JPH0630785A - Monoclonal antibody for alpha-cathenine - Google Patents

Abstract

PURPOSE:To provide a monoclonal antibody capable of recognizing alpha-cathenine, to provide a hybridoma capable of producing the monoclonal antibody, and to provide a reagent for immunochemical assay containing the monoclonal antibody. CONSTITUTION:The monoclonal antibody can be obtained by producing monoclonal antibody using alpha-cathenine as antigen followed by screening. The resultant monoclonal antibody is reactive with the normal cells of various kinds of animals and alpha-cathenine in human cancerous cells, being also applicable to immunohistology. The screening and raising the second objective hybridoma can be performed in a 10-2070 unit bovine serum-contg. medium for cell culture by addition of hypoxanthine, aminopterin and thymidine.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that binds to α-catenin, a hybridoma producing the monoclonal antibody, and an immunochemical assay reagent containing the monoclonal antibody, which is used in the field of medicine.

[0002]

2. Description of the Related Art Recently, much attention has been paid to the importance of cell adhesion molecules and their functions involved in cell-cell or cell-extracellular matrix interactions. Adhesion molecules are classified into cadherin family, immunoglobulin family, integrin family, selectin family, etc. due to their structural similarity, and through adhesion, cell development, differentiation, inflammation, regulation of life phenomena such as cancer metastasis, etc.
It is closely related to maintenance.

Cadherin, one of the adhesion molecules, is a transmembrane cell adhesion molecule that functions depending on calcium. Since it is specifically expressed in the fetal period, it is considered to be an important molecule for morphogenesis. To date, four types of E-type (epithelial tissue), N-type (nerve tissue), P-type (placenta), and L-CAM Is being analyzed (Takeichi, M. Science, 251,
1451-1455, 1991). Immunohistochemistry using anti-cadherin antibodies has shown from the early days that cadherin molecules are concentrated in an intercellular adhesion device called an adherence junction (hereinafter abbreviated as AJ). In addition, due to recent advances in genetic engineering, cadherin cDNA has been isolated, its primary structure has been clarified, and it has become possible to elucidate the mechanism of adhesion at the molecular level (Nagafu
chi, A. et al., Nature, 329, 341-348, 1987). Immunoprecipitation experiments using an antibody against E-cadherin have long been known to bind to this molecule three proteins with molecular weights of 102 kd, 88 kd and 80 kd (Peyr
ieras, N. et al., Proc. Natl. Acad. Sci. USA, 82,
8067-8071,1985), in order from the largest molecular weight α,
It is called β and γ catenin (Ozawa, et al., EMBO
J., 8, 1711-1717, 1990).

Although the molecular construction of the cadherin-catenin complex has been meticulously analyzed, since the studies so far have been conducted on the solubilized cadherin molecule, the cadherin molecule actually functioning. Little information is available on the relationship between and catenin, and how these complexes play a role in cadherin function.

The present inventors established a method for isolating an AJ fraction from rat liver and reported that the isolated AJ had a large concentration of a cadherin-binding protein (CAP 102) having a molecular weight of 102 kd. Furthermore, we succeeded in purifying this protein, determining its partial amino acid sequence, preparing a polyclonal antibody, and then isolating mouse CAP 102 cDNA. This molecule has homology with vinculin and is actually functional with cadherin. It was suggested for the first time that they are bound (Na
gafuchi, A. et al., Cell, 65, 849-857, 1991). After that, the isolation of α-catenin cDNA was reported, and it was revealed that it was the same molecule as CAP 102 (Herrenknecht,
K. et al., Proc. Natl. Acad. Sci. USA, 88, 9156-916
0, 1991). However, the structure of human α-catenin is still unknown.

Vinculin is a protein localized in the adhesion device between cells and between cell substrates, and has been vigorously studied so far. Since the primary structure of α-catenin has homology with vinculin, it has been speculated that the function of the α-catenin molecular region and the possibility of interaction with other AJ constituent factors are various. However, the factors that α-catenin interacts with, the region required for interaction with cadherin, the regulatory mechanism of cadherin-mediated cell adhesion, the mechanism of α-catenin induction, and the role that it plays in vivo are still poorly understood. Not, and the analysis is awaited.

On the other hand, basic studies on metastasis of cancer cells have been extensively studied at the cell level and the molecular level, and their complicated mechanism is being elucidated, but it is still unknown. One of the factors thought to be involved in the metastasis of cancer cells is a cell adhesion molecule. Much research on the correlation between various adhesion molecules of the adhesion molecule family and cancer cell metastasis,
Although there are reports, no clear answer has been obtained. In the field of cancer research, both in order to elucidate the mechanism of metastasis of cancer cells and to prevent metastasis, there is a strong demand for the development of a definite Mercumar for judging the metastasis of cancer in the clinical field.
Further, the present inventors have prepared an anti-rat α-catenin polyclonal antibody and reported its use in Western blotting, but this polyclonal antibody had a major drawback that it could not be applied to immunohistological staining. As a means to elucidate the physiological significance of α-catenin, it can be applied to the immunohistological staining method, and it has been desired to prepare an antibody having reactivity with human α-catenin.

