JP2002020400A - Anti-active keratinocyte transglutaminase antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce an anti-active keratinocyte transglutaminase antibody and to easily detect active keratinocyte transglutaminase using various immunological techniques such as immunostaining. SOLUTION: This antibody specifically recognizes keratinocyte transglutaminase, and has an affinity for active keratinocyte transglutaminase higher than that for a keratinocyte transglutaminase inactive precursor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibody which specifically recognizes the activation of keratinocyte transglutaminase produced by keratinocyte, which is a major cell present in the skin epidermis, and which has been transformed and has high properties of normal cells. The present invention relates to a prolonged life keratinocyte which enables easy and long-term subculture as it is, and a method for easily searching for various substances that maintain healthy functions of human skin epidermis using the same.

[0002]

2. Description of the Related Art Transglutaminase (amine γ-glutamyltransferase: EC 2.3.2.13.
A series of enzymes, hereinafter referred to as TGase), are mostly Ca ²⁺ -dependent, covalently introducing a primary amine into a glutamine residue in a peptide chain, and epsilon between a glutamine residue and a lysine residue. Catalyzes post-translational modifications of proteins such as-(γ-glutamyl) lysine isopeptide bond-forming cross-links, stabilizes proteins, constructs biological structures, participates in signal transduction, participates in cell proliferation, differentiation and apoptosis, etc. It is involved in various physiological functions in the living body.

[0003] Among TGase isotypes in mammals, keratinocytes TGa mainly present in keratinocytes
se (hereinafter abbreviated as “TGK”, also known as Type I TGa
se, skin TGase, etc.) mainly exist in keratinocytes,
Its function is mainly to construct the epidermis by cross-linking keratinocyte envelope constituent proteins. In recent years, it has been reported that many of the causes of Lamellar Ichthyosis, a serious genetic disease accompanied by a drastic decrease in epidermal barrier function, are due to mutations in the TGK gene (E. Candi et.
al; J. Biol. Chem. Vol 273 No. 22 13693-13702 1998),
Findings of TGK knockout mice in which transepidermal water loss is abnormally high and is lethal (M. Matsuki et.al; Proc. Natl. Acad.
Sci. USA Vol. 95 1044-1049 1998) In addition, ceramide, an endogenous lipid that is important for moisturizing in the stratum corneum, is tightly bound to keratinocytes by TGK catalysis, and the stratum corneum focusing on moisturizing The barrier function is adjusted (ZN
emes et.al .; Proc. Natl. Acad. Sci. USA Vol. 96 8402-84
07 1999) and the like, and the role of TGK in maintaining healthy skin physiology has been emphasized.

[0004] Therefore, if a method for simply detecting the normal activation state of TGK and its abnormality and a method for searching for a substance capable of improving the abnormality can be established, a new and important skin function will be developed. It was expected that the search for improved materials would be possible.

However, to detect TGK activation, a method of extracting TGK from a tissue or the like and measuring its enzymatic activity, a method of detecting a protein cross-linked by a TGK catalyst, and the like have hitherto been used. However, these have complicated steps because TGK is an enzyme that is unstable in the extraction operation, and indirectly activate T-type.
Only GK was detected.

[0006] A monoclonal antibody prepared using a protein having TGase activity present in the epidermis as an antigen is commercially available, but it has been pointed out that the specificity for TGK is incomplete (SYKim et al., J.Invest.Der
matol. 104: 211-217 1995), and it was not possible to recognize activated TGK more specifically than inactive precursors.

[0007] Due to these causes, the study of TGK activation at the protein level does not proceed, and the activation mechanism,
There are many unclear points about the identity of activated TGK.

[0008] On the other hand, normal human keratinocytes are widely used in cultured cell experiments for activating TGK in human keratinocytes and searching for a material for improving the function thereof. The number of possible replacements is limited to about the fourth to fifth generations, and there are many disadvantages due to problems such as reproducibility, simplicity, and cost. In addition, there are extremely few types of human keratinocyte cell lines on the market, and when cell lines are used, subculture can be performed semipermanently, so that the culture operation is easy, Due to its superiority in proliferation, there is a problem that TGK activation, which is closely related to differentiation, may not normally occur.

[0009] In view of the above, it has never been possible to simply and accurately detect the activity of TGK, which is important for maintaining healthy skin functions, and to simply search for a material that improves the abnormality. It was possible.

[0010]

Accordingly, an object of the present invention is to produce an anti-active TGK antibody, and to easily detect active TGK using various immunological techniques such as immunostaining. And Furthermore, to extend the number of passages while maintaining the properties closer to normal human keratinocytes than cell lines, to produce a life-extended human keratinocytes that became easier to culture,
Using these, we have established a method for analyzing the mechanism of TGK activation, constructing a model of human skin disease, etc., and selecting a substance that improves skin dysfunction associated with TGK activation modulation. The purpose is to select materials, cosmetic materials and food materials that maintain healthy skin.

[0011]

Means for Solving the Problems The anti-active TGK antibody according to the first aspect of the present invention is an antibody that specifically recognizes TGK, and has an affinity for active TGK of TK.
It has a higher affinity for the GK-inactive precursor.

[0012] The anti-active TGK antibody according to the second aspect of the present invention specifically recognizes a limited cleavage site accompanying the activation of the TGK-inactive precursor according to the first aspect. .

The anti-active TGK antibody according to the third aspect of the present invention specifically recognizes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a peptide having a sequence complementary to the amino acid sequence. Things.

The anti-active TGK antibody according to the invention described in claim 4 is a monoclonal antibody.

[0015] The life-extended human keratinocyte mixture according to the invention described in claim 5 comprises a life-extending human keratinocyte cell having an extended number of passages by subjecting a plurality of normal human keratinocytes to life-extending treatment. It is a thing.

[0016] The life-extended human keratinocyte mixture according to the invention described in claim 6 has been transformed with the SV40 virus.

