WO1994000591A1

WO1994000591A1 - Monoclonal antibody, production process, antibody-producing cell, and diagnostic method

Info

Publication number: WO1994000591A1
Application number: PCT/JP1993/000854
Authority: WO
Inventors: Hisaaki Kawakatsu; Yumiko Takagaki; Junichi Yano
Original assignee: Nippon Shinyaku Co., Ltd.
Priority date: 1992-06-26
Filing date: 1993-06-24
Publication date: 1994-01-06

Abstract

A monoclonal antibody which specifically recognizes the site for controlling phosphoenzyme activity present at the C-terminus of an src family gene product acting as a carcinogenic gene precursor, said antibody being obtained by using a specified peptide present at the C-terminus of c-src as the antigen; a cell producing said antibody; a process for producing said antibody; and a method of pathologically diagnosing cancer or precancer with said antibody.

Description

Specification

Monoclonal antibodies, manufacturing methods, antibody-producing cells, and diagnostic techniques

The present invention relates to a monoclonal antibody specifically recognizing a site present at the C-terminus of a src family gene product and having a function of controlling the activity of a phosphatase.

Background technology

The src gene was first known as an oncogene of Rous sarcoma virus (v-src). The corresponding oncogene precursor in cells of higher organisms was called the c-src gene. The src gene is located at human chromosome locus 20ql3.3. The src gene has an analog called the src family. For example, the yes gene is located at human chromosome 18q21.3, the fgr gene is located at 1P36.1, and the fyn gene is located at 6q21.

At present, about ten types of tyrosine kinase belonging to src family are known, and most of them are activated by phosphorylation at C-terminal domain. Has a tyrosine residue that decreases. It has been found that c-src, obtained by substituting this tyrosine with another amino acid by genetic engineering techniques, has a carcinogenic potential.Research on the src gene has only just begun, From the study of the amino acid sequence and the study of deficiency, the potential for application to pharmaceuticals and diagnostics is expected.

The amino acid sequence of c-src is already known. Recent studies have shown that proteins with SH2 (src homologous domain 2) bind to phosphotyrosine using this region. SH2 is not only a src family, but also phosphatidylinositol-13-phosphoryl oxidase (PI 3-kinase), phospho lipase Cr (PLC-r), GTPase It has been found in the sequence of various proteins such as the base protein (GAP), and its interaction with the C-terminal region of the src family is known as cross talk in signal transduction. Is a direct route to

In such a mechanism, extracellular signals are transmitted via receptors to the src family, which hangs inside the cell membrane, and the information is transmitted by the SH2-containing protein. Propagated to signaling in the tall (IP), diglyceride (DG), and GTPase systems, causing a variety of responses.

For this reason, it is desirable to use a monoclonal antibody that specifically recognizes the analysis of information transmission after the src family. Tissue, developmental (spatio-temporal) phosphorylation, and the ability to track changes in binding proteins.

According to another study, an oncogene called crk has only the SH2 region without tyrosine phosphatase activity. crk is thought to cause cell carcinogenesis by binding to the product of the src family, which has thi-sin lin oxidase activity, using the SH2 region (Mayer et al. Nature, 332, 272-275 (1988)).

Specific tyrosin residues at the C-terminus of the src family named csk An enzyme that oxidizes has also been discovered (Noda et al. Ature, 351, 69-71 (1991)).

Among the src family gene products, c-src has been reported to be high in colorectal cancer, neuroblastoma, retinoblastoma, small cell lung cancer, colon cancer, breast cancer, and rhabdomyosarcoma (S Pahlman and U. Hammerling, Am. Rev.

Respir. Dis., 142, s54-s56). Especially for colorectal cancer, see J.B.

Bolen et al., Proc. Natl.Acad.Sci., 84, 2251-2255 (1987), CA Cartright et al., J. Clin.Invest., 83, 2025-2033 (1989), CA Cartwright et al. , Proc. Natl. Acad. Sci., 87, 558-562 (1990) also reported high c-src. However, due to technical limitations, none of them has detected src-family activation in adenomas that are pre-cancerous. In addition, it has been revealed that C-fgr is highly expressed in B-kit lymphoma and myelomonocytic leukemia, and c-yes is highly expressed in head and neck and kidney cancers.

