JPH0683676B2 - Anti-human-tenascin monoclonal antibody - Google Patents

Description

TECHNICAL FIELD The present invention relates to a monoclonal antibody against human-tenascin produced by human fibroblast.

(Prior Art) Tenascin is a glycoprotein discovered as an antigen of an antibody that shows a unique staining property different from type V collagen by chance when a monoclonal antibody against type V collagen is obtained. It is a substance expressed on the interstitial side. Tenascin is one of the extracellular matrix components,
It is becoming clear that it has various physiological effects.
That is, since tenascin has a stronger hemagglutination ability than fibronectin, in the field of tissue formation,
It has been suggested that it acts in a controlled manner (Oike et al., Biological Science, Vol. 39-August, 1988). As a non-covalent interaction with other molecules, the interaction with fibronectin has been clarified (Ticket et al., Cell, 53.
Vol.:383-390, 1988), and interaction with proteoglycans has also been suggested (Ticket et al., Journal of Cell Biology, 98: 1926-1936, 1984).
Year). Furthermore, it has a calcium-dependent cell adhesion activity (Bourdon et al., Journal of Cell Biology, 105: 138a), and is induced on the mesenchymal side with the development of epithelial cancer, Have been shown to have a growth promoting effect on (Ticket et al., Cell, 47: 131.
-139, 1986), it has various physiological actions and plays an important role in cell proliferation and morphogenesis.

The present inventor has previously developed a method for purifying highly pure human-tenascin from human-fibroblast using a pure biochemical technique. This method makes it possible to obtain extremely high-purity human-tenascin in a high yield,
Various experiments using human-tenascin became possible.

On the other hand, the cell fusion technology is a hybridoma obtained by fusing mammalian spleen cells and mouse myeloma cells with Köhler and Milstein (Nature: pp. 495-497, 1975). Since the production of a recognizing monoclonal antibody has been reported, attempts have been made to produce a hybridoma and a monoclonal antibody which produce a monoclonal antibody against a biological substance such as various proteins.

If high-purity human-tenascin can be used to obtain a monoclonal antibody that specifically reacts with human-tenascin, it is useful because it enables diagnosis of epithelial cancer and the like.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to produce a hybridoma cell that produces a monoclonal antibody that specifically reacts with human-tenascin, using high-purity human-tenascin that has not been obtained hitherto. And to supply the monoclonal antibody in a large amount.

(Means for Solving the Problems) The present inventor obtained by extracting human-tenascin from human fibroblast, removing fibronectin, and further purifying using anion exchange resin for biopolymer. When cell fusion was performed using rat spleen cells immunized with high-purity human-tenascin, a hybridoma producing a human-tenascin-specific monoclonal antibody was obtained, and it was found that the above problems can be solved, and the present invention is completed. Came to.

That is, the present invention provides a monoclonal antibody specific for human-tenascin and a hybridoma producing a monoclonal antibody specific for human-tenascin.

Hereinafter, the present invention will be described in detail. As a source of human-tenascin used in the method of purifying tenascin, human melanoma cells, human glioma cells, and the like can be used in addition to human fibroblast cells.

When human fibroblast cells or the like are used, a protein mixture containing tenascin may be extracted after the cells have reached confluence, using fetal bovine serum or the like as a culture medium.

From these cells, a cell surface extract can be extracted by a conventional method. For extraction, urea, guanidine hydrochloride,
Although guanidine isothiocyanate or the like can be used, generally, urea can be used at a concentration of 1M to 8M, preferably 2M, and guanidine hydrochloride and guanidine isothiocyanate can be used at a concentration of 3M or more. it can. Further, other additives such as phenylmethylsulfonium fluoride (PMSF) can be added and used.

When using human fibroblast cells, 2M urea / phosphate buffer saline (PB
S) / 2 mM-PMSF may be used for extraction.

Thereafter, a protein mixture containing tenascin can be precipitated from the extract using ammonium sulfate or the like. Ammonium sulfate may be usually used at 60-80%, and a protein mixture containing tenascin, collagen, proteoglycan, fibronectin and other unknown proteins can be precipitated by treating at 4 ° C. for about 2 hours.