A method for selecting a substance for improving skin function using cultured human keratinocytes according to the invention according to claim 7 includes the following steps (a) and (b). (a) causing the modulation of keratinocyte proliferation, differentiation or TGK activation in the presence or absence of the test substance; (b) in the presence or absence of the test substance in step (a). The TGK activity of the anti-active T according to any one of claims 1 to 4.
Detecting with a GK antibody and comparing the activities of the two,

The method for detecting a skin lesion according to the invention described in claim 8 includes the following steps (a) and (b). (a) reacting the antibody of claims 1 to 4 with a test skin section and visualizing it; (b) analyzing the result visualized in step (a);

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, there is provided an antibody which specifically recognizes TGK, wherein the antibody has an affinity for active TGK higher than that for an inactive precursor of TGK. A TGK antibody is provided.

The active TGK in the present invention is a protein produced by limited degradation of a TGK inactive precursor due to the influence of keratinocyte differentiation and the like. It has a significant effect on maintaining skin function. Here, the limited degradation site in the present invention refers to a report that limited degradation occurs at two places in response to TGK activation (J. Biol. Chem. Vol. 270 N
o.30 18026-18035 1995) (the arginine residue at the 93rd position and the arginine residue at the 573th position). However, up to now, details such as what kind of protease is responsible for this limited degradation and how it becomes active TGK after the limited degradation is still unknown.

The anti-active TGK antibody of the present invention is
Which specifically recognizes the activated TGK,
Either one that recognizes both K-inactive precursor and active TGK, or one that specifically recognizes only active TGK can be used. Is important, and in order to surely know the degree, the affinity for activated TGK must be T
It is necessary to use an antibody having a higher affinity than the GK-inactive precursor, and it is particularly preferable to use an antibody that specifically recognizes only active TGK.

A preferred embodiment of the anti-active TGK antibody of the present invention is one which specifically recognizes a limited cleavage site accompanying activation of a TGK inactive precursor. That is, the cleavage planes of the two limited cleavage sites accompanying the activation of TGK are considered to be parts that are not present in the precursor but are exposed only after activation, and specifically recognize the cleavage plane parts. By preparing an antibody that performs the above, it became possible to obtain an antibody that specifically recognizes the active form of TGK whose substance is still unknown.

Particularly preferred among the anti-active TGK antibodies is a partial sequence of the amino acid sequence at the cleavage site.
It was prepared according to a conventional method using the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a peptide having a sequence complementary to the amino acid sequence as an antigen. The use of these antigens makes it possible to prepare an antibody having high specificity for activated TGK. Antibodies obtained in this manner include anti-active TGK polyclonal antibodies 1084 and 1087, and anti-active TGK monoclonal antibody R1410.
H antibody and M911E antibody are exemplified. Note that R1410
Hybridoma cell R1410H that produces H antibody is No. 17903 from Seikoken, and hybridoma cell M911E that produces M911E antibody is No. 17902 from Seikoken.
Both have been deposited on June 14, 2000.

As a method for producing an antibody, for example, in the case of preparing a polyclonal antibody, a rabbit is immunized with the antigen and a polyclonal antibody having high specificity to active TGK is obtained from an affinity column purified fraction of the serum. When a monoclonal antibody is prepared, a mouse is immunized according to a conventional method, and a hybridoma cell obtained by fusing the spleen cell and myeloma cell is prepared.
A monoclonal antibody having high specificity for GK may be selected.

By using these antibodies in various immunological techniques, it is possible to further pursue the activation of TGK and the substance of activated TGK, which are still unknown. Examples of the immunological technique include immunoblotting (Western blotting), ELISA, immunostaining such as cell staining and tissue staining, immunoprecipitation, and fractionation of active TGK using an affinity column. Polyclonal antibodies are preferably used for immunoblotting (Western blotting) because of their stability, and monoclonal antibodies are preferred for cell staining because of their high specificity.
It is preferably used for tissue staining.

In the immunostaining method, a light fixing method such as paraformaldehyde or acetone is used, and as a labeling substance, a fluorescent dye such as FITC or rhodamine red, an enzyme such as peroxidase or alkaline phosphatase, or colloidal gold is used. Good. In the immunoprecipitation method, an antibody is labeled with beads having a magnetic force or weight, and activated TGK is used.
And sedimentation by sedimentation. Further, the present antibody can be bound to a carrier to prepare an affinity column for activated TGK.

Another object of the present invention is to provide a life-extended human keratinocyte mixture in which a plurality of normal human keratinocytes are subjected to a life-extending treatment so that the number of passages can be extended.

The life-extended human keratinocytes according to the present invention are cells derived from normal human keratinocytes, which have been passaged six or more times and have not been established. Prolonged keratinocytes can be created, for example, by transformation by introducing a T antigen gene of SV40 virus. This prolonged keratinocyte is characterized by the use of unstained cells without the cloning operation.It has a low growth advantage that does not lead to immortalization, and is not a single cell line group close to the environment of normal skin tissue. The environment enhances the normality of cells, including their differentiation potential.

That is, the life-extending keratinocytes of the present invention are:
Because the number of passages is longer than normal cells,
Not only is it possible to perform systematic long-term culture experiments with high reproducibility, but also to maintain the properties of normal cells higher than cell lines, so that toxicity tests, cytokine production, and various other keratinocyte bioactive substances It is possible to examine the activation of TGK, particularly from the normality of its differentiation ability.

The number of keratinocytes that can extend the life of keratinocytes is preferably about 20 to 300, especially about 30 to 180, and a proper use may be selected depending on the application. Since the number of passages of about 30 generations has a differentiation ability close to that of normal cells, it is preferably used when the examination is to be performed in a more normal situation. If the number of passages is 100 or more, the differentiation ability is slightly lower than that of about 30 generations. Is preferred.