C-fyn, c-lyn, c-lck, c-hck, c-blk, etc., which are mainly expressed in immunocompetent cells, are deeply involved in lymphocyte-specific signaling. Has been suggested. Among them, C-lck is said to be highly expressed in T lymphomas and colon cancer in the-part, and C-lyn is highly expressed in T cell leukemias.o

In non-cancer diseases, studies using transgenic mice have shown that c-src deficiency causes osteopetrosis. (Sariano et al. Cell. 64, 693-702 (1991)) _D Also, c-fyn may play an important role in skull morphogenesis. Disclosure of the invention

No specific antibody has been produced that can detect activation of the src family. In particular, there is no antibody that can stain formalin-fixed or paraffin-embedded sections, which is the mainstream in pathological diagnosis.

In 1983, J. Brugge et al. Immunized v-src with monoclonal antibody 327, which crosses only human c-src. Have been identified.

The binding site of this antibody has been identified as the SH2 region of src, but this has not been confirmed (Lipsiuli cill. J. vii ul. 8, 352-360 (1983)).

An antibody, GD11, derived from v-src and crossing c-src was identified by Parsons et al. (J. Virol. 5, 272- (1984)) and MGS-1 by Shoji et al. (BB. C. 170, 657-664 (1990)).

For some other src family proteins, there have been some reports of monoclonal antibodies, but none for the C-terminal region.

Although Kempridge (CRB) has marketed a polyclonal antibody using the C-src (4-1 to 485) peptide, it lacks specificity and binding ability, and it is also SH2. It has nothing to do with the region or the C-terminus of the src family.

In view of the above, the present inventors have made intensive studies and as a result, have discovered a monoclonal antibody that specifically exists at the end of the src family and has a function of controlling the phosphorylation of the enzyme. Successful acquisition resulted in the completion of the present invention.

The gist of the present invention is as follows: ① The phosphatase present at the end of the src family A monoclonal antibody itself that specifically recognizes a site having a function of controlling the activity; (2) the peptide of SEQ ID NO: 1 to obtain the monoclonal antibody; (3) an antibody-producing cell that produces the monoclonal antibody ④ The monoclonal antibody is used for pathological diagnosis.

The monoclonal antibody according to the present invention can be obtained as follows.

Production of antigen

First, the C-terminal peptide of the above C-src that serves as an antigen is chemically synthesized.

In the present invention, among the above C-terminal C-src peptides, for example, peptides having an amino acid sequence as described in Sequence Listing 1 described below can be synthesized. The peptide as shown in SEQ ID NO: 1 is a novel compound, and the present inventors are the first to obtain a monoclonal antibody of the c-src gene using this peptide as an antigen.

In the present invention, in order to enhance antigenicity, the peptide obtained as described above is converted into an appropriate carrier, for example, a keyhole limpet hemocyanin or the like. Can be immunized by binding to

For the coupling reaction between the peptide and the carrier, appropriate chemical reagents such as glutaraldehyde, carbodiimide, MBS (N- (m-maleimide benzoyloxy) succinimide) And SMCC (sulfosuccinimidyl 4- (N-maleimide) cyclohexan-1-carboxylate) and the like.

Hereinafter, the present invention is used as an antigen, A method for obtaining a monoclonal π-null antibody according to the above is described.

Preparation of immunized cells and myeloma cells

First, the preparation of immunized cells will be described.

This can be obtained by administering an antigen into the body of an animal and obtaining cells of the animal. As the animal, for example, an animal such as a mouse which has been subjected to an experiment as a conventional method can be used. It is desirable to administer the antigen into the abdominal cavity or the like.

It is appropriate to administer the drug by admixing it with Freund's complete adjuvant in a usual manner, but in the present invention, the administration may be repeated several times at intervals of several weeks after administration. preferable. An administration interval of 2 weeks is sufficient, and the number of administrations is preferably 2 to 4 times. Thereafter, the animal is sacrificed, for example, an organ such as a spleen is removed, and the cells are removed according to a conventional method.