It is necessary to remove fibronectin, as this protein mixture usually contains about 300 μg / ml, about 50 times more fibronectin than tenascin. The precipitated protein mixture is added to a solution containing 2M urea, preferably 4 ° C.
It is preferable to dissolve it in 2 M urea / 0.15 M sodium chloride / 50 mM Tris-hydrochloric acid buffer (pH 8.0) and then roughly purify by gel filtration.

As a filler used for gel filtration, Sepharose CL2B, CL4B, CL6B (manufactured by Pharmacia) and the like which are resins for gel filtration can be used, and Sepharose-CL4B is particularly preferable. In addition, Bio-gel
5m, 15m (manufactured by Bio-Rad) and Sephacryl (Sep
Hacryl) S500 and S1000 (Pharmacia) can also be used.

Tenascin eluting from the column can be detected by the tenascin-dependent cell adhesion activity (Methods in Enzymology, 82: 803-831, 1982).
Examples of cells that can be used in the cell adhesion test include human melanoma, human fibroblasts, human epithelial cancer cells, and the like. As another identification method, an enzyme-linked immunoassay (ELIS) using a polyclonal antibody against chicken tenascin is used.
A) method etc. can also be used. Fibronectin can be detected by measuring its antigenicity to fibronectin (Archives of Biochemistry and Biophysics: 399-406, 1981).
Year).

The crude purified tenascin can be obtained by collecting the fractions containing 20%, preferably 10% or more of tenascin. Since the crude purified tenascin thus obtained usually contains fibronectin at 50%, it is necessary to further remove fibronectin by gelatin gel after concentrating and dialysis by a usual operation.

For the concentration operation, for example, Centriflow membrane (manufactured by Amicon)
It is preferable to centrifuge and concentrate at 2500 × g at 4 ° C. using dialysis, and for dialysis, a buffer containing 0.5 M urea, preferably 0.1%.
5M Urea / 0.15M Sodium Chloride / 50mM Tris Hydrochloric Acid (pH7.4)
It may be carried out at 4 ° C. for 12 hours.

Examples of the gelatin gel include gelatin sepharose (manufactured by Pharmacia), immobilized gelatin (manufactured by Pierce), gelatin agarose (manufactured by Sigma), etc. These are affinity chromatography in which gelatin is covalently bonded. . By using the gelatin column having a high affinity for fibronectin, tenascin is eluted in the flow-through fraction, and fibronectin can be removed to 1% by weight or less based on the solute. Fibronectin can be identified by an ELISA method using an anti-human fibronectin polyclonal antibody, and tenascin can be similarly identified by an ELISA method.

The fraction thus obtained was concentrated by a conventional operation and dialyzed against a solution containing 2M urea, preferably 2M urea / 50mM Tris-HCl (pH8.0) to contain tenascin at a high concentration. A solution can be obtained.

The solution usually contains 100 μg / ml of tenascin, but an extremely high-purity tenascin can be obtained by further purifying using anion-type ion exchange resin for biopolymer separation.

Examples of the method include a method of purifying the solution by ion exchange high performance liquid chromatography using a DEAE-5PW column (manufactured by Tosoh Corporation) having a DEAE (diethylaminoethyl) group as an exchange group. Furthermore, DEAE-Toyopearl 650 (Tosoh Corporation)
Made), Mono-Q (made by Pharmacia), DEAE-CL6B, DE
Columns such as AE-Sephacel (Pharmacia) and DEAE-Cellulofine (Seikagaku Corporation) can also be used. Of these, DEAE-5PW is preferably used.

The solvent used for elution is 1 to 8 M urea, preferably
Sodium chloride of 0 to 1M, preferably 0 to 0.4M in 2M urea may be used.