When the anti-active TGK antibody, life-extended human keratinocytes, and normal human keratinocytes of the present invention are used,
It is possible to easily detect the modulation of TGK activation, which is important for maintaining healthy human skin function, and to detect a material that improves the modulation. That is, it is possible to easily detect the health condition or lesion of the skin, and to prevent and treat the skin lesion. Specific examples of such a method include the following. (1) A method for selecting substances having the ability to maintain healthy skin function (2) A method for detecting skin lesions

That is, another object of the present invention is to provide a method for selecting a substance having the ability to maintain healthy skin function related to TGK activation, which comprises the following steps (a) and (b): It is. (a) causing the modulation of keratinocyte proliferation, differentiation or TGK activation in the presence or absence of the test substance; (b) in the presence or absence of the test substance in step (a). Detecting TGK activity with the anti-active TGK antibody and comparing the activities of the two.

In the step (a) of the method for selecting a substance having the ability to maintain healthy skin function, first, in the presence or absence of a test substance, modulation of keratinocyte proliferation, differentiation or TGK activation is caused. . The keratinocytes to be used are not particularly limited, but the keratinocytes established as described above have a low differentiation ability compared to normal cells. It is particularly preferable to use keratinocytes having a prolonged life. The medium used for culturing keratinocytes is M, which is usually used for keratinocyte culture.
Bovine pituitary extract and EG in CDB153 basal medium
A serum-free medium or the like to which a growth factor such as F or insulin is added may be used.

The test substance herein refers to a substance that is expected to suppress the modulation of keratinocyte proliferation, differentiation, etc., and includes animal-derived substances, microorganism-derived substances, plant extracts, natural minerals, and chemically synthesized substances. Substances and the like.

The method of culturing is not particularly limited. In addition to normal monolayer culture on a culture dish or the like, three-dimensional culturing by vapor-liquid culturing, transplantation into mouse skin, or the like is performed. Can be. When it is desired to analyze TGK activation and its modulation in a state closer to the in-vivo environment, it is desirable to perform three-dimensional culture.

The step of causing the modulation of keratinocyte differentiation and the like includes, for example, UV irradiation and addition of various reagents. The conditions of UV irradiation are appropriately adjusted according to the properties of the keratinocytes used, the amount of the test substance, and the like. What is necessary is just to set, for example, what is necessary is just to perform irradiation of 0.025 J / cm < ² > of ultraviolet-B wave several times, and single irradiation of 0.1 J / cm < ² >. Examples of the reagent to be added include physiologically active substances such as calcium, retinoic acid, vitamin D, cholesterol sulfate, and TGFβ, and the amount of the added reagent is about 1 nM to 1 mM.

Next, as the step (b) of the method for selecting a substance having the ability to maintain healthy skin function, the activity of the keratinocytes treated as described above is detected by the anti-active TGK antibody of the present invention, and the activity is determined in the presence of the test substance. Alternatively, the difference in activity in the absence is compared. As a method of detecting the activity, for example, keratinocytes treated as described above are lightly fixed with paraformaldehyde or the like, then solubilized with urea, lithium dodecyl sulfate, mercaptoethanol, Triton X-100, etc. Perform a blot. That is,
SDS polyacrylamide gel electrophoresis (SDS-PA
GE) and further convert the protein to poly vinilidene di
It is transferred to a fluoride membrane, a nitrocellulose membrane, or the like, reacted with activated TGK using the present antibody, and further reacted with, for example, a secondary antibody labeled with alkaline phosphatase, and developed with a substrate for alkaline phosphatase coloring. Bands which are considered to be those of activated TGK obtained by these methods may be quantified using inspection and an image analyzer. Alternatively, the keratinocytes treated as described above are lightly fixed with paraformaldehyde, and then reacted with an anti-active TGK antibody as a primary antibody.
After reacting the C-labeled secondary antibody and encapsulating it with an encapsulating agent such as glycerin, it may be observed with a fluorescence microscope and its fluorescence intensity may be measured.

From the results thus obtained, if a test substance that suppresses the normal modulation of TGK activation of keratinocytes is selected, it can be expected that the test substance is used as a substance having the ability to maintain healthy skin function.

Still another object of the present invention is to provide a method for detecting a skin lesion, comprising the following steps (a) and (b). (a) reacting the antibody of claims 1 to 4 with a test skin section and visualizing it; (b) analyzing the result visualized in step (a);

Step (a) of the method for detecting a skin lesion includes:
Specifically, an antibody of the present invention is allowed to react with a skin section of a test subject and visualized. Specifically, a test skin section is obtained by embedding a skin section of about 1 cm ² of a subject generated by a biopsy or a surgical operation with an OCT compound, and then thinly cutting it into a frozen section according to a conventional method. Good. After lightly fixing the frozen section using acetone or the like, a blocking operation is performed with horse serum or the like, and the present antibody is reacted as a primary antibody.
Further, after reacting with a secondary antibody labeled with a fluorescent substance such as FITC, it is encapsulated with glycerin or the like.

Step (b) of the method for detecting a skin lesion includes:
Specifically, the specimen of the skin section visualized in the step (a) is analyzed. For example, a FITC-labeled specimen is observed for its fluorescence, and the degree of modulation of TGK is compared with that of a normal TGK based on its intensity and range to make a diagnosis. Alternatively, a score may be obtained by using appropriate image analysis software.

From the results thus obtained, the state of TGK activity in the skin of various disease patients can be accurately detected, and the effect of diagnosis and treatment of each disease can be examined.

[0043]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Embodiment 1 TGK is a protein having an amino acid sequence of 817 residues, and a protein having an apparent molecular weight of 106 k on SDS-polyacrylamide electrophoresis (SDS-PAGE), which is further post-translationally modified with sugar chains and fatty acids. is there. The amino acid sequence of TGK is shown in SEQ ID NO: 3. According to SY Kim et al. (1995), TGK in keratinocytes induces differentiation between arginine residues 93 and 94, and arginine residues 573 and 574 during cell differentiation induction. It is said that it undergoes limited decomposition with the group.