Next, cell fusion with myeloma cells to impart a proliferative function to immunized cells will be described.

As the myeloma cells used here, for example, the SP2 / 0-AgI4 strain or the like is cultured in a medium containing, for example, PCS (fetal calf serum), and preferably cells in a logarithmic growth phase are used.

Cell fusion, measurement of antibody titer, and selection of antibody-producing cells

The cell fusion method involves the ratio of immunized cells to myeloma cells in a 1: 1 ratio of cell numbers.

The mixture may be mixed in the range of 10 to 1 and a fusion agent such as polyethylene glycol or a method such as electric stimulation may be used.

After fusion, cells are immediately or after preculture in a normal medium, and hypoxan. By culturing in a so-called HAT medium supplemented with tin, ryminopterin and thymidine, only cells fused with a combination of immunized cells and myeloma cells can be selected.

In the present invention, antibody-producing cells can be selected while confirming the antibody titer by, for example, enzyme immunoassay (ELISA).

The cells for which the antibody titer could be confirmed were repeatedly cultured in a well plate and a plate according to a conventional method such as an ultra-dilution method or a soft agar method. Screening can be performed by conducting an immunochemical test of the antibody, an antigen specificity test, or the like. In addition, sorting can be performed using a micro-manipulator or a cell sorter.

Obtaining antibodies

The antibody producing cell according to the present invention can continuously produce the monoclonal antibody according to the present invention in a normal state. Therefore, when the monoclonal antibody according to the present invention is used, the supernatant of the culture solution of the cell according to the present invention can be directly used as the monoclonal antibody solution according to the present invention as it is. .

In addition, the monoclonal antibody according to the present invention is obtained by administering antibody-producing cells to an animal body (for example, intraperitoneally), which is usually subjected to an experiment, such as a mouse, and transforming an animal body fluid such as ascites. It can also be produced by sampling.

In order to purify and obtain the monoclonal antibody D-null according to the present invention from the culture solution or ascites fluid of the cell according to the present invention, it can be obtained by, for example, a method such as ammonium sulfate precipitation, ion exchange chromatography, or the like. . The monoclonal antibody thus obtained is stored as a stable substance by adding various buffers, salts and, if necessary, azide, or the like, or by freeze-drying, for example. can do. Identification of each class

Identification of each class of immunogenic n-purine of these antibodies can be performed by the BLISA method using class-specific antibodies.

The monoclonal antibody obtained in this manner can be confirmed to specifically bind to each protein by the following immunobiochemical technique.

An identification method using the monoclonal antibody according to the present invention and the method of easter plotting will be described.

This technique can be applied to the identification and quantification of such peptides in biological specimens. That is, a sample derived from a living organism under the peptide or containing the peptide is fractionated by SDS-polyacrylamide gel electrophoresis according to a conventional method, and then subjected to nylon membrane or nitrose cellulose measurement. Plot into a blank. To bind the peptide was transferred onto main assembler emissions and the mono-click b Naru antibody, then, ³⁵ S or ^'25 1 is combined with-labeled was Puroti down A (protein A). At this time, excess monoclonal antibody and protein A should be thoroughly removed by washing each time.

Thus, the position of the base petit de depending on the specificity of the mono-click polyclonal antibody used, will be the ^{3 S} S or ¹²⁵ I present in accordance with the amount, ³⁵ S or on main assembler down ¹²⁵ Identify and quantify peptides by detecting 1 with RI scanner or autoradiography. Can be.

Next, a method for identifying such peptides, which is characterized by applying the RIP (radio-immuno-precision) technique, is described.

In RIP, for example, in ^{3 5} S or ^{3 2} P Oru' off O Sufuwei preparative like medium containing labeled Mechionin such, the incorporation of cells of a specimen in cells of a specimen by culture. Then, after adding a buffer for solubilization, a lysate is obtained. Then, the antigen-antibody reaction is completed by adding and mixing the monoclonal antibody according to the present invention.

Thereafter, protein A-Sepharose or the like is added according to a conventional method, and centrifugation and washing with a buffer solution are repeated to remove the contaminants other than the peptide of the present invention. After that, electrophoresis is performed according to a conventional method to detect radioisotope. If the peptide is present in the sample cells, a band corresponding to the specificity of the monoclonal antibody used will appear.

