurement was subjected to final immunization. However, in the final immunization, Freund's incomplete adjuvant (FIA) was used instead of FCA.
Three days after the final immunization, spleen cells were extracted from this mouse,
It was washed with RPMI1640 medium. On the other hand, mouse myeloma cells P3 × 63-Ag. 8.6.
5.3 was collected and washed with RPMI1640 medium. A suspension of 1 × 10 ⁸ spleen cells and a suspension of 2 × 10 ⁷ mouse myeloma were mixed, and the medium was removed by centrifugation. To the mixed cells, 1.5 ml of 50% polyethylene glycol 4000-75 mM HEPES buffer heated at 37 ° C. was gradually added over 1 minute to perform fusion by gently stirring for 1 minute. 10 ml of RPMI 1640 medium was added over 5 minutes with gentle stirring. Remove the medium by centrifugation,
After adding 100 ml of HAT medium containing 5 × 10 ⁶ mouse thymocytes / ml (the same composition as in Example 2) to the cells, 0.1 ml was distributed per well in a 96-well plate. 4 days later
0.1 ml of HAT medium was added to each well.

(5) Selection of hybridoma: The antibody in the culture supernatant of the hybridoma was searched for by the same method as shown in Examples 1 and 2. 10% BM-Condimed H1 (Boehringer Mannheim Yamanouchi) for wells in which antibody activity against various antigens (any one of healthy hair powder, perm hair powder, healthy fur powder, and healthy hair skin powder) was detected ) IM containing 10% fetal bovine serum
Cloning by the limiting dilution method was repeated using DM medium to obtain 9 stable hybridoma strains.

Table 1 shows the monoclonal antibody produced by the above hybridoma and its type, and the recognition site of the monoclonal antibody using the immunohistological method described in Example 2.

[0040]

[Table 1]

[0041]

[Brief description of drawings]

FIG. 1 is a drawing showing the reactivity of the monoclonal antibody obtained in Example 2 on healthy hair and permed hair.

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Claims (4)

[Claims] 1. A monoclonal antibody against the hair shaft after keratinization. 2. A hybridoma obtained by fusing mammalian antibody-producing cells immunized with hair powder obtained from keratinized hair shaft and mammalian myeloma cells. The monoclonal antibody according to claim 1. 3. A hybridoma producing the monoclonal antibody according to claim 1. 4. The method according to claim 3, which is obtained by fusing mammalian antibody-producing cells immunized with a hair powder obtained from the hair shaft portion after keratinization and mammalian myeloma cells. Hybridoma.