Since these limited degradations are similar to the limited degradation sites associated with the activation of blood coagulation factor XIII, which is the same TGase isotype, it is presumed that these limited degradations are important for TGK activation. . Further, as described above, the cut surface resulting from this limited decomposition is TG of the inactive precursor.
This sequence is exposed only when K is activated.
It is a part specific to GK. Therefore, considering the antigenicity of the sequence, 15 residues from the 94th glycine residue to the 108th glycine residue, and 12 residues from the 574th glycine residue to the 585th alanine are used as antigens. Then, a polyclonal antibody specific to activated TGK was prepared.

That is, 1 mg of the synthetic antigenic peptide shown in SEQ ID NO: 1 and SEQ ID NO: 2 was added to rabbit (Japanese native species II) and turbid in Freund's complete adjuvant (DIFCO).
/ Rabbits were injected subcutaneously in the back. Three weeks later, 1 mg each of the turbid antigen peptide was injected subcutaneously in Freund's incomplete adjuvant (DIFCO), and thereafter, every 10 days, a total of two to three booster immunizations were performed. When the antibody titer in the serum became stable, a large amount of blood was collected from the auricular artery, and a serum fraction was collected by centrifugation. The serum fraction was purified using an affinity column prepared by adsorbing an antigen-synthesizing peptide to FMP-activated Cellulofine (Seikagaku Corporation), and 1084 antibody (the antigen is SEQ ID NO: 1).
87 antibodies (the antigen is SEQ ID NO: 2) were obtained.

Embodiment 2 FIG. The immunoblotting subconfluent normal human keratinocytes (Kurabo) using polyclonal antibodies, high calcium concentration media (1 mM CaCl ₂₎
And treated with 2% paraformaldehyde, 9M urea, 2% Triton-X100, 5% 2
-Solubilized with mercaptoethanol and subjected to SDS polyacrylamide gel electrophoresis with lithium dodecyl sulfate (SD
S-PAGE).

SDS-PAGE was performed according to a conventional method at 7.5%.
Performed on acrylamide gel. Plotting is performed using a semi-dry transfer device (BE-330; Biocraft) with a poly vinilidene difluoride membrane (Millipore).
And transferred.

The membrane after the transfer was blocked with a 2% bovine serum albumin solution, and then neutralized by mixing with an anti-active TGK polyclonal antibody 1084 antibody and an excessive amount of an antigen peptide as primary antibodies for 1 hour at room temperature. An alkaline phosphatase-labeled anti-rabbit IgG antibody (Promega) is allowed to react as a secondary antibody, color is developed using BCIP / NBT chromogenic substrate (Promega), and an approximately 85k band which is specifically expressed depending on the differentiation induction treatment is amplified. Was quantified using an image analyzer.

FIG. 1 is a graph showing the results of detection of TGK activation modulation by immunoblotting using the 1084 antibody. In the figure, NONE on the horizontal axis is untreated, CaCl ₂
Indicates the treatment with 1 mM CaCl ₂ , RA indicates the treatment with 3 μM retinoic acid, and AVERAGE OD on the vertical axis indicates the result of quantifying the amplification amount of the band of about 85 k with an image analyzer.

It is known that normal human keratinocytes are induced to differentiate by culture in a medium having a high calcium concentration. At this time, TGK in keratinocytes is expected to be in an active form. In this study, the cells subjected to these differentiation-inducing treatments were examined by immunoblot using the 1084 antibody. As shown in FIG. 1, the 1084 antibody was dependent on the differentiation-inducing treatment at about 85 k. And the band which was expressed was detected. Since each antibody neutralized with each antigen peptide did not detect this band, it was shown that these were bands derived from activated TGK. Thus, by using this antibody for immunoblotting, normal human keratinocytes and TGK associated with differentiation induction can be obtained.
It was possible to examine the specific changes of.

Embodiment 3 FIG. Preparation of Hybridoma and Production of Ascites Antibody Synthetic peptide (SEQ ID NO: 1 and SEQ ID NO: 2) with hemocyanin bound to Balb / c mouse was turbidized in Freund's complete adjuvant (DIFCO) to 100 μg.
/ Mice were injected subcutaneously. Thereafter, every 14 days, 100 μg of the turbid antigen peptide in Freund's incomplete adjuvant (DIFCO) was injected subcutaneously and intraperitoneally, and a total of 4 to 5 booster immunizations were performed. Five days before the removal of the spleen, a solution obtained by dissolving 100 µg in physiological saline was intravenously injected into the tail to obtain the final immunization.

The removed spleen cells were turbid in RPMI 1640 medium (Sigma). Myeloma cells were grown in EPMI1640 medium containing 15% fetal bovine serum (Dainippon Pharmaceutical), and then opaque spleen cells and myeloma cells were mixed at a ratio of 4: 1 to obtain polyethylene glycol 60.
The cells were fused for 5 minutes at 37 ° C. with RPMI 1640 medium containing 45% of OO. The obtained cell mixture was added to RPMI1640 medium containing 15% fetal bovine serum,
The cells were suspended at 1 × 10 ⁵ cells / ml, and 0.1 ml thereof was dispensed into a 96-well plastic microtiter plate and cultured in a 37 ° C. incubator in the presence of 7% carbon dioxide.

On the first day of the culture, 0.1 ml of HAT (hypoxanthine-aminopterin-thymidine) medium was added, and on the second, fourth, seventh and tenth days, the culture supernatant was added to 0.1 ml. Each 1 ml was aspirated and fresh HAT medium was added instead. By the above operation, an average of 1 to 2 hybridoma colonies per well grew in a culture well of a 96-well plastic microtiter plate of 30 to 60%. When these hybridomas were sufficiently grown, the antibody titer of the culture supernatant against the antigen was assayed by ELISA, and the cloning operation was performed by limiting dilution of the hybridoma in each well producing the desired antibody.