Next, a method of detecting the peptide in an organism, which is characterized by applying the technique of immunocytochemistry, will be described.

In the case of cultured cells, the cells can be fixed with an appropriate fixing solution such as a solution of formaldehyde phosphate buffer (PBS) and, if necessary, subjected to a triton treatment to prepare a sample. After the monoclonal antibody according to the present invention is added to these samples to complete the antigen-antibody reaction, the sample is thoroughly washed with a phosphate buffer. After reacting piotin-conjugated anti-immunoglobulin as a secondary antibody, wash thoroughly. Fluorescence labeling of the resulting peptide-monoclonal antibody-piotin-conjugated secondary antibody complex was performed. By reacting vidin and observing the fluorescence with a fluorescence microscope, peptides in the cells can be detected.

In the case of pathological tissues, sections were fixed and fixed with formalin or other appropriate fixative, or frozen sections were fixed with formaldehyde and treated with a surfactant, if necessary. Using the specimen as a sample, immunocytochemistry can be performed by the same operation. Pathological diagnosis

By using the monoclonal antibody according to the present invention, a human or other mammalian src family gene product can be identified with high sensitivity. Therefore, the monoclonal antibody according to the present invention can be used for pathological diagnosis of human or other mammalian src family gene-derived diseases.

Examples of the disease derived from the src gene include colorectal cancer, lung cancer, breast cancer, neuroblastoma, rhabdomyosarcoma, retinoblastoma, and other cancers, osteolithiasis, and the like.

As the pathological diagnostic method using the monoclonal antibody according to the present invention, a general pathological diagnostic method using a monoclonal D-nal antibody can be applied as it is. Specifically, for example, Western blotting

(Western blotting) method, immunoprecipitation method

Immunopreci.pitation) method, immuno site chemistry

Immunocytochemistry) method and the like. These techniques can be used alone or in combination.

More specifically, for example, tissues such as colon and lung obtained by biopsy or surgery are fixed in formalin and paraffin-embedded sections are used in the present invention. By reacting with a monoclonal antibody, staining with an ordinary method, and detecting activation of the c-src family, pathological diagnosis can be performed. In addition, lung cancer can be diagnosed by performing a sputum test. Specifically, sputum is collected from the subject, spread on a slide glass by smearing, and then immersed in, for example, -20: acetone or methanol. It can be diagnosed by fixing, reacting with the monoclonal antibody of the present invention, staining by a conventional method, and detecting activation of the c-src family. Confirmation of activated c-src along with morphological observation will facilitate definitive diagnosis.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples and the like according to the present invention. However, these Examples and the like are merely examples of the present invention, and the present invention is not limited thereto. is not.

Example 1 Preparation of Monoclonal Antibody Against C-Terminal of c-src (1) Preparation of Antigen

A peptide having a sequence consisting of 19 amino acids was chemically synthesized according to a conventional method. The amino acid sequence of this peptide is as shown in SEQ ID NO: 1.

10 mg of this peptide dissolved in 2 ml of purified distilled water was mixed with 5 mg Zml of KLH solution (manufactured by Calbiochem), and sulfo-SMCC (manufactured by Pierce) was used as a coupling agent. The concentration was adjusted to 0.2M.

The peptide and the carrier conjugate were purified using a disposable gel filtration column (manufactured by Biorad). In this way, 12 mg of the peptide-KLH conjugate was obtained.

(2) Preparation of immunized cells

In a 10-week-old female BALB / C mouse, 0.1 ml of a solution containing 100 mg of the substance obtained in (1) and complete adjuvant of Freund (DIFC0) were intraperitoneally D A mixture with 1 ml was administered. Thereafter, a mixed solution of the above solution and incomplete adjuvant (manufactured by DIPC0) was intraperitoneally administered twice at two-week intervals. Thereafter, the mouse was killed by cervical dislocation and the spleen was aseptically probed.

Place about 1 g of the above spleen on Selector 1 (manufactured by BBLC0) and pass through about 10 meshes while supplying Dulbecco's modified MBM (D-MBM) medium.