In ion exchange high performance liquid chromatography using a DEAE-5PW column, when elution is performed with a linear concentration gradient of 0-0.4M sodium chloride in 2M urea, it is usually 0.2
Tenascin is eluted with -0.3M, preferably about 0.25M sodium chloride. The eluted tenascin can be identified by ultraviolet absorption and cell adhesion specific to tenascin.

By such a purification method, tenascin having a purity of almost 100% can be obtained. Human-tenascin is SDS-PAGE
Analysis revealed that the bands characteristic of human-tenascin were clearly defined at 250K and 200K. The yield of tenascin by the above purification method is usually 80% or more.

Furthermore, a crude tenascin fraction can be obtained by using 30-35%, preferably 33.3% ammonium sulfate from the culture medium, and purified tenascin can be obtained by the same method as the above operation.

A hybridoma producing a monoclonal antibody specific to human-tenascin thus purified is produced, for example, as follows.

To purify antibody-producing cells from an animal immunized with human-tenascin as an antigen, it suffices to carry out according to a conventional method. Immunize an animal with human-tenascin as an antigen and obtain antibody-producing cells of the animal. The method to do is good. Examples of animals that can be used include mice, rats, rabbits, guinea pigs, sheep and the like. In the case of rats among these animals, Wistar rats can be used, for example. As the antibody-producing cells, cells isolated from spleen, lymph nodes, peripheral blood and the like can be used, but spleen cells are preferably used. To immunize the animal, Freund's complete adjuvant can be used in combination. For example, Freund's complete adjuvant can be mixed with the purified human-tenascin. When immunized with Wistar rats, human-tenascin 20 μg (protein amount)
The desired immunized animal can be obtained by immunizing 3 to 4 times.

As myeloma cells that can be used for cell fusion, cells derived from various mammals can be used. In this case, myeloma cells derived from an animal of the same species as the animal used to prepare the antibody-producing cells can also be used. Since these cells cannot survive in the particular selective medium, unfused myeloma cells can be removed by a culturing procedure after cell fusion. For example, 8-azaguanine-resistant cells have a property that they cannot grow in HAT medium (hypoxanthine-aminopterin-thymidine medium), and therefore hybridoma cells can be separated from unfused myeloma cells. When using mouse myeloma cells, for example, PAI, P3X63Ag
8, P3X63Ag8U1, P3NSI / 1Ag41, X63Ag8,653, SP2 / 0Ag1
4, FO, S194 / 5XXOBU1, MPC11456TG17, etc. can be used.

Cell fusion is usually performed by mixing the above-mentioned myeloma cells with antibody-producing cells using a medium such as MEM medium or IMEM medium. The myeloma cells and the antibody-producing cells can be fused by mixing them at a ratio of 1:10 or less, preferably 1: 5, and preferably using polyethylene glycol as a fusion promoter. Polyethylene glycol having an average degree of polymerization of 1,000 to 6,000 can be used, and usually 30 to 50% can be used. When X63Ag8,653 is used as mouse myeloma cells, polyethylene glycol having an average degree of polymerization of 3,800 is set to 42.8% and fused with human-tenascin antibody-producing rat spleen cells at 37 ° C. for 1 to 3 minutes to obtain the desired mixture. Fused cells can be obtained. Furthermore, other methods known as cell fusion methods,
For example, an electrofusion method, a method of using biotin-avidin crosslinking and electric pulse in combination, or the like can be used.

The cells after cell fusion can be obtained by diluting with an appropriate medium and then centrifuging. After that, only the fused cells can be selectively cultured using the HAT medium. HAT
It is also possible to use a medium such as HIAT medium in which insulin is added to the medium. Hybridoma cells formed by fusing human-tenascin antibody-producing cells with mouse myeloma cells have HA
The T medium can be used to selectively culture at 37 ° C. under 5% carbon dioxide. As an example of a hybridoma that produces a monoclonal antibody specific for human-tenascin, HTN2-9B2 (Microtechnology Research Institute No. 10547, FERMP-105
47) can be mentioned.

(Effect of the invention) According to the present invention, a hybridoma cell producing a monoclonal antibody against human-tenascin can be obtained, and a monoclonal antibody against human-tenascin can be obtained at a high yield.

Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to this embodiment.

(Example) Human fibroblast cells were prepared by using Dulbecco's modified Eagle medium / Ham's F12 medium and 10% fetal bovine serum as culture medium.
Using 100 roller bottles (850 cm ² ), 37 ℃, 110 ml /
The cells were continuously cultured at 1.1 rpm, and the medium was replaced about day 5 after the cells reached confluence. This medium 100 was cooled to 4 ° C, ammonium sulfate was added to 33% saturation, the mixture was gently stirred, and then left at 4 ° C for 120 minutes,
A protein mixture containing human-tenascin, collagen, proteoglycan, fibronectin, and other proteins was precipitated.

The protein mixture was separated by centrifugation at 20,000 xg for 60 minutes at 4 ° C, the supernatant was decanted and the precipitate was washed with 4
The solution was dissolved in 750 ml of 2 M urea / 0.15 M sodium chloride / 50 mM Tris-hydrochloric acid (pH 8.0) at ℃. The amount of protein was 3.9 g by BCA protein assay (manufactured by Pierce).

25 ml of the protein mixture solution was applied to a Sepharose CL6B column (Pharmacia, about 500 ml), and 2 M urea /
Tenascin and fibronectin were separated and eluted at 15 ° C using 0.15M sodium chloride / 50mM Tris-HCl (pH8.0).

The tenascin-dependent cell adhesion activity was according to the method described below. Put 200μ sample from each screen into wells of ELISA plate (Libro, 76-203-05, Titertek), and add 20μ of 50mM sodium carbonate buffer (pH 9.6) to each well at 4 ° C for 12 hours. I coat for hours. Thereafter, the wells were washed 3 times with PBS / 0.5% -BSA, and then an anti-fibronectin antibody (Cappel, PBS / 0.5% -5% in BSA) 20
Add 0 μm and incubate at 37 ℃ for 2 hours.
Washed with BS / 0.5% -BSA. On the other hand, human fibroblast cells were maintained in Dulbecco's modified Eagle medium / Ham's F12 medium and 10% fetal bovine serum until they became confluent.
Trypsin without DTA (Type III, Sigma, 0.
1mg / ml) solution, keep the cells at 37 ℃ for 1 hour and peel off the cells.
A cell solution was prepared at × 10 ⁶ cells / ml (PBS / soybean trypsin inhibitor 0.5 mg, Sigma). This cell solution was added to the wells of a microtiter plate by 100 μm and incubated at 37 ° C. for 1 hour. Then plate the PBS /
0.5% -Wash once with BSA to remove 3% of cells attached to the plate.
Was fixed with 1% -toluidine blue (manufactured by Merck) / 3% -paraformaldehyde, and the number of adhered cells was observed under a microscope.

Fibronectin was detected using the following method by measuring the antigenicity to fibronectin. Take a 20μ sample from each fraction and use an ELISA plate (3912
Falcon) well, add 180 mM of 50 mM sodium carbonate buffer (pH 9.6) to each well, and add 12 at 4 ℃
I kept it warm for an hour. After washing each well three times with PBS / 0.5% (W / W) -Tween 20 / 0.05% (W / W) -BSA, rabbit anti-fibronectin antibody (0.4%, Cappel) 2
00 μm was added and the mixture was kept at 20 ° C. for 1 hour. Furthermore, the antibody solution is drained and PBS / 0.5% (W / W) -Tween 20 / 0.05% (W
Each well was washed with / W) -BSA, and a peroxidase-labeled goat anti-rabbit antibody (0.4%, manufactured by Cappel) was added and the reaction was continued for 1 hour. PBS / 0.5% (W / W) -Tween 20 / 0.05% (W / W) -Wash each well with BSA, and
The peroxidase activity was measured by absorption at 492 nm using phenylenediamine hydrochloride as a substrate.