Thus, a clone stably producing an antibody was isolated, and R1410H (the antigen was SEQ ID NO: 1)
M911E (the antigen is SEQ ID NO: 2).
The isotype of clone R1410H was IgG2
a, The isotype of clone M911E was IgG1. These clones were obtained from R1410H-producing hybridoma cells R1410H.
No. 03 and M911E, a hybridoma cell that produces the M911E antibody, have been deposited on June 14, 2000 under the name of Seikoken No. 17902.

For these clones, monoclonal antibodies R1410H and M911E specific to each antigen peptide were obtained from the protein G affinity column purified fraction of the culture supernatant and the ascites of the mouse into which these clones were transplanted.

Embodiment 4 FIG. Confirmation of monoclonal antibody activity and specificity by ELISA method Each of the antigen peptides was diluted to about 50 μg / ml, and
Plate for hole ELISA (NUNC immunoplate)
Was coated at 4 ° C. overnight. Each well on the plate was washed three times with a washing solution for ELISA (0.05% Triton X-100 added to a phosphate buffer), and a 5% bovine serum albumin solution was added thereto to remove the antigen-unadsorbed portion at room temperature. For 2 hours. This was washed three times with a washing solution for ELISA, and antibody R1410 was used as a primary antibody.
H and antibody M911E were reacted at room temperature for 1 hour. However, as a control, an antibody neutralized by mixing with the antigen peptide of each antibody at room temperature for 1 hour was used. EL
After washing three times with a washing solution for ISA, a peroxidase-labeled anti-mouse immunoglobulin antibody (Cap
pel) at room temperature for 1 hour, and after washing three times,
Add a chromogenic substrate (3,3 ', 5,5', Tetramethyl Benzidine), stop the reaction with 2M sulfuric acid when sufficient color is developed, and confirm the reactivity of each antigen and each antibody from the degree of color development. did.

FIG. 2 is a diagram showing the reactivity of each antibody to the antigen. In the figure, r on the horizontal axis represents synthetic peptide SEQ ID NO: 1 (R1410H antigen), m represents synthetic peptide SEQ ID NO: 2 (M911E antigen), and BSA represents bovine serum albumin. In each bar graph, R represents antibody R1410H, R + m represents an antibody R mixed with an excess amount of peptide m and treated for 1 hour at room temperature, and R + r represents an antibody R mixed with an excess amount of peptide r and treated for 1 hour at room temperature. , M is antibody M911
E and M + r show the results obtained by mixing an excess amount of peptide r with antibody M and treating at room temperature for 1 hour, and M + m show the results obtained by mixing antibody M and excess amount of peptide m and treating at room temperature for 1 hour.

As shown in the figure, the R1410H antibody strongly reacted with the corresponding antigen peptide, but did not react with the non-corresponding antigen peptide and BSA. In addition, the reactivity did not change significantly when mixed with the corresponding antigen peptide, but the reactivity was significantly reduced when mixed with the corresponding antigen peptide. Similarly, the M911E antibody strongly detected only the antigen peptide, and its reactivity was reduced only when it was mixed with the corresponding antigen peptide. From the above, the antigen specificity of each antibody was shown.

Embodiment 5 FIG. Detection of Differentiated Cells by Cell Staining Using Monoclonal Antibodies Normal human keratinocytes (Kurabo) are grown on a collagen-coated cover glass with keratinocyte growth medium (Kurabo) to reach subconfluence, and calcium in the medium The cells were cultured at a concentration of 1 mM for 48 hours to induce differentiation. Cells after differentiation induction were fixed with 2% paraformaldehyde, equilibrated with 20% sucrose, and
The blocking operation was performed for 1 hour at room temperature with% bovine serum albumin (BSA). The R1410H antibody and the M911E antibody were reacted at 37 ° C. for 1 hour as primary antibodies.
After washing three times with 0.1% BSA solution, as a secondary antibody,
Fluorescent (FITC) labeled anti-mouse immunoglobulin antibody (J
ACKSON) was reacted at 37 ° C. for 1 hour.

After further washing three times with 0.1% BSA solution, 100 μg / ml propidium iodide (P
I) Cell nuclei were stained with the solution. After staining, the cells were sealed with non-fluorescent glycerin and observed with a fluorescent microscope (Olympus). The fluorescence intensity was measured using a CCD camera system (HC-2
(500: Fuji Film), the FITC fluorescence intensity within a fixed field of view was measured using image analysis software Image-Pro Plus (Planetron), and converted per cell.

FIG. 3 is a diagram showing the results of detection of cells induced to differentiate by the cell staining method. As described above, cultured normal human keratinocytes have a high calcium concentration (1 m
Culture in M) induces differentiation and, consequently, TGK
It is expected that differentiation-induced cells can be specifically detected by using the present antibody by a cell staining method because of the activation of. As shown in FIG. 3, the fluorescence intensity per cell of the cells induced to differentiate was higher than that of the cells undergoing proliferation, and each of the present antibodies specifically detected the cell group subjected to the differentiation induction treatment.

Embodiment 6 FIG. Establishment of prolonged human keratinocytes Normal human keratinocytes (Kurabo) are cultured in a keratinocyte growth medium (Kurabo) until the second passage, and then a linear SV40 virus T antigen gene clone previously prepared (VG026: Human Science Research Resource Bank) was introduced into cells by a lipofection method (DOSPER: Boehringer Mannheim). After gene transfer, the cells were split, cultured for about 3 weeks, and the growing colonies were collected and subcultured.