Spleen cells suspended in D-MBM were obtained. This was centrifuged at 100 G for 5 minutes to collect spleen cells.

The cells were put into 1 ml of a solution obtained by adding 20 mM HBPES buffer (PH 7.4) to a 0.84% ammonium chloride solution and hemolyzed. Cells were collected by centrifugation at 1,000 G for 5 minutes. It was suspended again in D-MBM medium.

(3) Preparation of myeloma cells

The mouse myeloma cell line SP2 / 0 strain, 20% © shea calf serum (FCS) including D - in MBM medium, and cultured in 10% C_〇 ₂ · 37 t i Nkyubeta within one, the logarithmic growth phase The cells were collected and used for the next operation.

Only the cells were obtained by centrifugation at 1,000 G for 5 minutes, suspended in a D-EM medium, and the number of cells was counted using a hemocytometer. After centrifugation again at 1,000 G for 5 minutes, the cells were suspended in D-MBM medium.

(4) Cell fusion And D-MBM medium containing immunizing cell ^{1 0 β ~ 3 X 1 0} 8 pieces obtained in (2),

After mixing with a D-MBM medium containing 10 ^β myeloma cells obtained in (3), the mixture was homogenized, and centrifuged at 1,000 G for 5 minutes. The supernatant was removed and the sediment was collected. To this was added 1 ral of a solution containing 25% (/ v) polyethylene glycol 1500 (PBG 1500, manufactured by Behringer) and 37.5 m of Hex buffer. It was added dropwise over one minute. Thereafter, the whole was diluted with D-MBM medium to make 10 lOral.

To this, 10 ml of D-MBM medium containing 20% PCS was added, and the mixture was centrifuged at 1,000 G for 5 minutes. The resulting cells by the addition of D-MEM containing 20% PCS 1 0 in such a manner that the ⁶ cell / ml, so that a ratio of 1 ml / well in 24-well culture plate over preparative co-learning Co. On top. In 10% C 0 ₂ 'in 37t I Nkyubeta one were cultured for 24 hours.

Thereafter, a HAT solution was added to remove cells other than the fused cells, and the culture was continued for another 2 weeks.

(5) Measurement of antibody titer by SA method

Each well of Falcon's BLISA plate was coated with 10 ug Zml of the peptide obtained in (1). The supernatant 1001 containing the antibody to be measured was taken in a well, and left in an incubator 371 for 2 hours. Thereafter, the plate was washed with PBS (phosphate buffer). To this, peroxidase-conjugated anti-mouse whole antibody was added, and the mixture was left to stand in an incubator for 1 hour. After washing with PBS, a color former (ABTS) was added for 15 minutes to develop color. Stop solution (0.1 M quencher) The reaction was stopped by adding 0.02% of sodium acid (acid sodium), and the antibody titer was calculated by measuring the absorbance of the well with a titantech multiscan. (6) Selection of antibody-producing cells

The cells in the wells confirmed by the BLISA method were spread on a soft agar medium in a 60 mm petri dish. The cells were cultured for 2 weeks in a 10% CO ₂ · 37t incubator to form colonies.

The resulting colonies were picked and placed on 24-well culture plates. Again, the antibody activity was confirmed by the BLISA method. Cells in the well in which the antibody activity was confirmed were seeded on a soft agar medium in a 60 mm0 dish. The cells were cultured in an incubator with 10% C〇 ₂ · 37 for 2 weeks to form colonies. The resulting colonies were picked and placed on 24-well culture plates. Again, antibody activity was confirmed by the BLISA method, and useful cells were selected.

The antibody-producing cells selected in this way (Antibody No. 28) are from the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-3 Tsukuba, Higashi, Ibaraki, Japan (zip code: 2005)). International Deposit (Accession No .: PBRM BP-4253. Deposit date: March 30, 1993).

(7) Obtaining antibodies

(1) Since the antibody-producing cells obtained in (6) always produce the antibody of the present invention, the supernatant of the culture solution in which the antibody-producing cells are cultured should be used directly as the antibody solution of the present invention. Was completed.