The above operation was repeated 30 times. The elution patterns of both are shown in FIG. As is clear from the figure, tenascin and fibronectin were slightly separated by the Sepharose column, and tenascin was eluted at the Kd value of 0. By collecting fractions No. 25 to No. 30 (each fraction is 5 ml), 900 ml of a solution containing tenascin at a high concentration was obtained. The protein yield was 90.0 mg.

Using a Centriflow membrane (manufactured by Amicon), the solution is
After centrifuging at 2500 xg at 4 ℃ and concentrating to 100 ml, 0.5M urea / 0.15M sodium chloride / 50mM Tris-HCl (pH7.4) 2
Against it, it dialyzed at 4 degreeC for 12 hours.

120 ml of the solution was applied to a gelatin column (gelatin sepharose, manufactured by Pharmacia, 30 ml) to specifically adsorb fibronectin. Tenascin was eluted in the flow-through fraction, and 220 ml of a tenascin solution containing no fibronectin could be obtained.

The solution was mixed with a Centriflow membrane (Amicon) 4
After centrifuging at 2500 xg at 40 ° C and concentrating to 40 ml, the mixture was dialyzed with 2M urea / 50 mM Tris-HCl (pH 8.0) of 2 at 4 ° C for 12 hours.

After dialysis, the solution was purified by high performance liquid chromatography using a column of DEAE-5PW (manufactured by Tosoh Corporation, column diameter 7.5 mm, column length 50 mm). After injecting 1 ml of the solution into the column, a linear gradient solution of 0-0.4 M sodium chloride in 2 M urea was used to separate at a flow rate of 0.5 ml / min, a column pressure of 15 kg / cm ² , and a column temperature of 15 ° C. The target tenascin was eluted with 2M urea / 0.25M sodium chloride at 42-45 minutes. Tenascin was detected by absorption at 280 nm and cell adhesion activity specific to tenascin. The results are shown in FIGS. 2 and 3. As is clear from the figure, tenascin was separated as a sharp single peak.

100 roller bottles (approximately 200 x 10 ⁸ cells), 100 culture media
It was possible to obtain a total yield of 6.6 mg of human-tenascin.

5 μg of the obtained human-tenascin was reduced with 1% -2-mercaptoethanol / 2% -SDS / 50 mM Tris-HCl (pH 6.8) at 100 ° C. for 3 minutes, and then 4% -20% SDS- PA
It was analyzed by GE. 250K and 200K characteristic of human-tenascin
The band could be detected. No other bands were detected, and the purity of the obtained human-tenascin was almost 100%.
It was confirmed that

20 μg (100 μ) of the above-mentioned human-tenascin was added with 100 μ of Freund's complete adjuvant (manufactured by Difco) and well stirred, and when it became an emulsion, Wistar rat (female, 4 weeks old, 2) lymph nodes At 10
Each injection was 0 μm. Thereafter, every two weeks, a similarly prepared human-tenascin-emulsion was injected to immunize a total of four times.

The final antibody titer against human-tenascin was measured by the following ELISA method. ELISA to 0.1 μg / well
A plate (3912, manufactured by Falcon) was coated, and a dilution series of the primary antibody shown in FIG. 4 was prepared and reacted, and the biotinylated secondary antibody (1: 250, rabbit-anti-rat IgG,
After reacting with Capel) for 1 hour, the plate was washed as described above, further reacted with peroxidase-avidin (1: 250, Capel) for 1 hour, and the plate was washed 3 times, and then each well was washed. Color was developed with 2% -ortho-phenylenediamine (manufactured by Sigma) /0.001%-H ₂ O ₂ per 150 μm. To each well, 50 µ of 2N-H ₂ SO ₄ was added to stop the reaction, and absorption at 492 nm was measured (MTP100, manufactured by Corona) to measure the antibody titer.

The spleen of the rat is removed, a hole is made with an injection needle, and lymphocytes are removed.
Prepare the cells in RPMI 1640 medium and repeat the cells three times (1200 × g, 8
Washed). Cell yield is 2.5 x 10 ⁸ per rat
Met.