As a result, when the cells were cultured under the same conditions, several types of life-prolonged human keratinocytes that can be passaged up to about 30 times, and several types of life-extended human keratinocytes that can be prolonged beyond 30 times were obtained. . The main ones are shown in Table 1 below.
Shown in

[0065]

[Table 1]

All of the obtained cells could be stored frozen, thawed, and re-cultured. In addition, cells up to about 30 generations
The tumor necrosis factor α (TNFα) exhibited an excessive secretion ability by ultraviolet irradiation, which has a differentiation ability similar to that of normal cells and is generally observed in normal cells but not reported in established cells. In addition, most life-prolonging cells had hyaluronic acid-producing ability generally found in normal cells.

As described above, it was found that the life-prolonged human keratinocytes, while maintaining the properties close to normal cells at a high level, are significantly extended in the number of passages as compared to normal cells. In addition, in view of the results such as differentiation ability and proliferation stability, the number of passages is more than 20 passages, especially 30 to 18 passages.
It was considered that those of about 0 generations were easy to use for various tests.

Embodiment 7 FIG. Detection of TGK activation using life-extending cells Subconfluent normal human keratinocytes (Kurabo) and the life-extending keratinocytes obtained in Example 6 were cultured in a high calcium concentration medium (1 mM CaCl ₂ ).
After culturing for 48 hours with the addition of retinoic acid and 3 μM, immunoplotting was carried out using the 1084 antibody in the same manner as in Example 2. Further, the amplification of a band of about 85 k expressed in association with the above-described differentiation induction treatment was quantified using an image analyzer.

FIG. 4 is a graph showing the results of detection of TGK activation modulation by immunoblotting using life-prolonging cells. In FIG, RKC3-3 is passaged permitted number 30s about survival keratinocytes, RKC3-2 is passaged permitted number 150 generations or more extended life keratinocytes, NONE untreated, CaCl ₂ is 1 mM CaCl ₂ treatment, RA Is 3
μM retinoic acid treatment, AVERAGE OD on the vertical axis is about 8
The results of quantifying the amount of amplification of the 5k band with an image analyzer are shown. As shown in FIG. 4, in the life-prolonged human keratinocytes whose number of possible passages is about 30 generations, the band is amplified with the differentiation induction treatment, and the degree is almost similar to that of normal cells (FIG. 1). Therefore, it was found that the life-extending keratinocytes with respect to the activation of TGK retained the properties of normal human keratinocytes at a high level.

In the case of keratinocytes having a prolonged life of 150 or more passages, the amount of band amplification accompanying the differentiation induction treatment was smaller than that of normal cells. This result suggests that TGK activation may not be as likely to occur in fully immortalized and cancerous cells as normal.
In order to examine the activation of TGK, the number of passages can be greatly extended compared to normal cells, and the culture operation is easy.
The usefulness of using keratinocytes having a life-extending property that retains properties close to that of normal cells was demonstrated.

Furthermore, when both normal cells (FIG. 1) and life-prolonging cells (FIG. 4) are treated with retinoic acid, which is a substance that suppresses TGK activity and suppresses keratinocyte differentiation, the cells of activated TGK Since almost no band was thought to be detected, it was found that the present antibody well detected modulation of TGK activation.

Example 8. Experimental Example of Three-Dimensional Culture Normal culture and 0.025 J / cm ² / day 3
A normal human three-dimensional model (EPI-606 :) irradiated with a 12 nm ultraviolet B wave over time (UV lamp manufactured by VILBERLOURMAT)
(Kurabo) was cultured for 4 days in a 5% CO ₂ incubator at 37 ° C. using a dedicated maintenance medium (EPI-100 maintenance medium: Kurabo), and the upper layer to the stratum corneum of the granular layer were collected without being fixed. After embedding with an OCT compound according to a conventional method, the sections were sliced and used as frozen sections. Frozen section is -2
After fixing in acetone at 0 ° C, blocking was performed with 10% normal horse serum, and a commercially available anti-transglutaminase (BC1 clone: HARBORBIO-PRODUCTS), R14
The 10H antibody and M911E antibody were reacted at room temperature for 2 hours. After washing with 0.1% BSA solution, furthermore, fluorescence (FITC)
A labeled anti-mouse immunoglobulin antibody (JACKSON) was reacted at room temperature for 1 hour. After washing with 0.1% BSA solution, the cells were sealed with non-fluorescent glycerin, observed with a fluorescence microscope (Olympus), and the images were taken using a CCD camera system (HC).
-2500: Fujifilm).

FIG. 5 is a photograph showing the result of detection of TGK modulation by ultraviolet irradiation using a normal human three-dimensional model.
Fig. a shows the reaction with a commercially available anti-TGase antibody without UV irradiation, Fig. b shows the reaction with a commercially available anti-TGase antibody without UV irradiation, and Fig. c shows the reaction with the R1410H antibody without UV irradiation. Figure d shows R1410 with UV irradiation
Reaction with H antibody, e shows M9 without UV irradiation
Reaction with 11E antibody, f figure shows M
The figure shows a reaction with the 911E antibody.

As shown in FIG. 5, in the normal human three-dimensional model, TGase expression was detected by the commercially available anti-TGase antibody in the epidermis and stratum corneum, and TGK was activated by the R1410H antibody and the M911E antibody. Were detected respectively. Furthermore, the commercially available anti-TGase antibody did not detect remarkable TGase modulation caused by ultraviolet irradiation, whereas the R1410H antibody and M911E antibody both detected that TGK activation was significantly suppressed by ultraviolet irradiation. did.

Embodiment 9 FIG. Diagnosis by human disease skin tissue staining method Human disease skin tissue, specimen a: 53-year-old male, face, senile bruise, and specimen b: 53-year-old female, anterior thorax, skin tumor were embedded with an OCT compound according to a conventional method. Thereafter, the slices were cut into frozen sections. The frozen sections were fixed in acetone at −20 ° C., and then blocked with 10% normal horse serum.
The 1410H antibody and the M911E antibody were reacted at 37 ° C. for 1 hour. Furthermore, a fluorescence (FITC) -labeled anti-mouse immunoglobulin antibody (JACKSON) was reacted at 37 ° C. for 1 hour, washed with 0.1% BSA solution, sealed with non-fluorescent glycerin, and observed with a fluorescence microscope (Olympus). went.