2) Sulfonated ammonium was added to the antibody-producing cell culture obtained in (6) to a final concentration of 30%. The precipitate was collected by centrifugation, and 20 πιΜ of pH 7.4 phosphate buffer was added thereto, followed by dialysis with the same phosphate buffer (containing 0.02 sodium sodium azide) to remove ammonium sulfate. The dialyzed liquid was freeze-dried to obtain a white powder.

③ Inject 0.5 tnl of pristane into the abdominal cavity of BALB / C male mice for 2 weeks した bred. Injected with antibody-producing cells obtained in (6) such that ^{1 0 6 ~ 3 x 1 0} 6 cell / mouse, were raised for 10 days. About 10 ml of ascites collected in the abdominal cavity was collected using a syringe.

The ascites was diluted approximately 10-fold with an aqueous solution containing Tris 50 mM (pH 8.2) salt 150 mM NaN ₃ 0.02%. This solution was diluted with monoclonal anti-mouse immunoglobulin (available from Ricoh Corning Rex, Inc.) if the subclass of the antibody was IgG. Those with a subclass of IgG ₂ were purified using a brotin A-Sepharose CL-4B column (manufactured by Pharma Ryo). Elution was performed with IgGJ ^ glycine monohydrochloride 0.1M (pH2.8).

IgG ₃ was carried out with an Imuno View Buffer (Pierce).

(8) Identification of each class of immunog α-brin

Identification of Lee Mi Interview Roh Grob Li down each class is class specific antibodies _{(IgA, IgG ,, IgG 2a IgG} 2b, IgG 3, IgM has had use those Baiorai de Co.) was added, 37 After washing for 2 hours, the plate was washed thoroughly with PBS. To this, peroxidase-conjugated anti-mouse whole mouse antibody was added, and the plate was allowed to stand for 1 hour in a 371 incubator. After washing with PBS, the color former ( (ABTS) was added for 15 minutes for color development, and each class was judged. Table 1 shows some of the results. table 1

Test Example 1 RIP (immunoprecipitation method)

Mono click b Naru antibody according to the present invention, since the switch port Thin residues is a site that receives the re-phosphorylation, to label the proteins in the cell ³² .rho. d'soft O Sufuwei preparative or ³⁵ S- Mechionin Was used.

After culturing the cells to a confluent state, the cells were cultured in a medium free from phosphoric acid or methionine for 2 to 3 hours (starvation). After this, the medium was replaced again, and ³² P-orthophosphate (NBX-053 370 MBq / ml manufactured by NB) or ³⁵ S-methionine (SJ1015 370 MBq / ral manufactured by Amersham) was added. Culture was continued for another 2 to 4 hours. After incubation, the medium was removed, PBS (-) was washed with a radioactivity lysate obtained _c solubilized in RIPA buffer was measured by liquid shea inch Reshi Yo Lanka c printer over, immunoprecipitation one sample per 3-5 Use xl05 cpm. The antibody of the present invention was added to the lysate, and the mixture was stirred gently at 4 t for 2 hours to complete the antigen antibody reaction.

200 1 was prepared by suspending protein A-Galose (manufactured by Zymed) at 10% in RIPA buffer, and the mixture was reacted at 4: 1 for 1 hour. After the reaction, the mixture was centrifuged at 15,000 rptn for 1 minute to separate from the supernatant. RIPA buffer to wash precipitated antigen-antibody-protein A-sepharose complex 0.75 ml of the buffer solution was added, and the mixture was stirred well, centrifuged at 15, OOO rpm for 1 minute, and separated from the supernatant again. This operation was repeated five times. to this

The sample buffer 30/1 for SDS-PAGB electrophoresis was added, and the mixture was heated with lOOt for 2 minutes to denature the protein. The electrophoresis was performed on a 10% poly-middle gel. After electrophoresis, the proteins in the gel 5% METHANOL, after fixing with a solution containing 7.5% acetic acid, ³⁵ S sensitizer when the label (NBN Co., ENLIGHTIUNG) were sensitized with . The gel was dried, exposed to X-ray film or an imaging plate (for Fuji, BAS2000), and the band specifically recognized by the antibody was detected.