On the other hand, X63Ag8,653 myeloma cells, which are parental cells, contain RPMI 1640/10% containing 10 mg / 8-azaguanine.
-Maintaining in FCS for 1 week, 5 x 10 ⁷ cells were treated with RPMI 16
It was washed 3 times (1200 × g, 8 minutes) in 40 medium.

The lymphocytes and X63Ag8,653 myeloma cells were mixed and RPMI
It was washed once with 1640 medium. The cell fusion operation was performed as follows. 500 μ of 42.8% -polyethylene glycol (molecular weight 3800, manufactured by Sigma) / 15% -dimethyl sulfoxide (manufactured by Sigma) / PBS was gently added to the mixture of cells at 37 ° C. for 90 seconds, and then for another 60 seconds. I let it stand. The reaction was stopped by dropping RPMI 1640/10% -FCS (37 ° C) drop by drop over 1 minute (1 ml in total), and adding the remaining 24 ml in the following 2 minutes.

RPMI 1640/10% -FCS / HAT was added to the reaction product that had been precipitated by centrifugation at 500 xg for 5 minutes, and a 96-well plate was prepared.
Using 30 sheets (per animal), 100 µ was inoculated per well. One week later, 100μ RPMI 1640/10% -FCS /
HAT was added to each well. Then, on the third day, the seventh day, the fourteenth day, and the twenty-first day, 100 μm was sampled from each well, and it was determined by ELISA whether or not the hybridoma cells were producing antibodies against human-tenascin. . Approximately 150 of all holes in the plate in the first fusion
In the second and third fusions, about 500 human-tenascin-positive hybridomas were similarly obtained.

Among the hybridomas, 14 clones having a good antibody-producing ability were selected, inoculated into a 96-well plate so as to have 1 cell / well, and cloned in HAT medium.
When the colonies of hybridoma cells grew,
ISA was performed to select two clones producing the antibody per original clone, and the cells were further inoculated into a 96-well plate at 1 cell / well, and cloned in HAT medium. As described above, antibody-producing strains were fixed by ELISA, and stable hybridoma cells (14 clones) were RPMI.
Transferred to bulk culture in 1640 medium / 10% -FCS. Some of the clones were transplanted to nude mice to prepare ascites antibody. Table 1 shows the results of determining the subclass of the antibody produced by the clone using the rat-immunoglobulin-subtype-typing kit (manufactured by Zymet). The hybridoma cell producing the monoclonal antibody 9B2 is HTN2-9B2, which has been deposited as Microindustrial Research Institute No. 10547 and can be obtained by any person.

Immunostaining for human tumors was performed by the following method.

Paraffin-embedded sections of various formalin-fixed tissues of the body pathologically dissected as specimens were used for VECTASTAIN ABC K
A rat IgG kit from IT (Vector) was used for staining with an enzyme antibody method using peroxidase, and DAB was used for color development using the avidin-biotin binding method as a color development method.
PBS was used as the buffer solution.

To suppress the activity of endogenous enzymes such as peroxidase,
It was immersed in a methanol-0.6% H ₂ O ₂ solution for 30 minutes, and then washed with PBS for 5 minutes three times. Moreover, in order to block the tissue that electrically binds nonspecifically with the antibody, the rabbit primary serum in the VECTASTAIN ABC KIT was used to block the primary antibody prepared in the rat. It was allowed to work for 30 minutes at room temperature in an incubator, and the next step was performed without washing in order to maintain the effect.

The cells were immersed in an anti-human-tenascin antibody (hybridoma cell culture medium) adjusted to 100 times with PBS at 5.5 ° C. overnight to act. Thereafter, the plate was washed with PBS three times for 5 minutes. Further, the biotinylated anti-rat antibody in VECTASTAIN ABC KIT was allowed to act for 30 minutes in a room temperature incubator, and then washed with PBS 3 times for 5 minutes. In addition, VECTASTAIN AB
After allowing ABC in C KIT to act in a room temperature incubator for 30 minutes, the cells were washed with PBS three times for 5 minutes each.