As a result, fluorescence was observed over a wide area in the epidermis in sample a using either the R1410H antibody or M911E antibody, but almost no fluorescence was observed in sample b using either antibody. This suggests that TGK activation occurs extensively in the skin of patients with senile bruises, whereas TGK is activated in the skin of patients with skin tumors.
It was found that almost no activation occurred.

[0077]

As described above, the present invention provides anti-active T
GK antibodies can be produced, and active TGK can be easily detected using various immunological techniques such as immunostaining. Furthermore, life-prolonged human keratinocytes were prepared by extending the number of passages while maintaining the properties closer to normal human keratinocytes than the cell lines, thereby facilitating culturing. Using these, analysis of the TGK activation mechanism, human skin Pharmaceutical materials effective for various skin diseases, etc., and cosmetic materials and foods that maintain healthy skin by establishing a model for disease and the like, and establishing a method for selecting a substance that improves skin dysfunction associated with TGK activation modulation There is an effect that the material can be sorted.

[0078]

[Sequence List] SEQUENCE LISTING <110> KABUSHIKI KAISHA YAKULT HONSHA <120> Anti-active keratinocyte transglutaminase antibody <130> PJ23710 <160> 3

<210> 1 <211> 15 <212> PRT <213> Artificial Sequence <400> 1 Gly Ser Gly Val Asn Ala Ala Gly Asp Gly Thr Ile Arg Glu Gly 1 5 10 15

<210> 2 <211> 12 <212> PRT <213> Artificial Sequence <400> 2 Gly Ser Ala Glu Asp Val Ala Met Gln Val Glu Ala 1 5 10 12

<210> 3 <211> 817 <212> PRT <213> Human <300> <301> MA Phillips et.al <302> Primary structure of keratinocyte transglutaminase <303> Proc. Natl. Acad. Sci. USA <304> 87 <306> 9333-9337 <307> 1990-12 <400> 3 Met Met Asp Gly Pro Arg Ser Asp Val Gly Arg Trp Gly Gly Asn Pro 1 5 10 15 Leu Gln Pro Pro Thr Thr Pro Ser Pro Glu Pro Glu Pro Glu Pro Asp 20 25 30 Gly Arg Ser Arg Arg Gly Gly Gly Arg Ser Phe Trp Ala Arg Cys Cys 35 40 45 Gly Cys Cys Ser Cys Arg Asn Ala Ala Asp Asp Asp Trp Gly Pro Glu 50 55 60 Pro Ser Asp Ser Arg Gly Arg Gly Ser Ser Ser Gly Thr Arg Arg Pro 65 70 75 80 Gly Ser Arg Gly Ser Asp Ser Arg Arg Pro Val Ser Arg Gly Ser Gly 85 90 95 Val Asn Ala Ala Gly Asp Gly Thr Ile Arg Glu Gly Met Leu Val Val 100 105 110 Asn Gly Val Asp Leu Leu Ser Ser Arg Ser Asp Gln Asn Arg Arg Glu 115 120 125 His His Thr Asp Glu Tyr Glu Tyr Asp Glu Leu Ile Val Arg Arg Gly 130 135 140 Gln Pro Phe His Met Leu Leu Leu Leu Ser Arg Thr Tyr Glu Ser Ser 145 150 155 160 Asp Arg Ile Thr Leu G lu Leu Leu Ile Gly Asn Asn Pro Glu Val Gly 165 170 175 Lys Gly Thr His Val Ile Ile Pro Val Gly Lys Gly Gly Ser Gly Gly 180 185 190 Trp Lys Ala Gln Val Val Lys Ala Ser Gly Gln Asn Leu Asn Leu Arg 195 200 205 Val His Thr Ser Pro Asn Ala Ile Ile Gly Lys Phe Gln Phe Thr Val 210 215 220 Arg Thr Gln Ser Asp Ala Gly Glu Phe Gln Leu Pro Phe Asp Pro Arg 225 230 235 240 Asn Glu Ile Tyr Ile Leu Phe Asn Pro Trp Cys Pro Glu Asp Ile Val 245 250 255 Tyr Val Asp His Glu Asp Trp Arg Gln Glu Tyr Val Leu Asn Glu Ser 260 265 270 Gly Arg Ile Tyr Tyr Gly Thr Glu Ala Gln Ile Gly Glu Arg Thr Trp 275 280 285 Asn Tyr Gly Gln Phe Asp His Gly Val Leu Asp Ala Cys Leu Tyr Ile 290 295 300 Leu Asp Arg Arg Gly Met Pro Tyr Gly Gly Arg Gly Asp Pro Val Asn 305 310 315 320 Val Ser Arg Val Ile Ser Ala Met Val Asn Ser Leu Asp Asp Asn Gly 325 330 335 Val Leu Ile Gly Asn Trp Ser Gly Asp Tyr Ser Arg Gly Thr Asn Pro 340 345 350 Ser Ala Trp Val Gly Ser Val Glu Ile Leu Leu Ser Tyr Leu Arg Thr 355 360 365 Gly Tyr Ser Val Pro TyrGly Gln Cys Trp Val Phe Ala Gly Val Thr 370 375 380 Thr Thr Val Leu Arg Cys Leu Gly Leu Ala Thr Arg Thr Val Thr Asn 385 390 395 400 400 Phe Asn Ser Ala His Asp Thr Asp Thr Ser Leu Thr Met Asp Ile Tyr 405 410 415 Phe Asp Glu Asn Met Lys Pro Leu Glu His Leu Asn His Asp Ser Val 420 425 430 Trp Asn Phe His Val Trp Asn Asp Cys Trp Met Lys Arg Pro Asp Leu 435 440 445 Pro Ser Gly Phe Asp Gly Trp Gln Val Val Asp Ala Thr Pro Gln Glu 450 455 460 Thr Ser Ser Gly Ile Phe Cys Cys Gly Pro Cys Ser Val Glu Ser Ile 465 470 475 480 Lys Asn Gly Leu Val Tyr Met Lys Tyr Asp Thr Pro Phe Ile Phe Ala 485 490 490 495 Glu Val Asn Ser Asp Lys Val Tyr Trp Gln Arg Gln Asp Asp Gly Ser 500 505 510 Phe Lys Ile Val Tyr Val Glu Glu Lys Ala Ile Gly Thr Leu Ile Val 515 520 525 Thr Lys Ala Ile Ser Ser Asn Met Arg Glu Asp Ile Thr Tyr Leu Tyr 530 535 540 Lys His Pro Glu Gly Ser Asp Ala Glu Arg Lys Ala Val Glu Thr Ala 545 550 555 560 Ala Ala His Gly Ser Lys Pro Asn Val Tyr Ala Asn Arg Gly Ser Ala 565 570 570 575 Glu Asp Val Ala Met GlnVal Glu Ala Gln Asp Ala Val Met Gly Gln 580 585 590 Asp Leu Met Val Ser Val Met Leu Ile Asn His Ser Ser Ser Arg Arg 595 600 605 Thr Val Lys Leu His Leu Tyr Leu Ser Val Thr Phe Tyr Thr Gly Val 610 615 620 Ser Gly Thr Ile Phe Lys Glu Thr Lys Lys Glu Val Glu Leu Ala Pro 625 630 635 640 Gly Ala Ser Asp Arg Val Thr Met Pro Val Ala Tyr Lys Glu Tyr Arg 645 650 655 655 Pro His Leu Val Asp Gln Gly Ala Met Leu Leu Asn Val Ser Gly His 660 665 670 Val Lys Glu Ser Gly Gln Val Leu Ala Lys Gln His Thr Phe Arg Leu 675 680 685 Arg Thr Pro Asp Leu Ser Leu Thr Leu Leu Gly Ala Ala Val Val Gly 690 695 700 Gln Glu Cys Glu Val Gln Ile Val Phe Lys Asn Pro Leu Pro Val Thr 705 710 715 715 720 Leu Thr Asn Val Val Phe Arg Leu Glu Gly Ser Gly Leu Gln Arg Pro 725 730 735 Lys Ile Leu Asn Val Gly Asp Ile Gly Gly Asn Glu Thr Val Thr Leu 740 745 750 Arg Gln Ser Phe Val Pro Val Arg Pro Gly Pro Arg Gln Leu Ile Ala 755 760 765 Ser Leu Asp Ser Pro Gln Leu Ser Gln Val His Gly Val Ile Gln Val 770 775 775 780 Asp Val Ala Pro Ala Pro Gly Asp Gly Gly Phe Phe Ser Asp Ala Gly 785 790 795 800 Gly Asp Ser His Leu Gly Glu Thr Ile Pro Met Ala Ser Arg Gly Gly 805 810 815 Ala 817