Figure 1 shows the protein bands detected using these antibodies.

As for the cells used, lane numbers 1 to 3 and 7 to 9 are neuroblastoma NB-1, 4 to 6 and 10 to 12 are acute lymphoblastic leukemia cells SKW3. Monoclonal antibodies used antibody numbers 28, 2, 5, 8.11 for antibody numbers 1, 4, 7, and 10, antibody number 29 for antibody numbers 3, and antibody number 40 for 3, 6, 9, and 12. Among the present invention, antibody ban Nos. 28 and 29 had particularly good detection sensitivity in the immunoprecipitation method.

Antibody No. 28 showed a band that was not detected in the ³² P lapel at the ³⁵ S label, and recognized that it recognized the vicinity of the thiacin residue, which is a phosphorylated site.

Antibody No. 29 showed the same pattern in both ³² ° and ^{3 s} S, and was proved to be an antibody recognizing other than phosphorylated sites.

In addition, antibody No. 40 detected a band different from the above two antibodies, and by these, it was possible to analyze the difference in information transmission due to the presence or absence of phosphorylation. 例 Example using the blotting method

The result is shown in figure 2. The antibody used was antibody number 29. The cell lines that produced lysate were as follows: lane number 1 is acute lymphoblastic leukemia cell ΜΰίΤ3, lane number 2 is S3, and lane number 3 is monocyte leukemia cell.

J111, Lane 4 with U937, Lane 5 with glial tumor

U251, lane number 6 is neuroblastoma SC-N-RT, and lane number 7 is NB-1.

Bands could be detected with higher sensitivity than each cell.

An example of segmentation by a tie-fetish column

The results are shown in Figure 3. Antibodies Nos. 28 and 29 were used as antibodies. Lane No. 1 indicates the whole protein solubilized rat brain. Lane No. 2 shows the No. 28 affinity column, and Lane No. 3 shows the protein eluted with 3M KSCN from the affinity column of 29 Ban.

Diagnosis example 1 Pathological diagnosis of colorectal cancer ''

Each colon section obtained by biopsy or surgery from 76 subjects was used as a paraffin-embedded pathological section, which was immersed in xylene to remove paraffin. These were then treated with 0.1% NaN ₃ sodium, 0.3% hydrogen peroxide methanol for 30 minutes, then 5% normal goat serum, 1% bovine serum albumin (BSA) Z PBS Blocking was performed with (-) for 20 minutes, and the mixture was allowed to react overnight with a monoclonal antibody (antibody number 28) of the present invention (antibody number 28) diluted 1500-fold at 4 in a moist chamber (moist chamber). Thereafter, these were washed three times with PBS (-), and reacted with a 500-fold diluted biotinylated anti-mouse immunoglobulin antibody for 1 hour at room temperature to develop color. The color was developed by the peroxidase-conjugated avidin-piotin method (ABC comp lex method) using 3,3'-diaminobenzyme. The test was performed using a combination of anzidine tetrahydrochloride (0.1% solution, containing 0.02¾ hydrogen peroxide and 50 mM Tris-HCl buffer). Hematoxylin was used for counterstaining. Observation of the color-developed site was performed with an optical microscope, and only a clear brown stain was made enteric. The results are shown in Table 2.

Table 2

(Number of positives Z Number of subjects) As is evident from Table 2, activation of the 髙ぃ src family gene product (positive staining) was observed in adenomas (pre-cancerous), leading to canceration. This reduced the number of positive cases. There were few enteric cases in advanced cancer, and there was no mosaic-like staining.

Therefore, the monoclonal antibody according to the present invention can be used particularly for pathological diagnosis of precancerous conditions. In addition, most of the adenoma stained enteric cases have mosaic-like staining, and the presence or absence of mosaic-like can be used as an indicator of a precancerous condition.