A solution of DAB 5 mg / PBS 10 ml / 3% H ₂ O ₂ 25 μ was allowed to act for 4 minutes in a room temperature incubator, and then immediately washed with PBS for 5 minutes. After being immersed in a hematoxylin solution for several tens of seconds for counterstaining, it was washed with running water for 10 minutes, dehydrated and mounted.

The monoclonal antibodies 8C9, 9B2, 13F6, 20D6 of the present invention stained only the mesenchyme of cancer in a tenascin-specific manner.

Western blotting was evaluated as follows.

100 μg / ml gelatin passage crude tenascin fraction (75 μ)
25μ 8% w / v SDS / 0.2M Tris-HCl (pH6.8) / 4%
A sample was prepared by adding v / v2-mercaptoethanol / 50% v / v glycerol and heating at 70 ° C for 30 minutes.

SDS-PAGE used a 4% -20% gradient gel plate (Daiichi Pure Chemicals). Electrophoresis of 25μ sample (15mA
4 hours) (Nature, 227: 680-685, 1970)
Year).

Western blotting is performed by the method of Towbin et al. (Proc. Natl. Acad Sci. USA, 76: 4350-4354).
P., 1979). When transferring, a nitroplus membrane (Micromembrane) was used instead of the nitrocellulose membrane. To detect the antigenic polypeptide after transfer, wash the membrane with 1% BSA / 50 mM Tris-HCl (pH7.4) /0.15M NaCl and then react with the primary antibody (monoclonal antibody) according to the ELISA method. Let
In addition, a secondary antibody (peroxidase-labeled anti-rat
Rabbit antibody, 1: 250). Staining was carried out with 4-chloro-1-naphthol (1 mg / ml) /0.1% H ₂ O ₂ , and the reaction was stopped by washing the membrane with water. Although the monoclonal antibodies 8C9 and 9B2 of the present invention showed high staining properties, 13F6
And 20D6 did not show stainability.

[Brief description of drawings]

FIG. 1 is a pattern in which human-tenascin obtained from a culture solution of human fibroblast cells was separated and eluted on a Sepharose CL-4B column. FIGS. 2 and 3 show DEAE of fractions passed through a gelatin column, respectively. 3 is a UV absorption pattern and a cell adhesion pattern of high performance liquid chromatography when separated using -5PW. FIG. 4 shows the antibody titer of rat anti-human-tenascin serum before cell fusion,
Fig. 5 shows the results of measurement by the ELISA method, and Fig. 5 shows the results obtained by analyzing the eluate fraction obtained by subjecting the crude tenascin fraction passing through gelatin sepharose to DEAE-5PW by the ELISA method by the antigenicity to rat anti-human tenascin serum. In FIG. 4, the horizontal axis shows the dilution ratio.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location G01N 33/53 V 8310-2J 33/574 A 8310-2J 33/577 B 8310-2J (C12P 21 / 08 C12R 1:91) Microorganism consignment number FERM P-10547 Pending pending examination (72) Inventor Teruyoshi Sakakura 3-1-1 Takanodai, Tsukuba-shi, Ibaraki Life Science Tsukuba Research Center (72) Inventor Yasuteru Oike 3-1-1, Takanodai, Tsukuba-shi, Ibaraki Life Science Tsukuba Research Center, Research Center for Science and Science (72) Hideki Hiraiwa 3-1-1, Takanodai, Tsukuba-shi, Ibaraki Institute of Life Sciences, Tsukuba Research Center (72) Inventor Kazutoshi Kawakatsu 3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture (56) Reference Journal of Call Biology, 105 (4part2), 138a (1987) Biological Science, 39 (4), 303-305 (1988) Cell, 53 (3) ), 383-390 (1988)

Claims (2)

[Claims] 1. A monoclonal antibody that reacts with human-tenascin in tissue embedded in paraffin-fixed paraffin. 2. A monoclonal antibody which is obtained by cell fusion of a mammal-derived antibody-producing cell and a mammal-derived myeloma cell and reacts with human-tenascin in a tissue embedded with paraffin-fixed paraffin. A hybridoma that produces.