[Brief description of the drawings]

FIG. 1 is a graph showing the results of detection of TGK activation modulation by immunoblot using the 1084 antibody.

FIG. 2 is a diagram showing the reactivity of each antibody to an antigen.

FIG. 3 is a diagram showing the results of detecting cells induced to differentiate by cell staining.

FIG. 4. T by immunoblot method using life-prolonging cells
It is a graph which shows the detection result of GK activation modulation.

FIG. 5 is a photograph showing a result of detecting TGK modulation by ultraviolet irradiation using a normal human three-dimensional model.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/573 C12N 5/00 B // C12P 21/08 15/00 ZNAC (72) Inventor Makoto Owaki Tokyo 1-19, Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Shinobu Oumi 5-8-38 Kamisagimiya, Nakano-ku, Tokyo F-term (reference) 4B024 AA11 BA53 CA02 DA03 GA03 HA15 4B063 QA01 QA05 QA19 QQ08 QQ26 QR48 QR51 QR77 QR80 QS11 QS24 QS33 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA92X AA93X AA95Y AB05 CA20 CA46 4H045 AA10 CA40 DA75 DA76 EA50 FA72 GA26

Claims (8)

[Claims] 1. An antibody that specifically recognizes keratinocyte transglutaminase, wherein the antibody has an affinity for active keratinocyte transglutaminase higher than that for a keratinocyte transglutaminase inactive precursor. Anti-active keratinocyte transglutaminase antibody. 2. The anti-active keratinocyte transglutaminase antibody according to claim 1, which specifically recognizes a limited cleavage site accompanying activation of the keratinocyte transglutaminase inactive precursor. 3. The method according to claim 1, which specifically recognizes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a peptide having a sequence complementary to the amino acid sequence. Anti-active keratinocyte transglutaminase antibody. 4. The anti-active keratinocyte transglutaminase antibody according to claim 1, which is a monoclonal antibody. 5. A life-extended human keratinocyte mixture comprising a life-extending human keratinocyte cell which has been extended in the number of passages by subjecting a plurality of normal human keratinocytes to life-extension treatment. 6. The life-extended mixture of human keratinocytes according to claim 5, which has been transformed with the SV40 virus. 7. A method for selecting a substance for improving skin function using cultured human keratinocytes, which comprises the following steps (a) and (b): (a) in the presence or absence of a test substance, causing a change in keratinocyte proliferation, differentiation or activation of keratinocyte transglutaminase, (b) in the presence or absence of the test substance in step (a) Detecting the activity of keratinocyte transglutaminase with the anti-active keratinocyte transglutaminase antibody according to claims 1 to 4, and comparing the activities of the two. 8. A method for detecting a skin lesion, comprising the following steps (a) and (b). (a) reacting the antibody of claims 1 to 4 with a test skin section and visualizing it; (b) analyzing the result visualized in step (a);