Diagnosis example 2 Pathological diagnosis of lung cancer

Each of the lung sections obtained by biopsy or surgery from nine subjects was used as a paraffin-embedded pathological section, which was immersed in xylene to remove the paraffin. Was. These were then treated with 0.1% NaN ₃ sodium, 0.3% hydrogen peroxide Z-metal for 30 minutes, followed by 5% normal goat serum, ^ bovine serum albumin

Blocked with (BSA) / PBS (-) for 20 minutes and diluted 1500 times with the monoclonal antibody of the present invention (Antibody No. 28) and in a humidifier (moist chamber) at 4 overnight. Reacted. Thereafter, these were washed three times with PBS (-), and reacted with a biotinylated anti-mouse immunoglobulin antibody diluted 500 times for 1 hour at room temperature to develop color. The color was developed by the peroxidase-conjugated avidin-piotin method (ABC comp lex method) using 3,3'-dimininobenzidine tetrahydrochloride (0.1% solution, 0.02% Hydrogen peroxide and 50 mM Tris-HCl buffer were combined). Hematoxylin was used for counterstaining. Observation of the color-developed site was carried out with an optical microscope, and those with a staining intensity of nerve fibers equal to or higher than £ 1 were judged to be enteric / 0

As a result, of the 9 subjects, 5 showed clear positives (3 in adenocarcinoma, 1 in epithelioid carcinoma, 1 in small cell carcinoma) and 3 in suspicious bowel (1 in adenocarcinoma) And two epithelioid carcinomas).

Therefore, it is clear that the monoclonal antibody according to the present invention can be sufficiently used for diagnosis of lung cancer. one

Arrangement

SEQ ID NO: 1

Array length: 19

Sequence type: amino acid

Topology I: Linear

Sequence type: peptide

Array

Leu Gin Aap Tyr Phe Thr Ser Thr Glu Pro Gin Tyr Gin Pro Gly 1 5 10 15

Glu Asn Leu Cys

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the detection band by immunoprecipitation. The horizontal axis represents the lane number, rate NShigerugo 1-6 lapel by ³² P, lanes trial No. 7 - the label 1 2 by ³⁵ S, represents respectively. The vertical axis indicates the direction of electrophoresis, the upper part indicates a higher molecular weight, and the lower part indicates a lower molecular weight. Lane number 1 _3, molecular weight Ma - representing the position of the car (^ c). Fig. 2 shows an example of use in the dust plotting method. The 攢 axis indicates the lane number, and the ordinate indicates the electrophoresis direction. Lane number 8 represents the position of the molecular weight marker ( ¹⁴ C).

FIG. 3 shows an example of separation using an affinity column. The horizontal axis represents the lane number, and the vertical axis represents the direction of the electric swing. Lane number 4 indicates the position of molecular weight markers ( ^'4 C).

Claims

The scope of the claims

1. A monoclonal antibody that specifically recognizes a site that exists at the C-terminus of the src family gene product and has a function to control the activity of phosphatase.

2. The monoclonal antibody according to claim 1, wherein the src family gene product is one selected from the group consisting of c-src, c-yes, c-fynII and c-fgr.

3. The monoc according to claim 1, wherein the src family gene product is c-src. Internal antibodies.

4. The Institute of Biotechnology and Industrial Technology (IIST, Tsukuba, Ibaraki, Japan, 1st Ban, 3rd (Postal Board No. 3 05)) received the accession number FBRM BP-4253 on March 30, 1993. Monoclonal antibodies produced by antibody-producing cells deposited internationally.

5. A peptide having the amino acid sequence of SEQ ID NO: 1.

6. The method for producing a monoclonal antibody according to any one of claims 1 to 3, wherein the peptide according to claim 5 is used as an antigen.

7. An antibody-producing cell that produces the monoclonal antibody according to claims 1 to 3.

8. Access to the Institute of Biotechnology, Institute of Industrial Science and Technology (Tsukuba, Ibaraki, Japan, 1-3-1, Higashi 1-chome (zip code: 3005)) with accession number PBRM BP-4253 on March 30, 1993 The deposited antibody-producing cells.

9. A pathological diagnosis method for a cancer or precancerous state, comprising using the monoclonal antibody according to any one of claims 1 to 4.

10. The method according to claim 9, wherein the cancer is colorectal cancer.

11. The pathological diagnosis method according to claim 9, wherein the cancer is lung cancer.

12. A method for diagnosing osteopetrosis, which comprises using the monoclonal antibody according to claims 2 to 4.