[0008]

In view of the above situation, an object of the present invention is to use a monoclonal antibody that recognizes α-catenin and a hybridoma producing the monoclonal antibody, which can be used for immunohistological staining, by further using the monoclonal antibody. An object of the present invention is to provide an immunochemical assay reagent for determining the metastatic ability of cancer cells by measuring the amount of α-catenin in the cancer cells.

[0009]

[Means for Solving the Problems] The present inventors have elucidated the structure of a protein α-catenin (derived from mouse) that is bound to E-cadherin, which is one of cell adhesion molecules. Next, as a result of the study of the interaction between cadherin and α-catenin, it was reported that α-catenin has a strong interaction with cadherin and other AJ components and is involved in the mechanism of cadherin-mediated cell adhesion. Furthermore, as a result of screening for producing a monoclonal antibody using mouse α-catenin as an antigen for its physiological research, a monoclonal antibody having reactivity with α-catenin of various animals including humans and applicable to immunohistochemical staining method was also found. It was made successfully. A study on human cancer cells has surprisingly found an α-catenin deficient strain that has never been identified in the normal cultured cells examined so far. This suggests that there is a possibility of correlation between changes in the expression level of α-catenin and cancer cell transfer, and completed the present invention. That is, the present invention relates to a monoclonal antibody that recognizes α-catenin, a hybridoma cell that produces the monoclonal antibody, and an immunochemical assay reagent for α-catenin that uses the monoclonal antibody.

The monoclonal antibody of the present invention is prepared by using α-catenin of various animals as an antigen, forming a complex with a carrier protein as necessary, and inoculating the animal with this to immunize. It is prepared by fusing antibody-producing cells obtained from the spleen or lymph node of the immunized animal with myeloma cells and selecting a hybridoma that produces an antibody showing a strong specificity for α-catenin. The operation may be based on a conventionally known method.

As the immunogen, any of naturally purified products, products produced by a gene recombination method or a chemical synthesis method can be used. Preparation of α-catenin by gene recombination method
The cDNA encoding α-catenin can be recombined by a known method using a restriction enzyme and DNA ligase downstream of the promoter suitable for the expression of α-catenin by a known method to prepare a recombinant expression vector. The vector is not particularly limited as long as it can be replicated and amplified in the host. The promoter and terminator are not particularly limited as long as they correspond to the host used for expressing the nucleotide sequence encoding α-catenin, and an appropriate combination is also possible depending on the host.

The recombinant expression vector thus obtained was obtained by the competent cell method (J. Mol. Biol., 53, 154, 197).
0), calcium phosphate method (Science, 221, 551, 1983
), Etc., is introduced into a host to produce a transformant. Escherichia coli and animal cells are used as the host, and the obtained transformant is cultured in a medium suitable for the host. Cultivation is usually carried out at a temperature of 20 ° C to 45 ° C and a pH range of 5 to 8, and aeration and agitation are carried out if necessary. Separation and purification of α-catenin from the culture may be carried out by appropriately combining known separation and purification methods. Examples of these known methods include salting out, solvent precipitation, dialysis gel filtration, electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography and the like. Naturally refined products are made from various animal organs
Tsukita et al. (Tsukita, S. et al., J. Cell Bio
l., 108, 31-41, 1989), α-catenin can be prepared. It is preferable that α-catenin is a pure product, but a fraction containing α-catenin may be used in some cases. As the raw material organ, any organ can be used, but the liver is preferred because of easy preparation. The type of animal is preferably human, but rat or mouse may be used because it is easily available.

The immunogen α-catenin preferably has the entire structure, but may be a fragment or peptide having a partial structure, and can be appropriately selected from the entire amino acid sequence of α-catenin. For the preparation of the fragment or peptide, a chemical synthesis method, the above-mentioned gene recombination method or a method for degrading a natural product is used. Various condensing agents can be used to prepare a complex of an antigen and a carrier protein, but glutaraldehyde, carbodiimide,
Maleimide active ester etc. can be used. The carrier protein may be a commonly used carrier protein such as bovine serum albumin, thyroglobulin, and hemocyanin, and a method of coupling at a ratio of 1 to 5 times is usually used.

Animals to be immunized are mice, rats,
Rabbits, guinea pigs, etc. are given, the inoculation method is subcutaneous,
It is administered intramuscularly or intraperitoneally. Upon administration, it may be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant, and the administration is usually performed once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph node of the immunized animal are fused with myeloma cells and isolated as hybridomas. As the myeloma cells, those derived from mice, rats, humans and the like are used, and it is preferable that they are derived from the same species as the antibody-producing cells, but it may be possible between different types.

The operation of cell fusion is known in the art, for example, the method of Kohler and Milstein (Nature, 256, 495, 197).
It can be carried out according to 5). Examples of fusion promoters include polyethylene glycol and Sendai virus,
Usually, polyethylene glycol (average molecular weight 1000-4000) with a concentration of about 20-50% is used at 20-40 ° C, preferably 30
Cell fusion can be carried out by reacting at a temperature of ˜37 ° C. for a ratio of the number of antibody-producing cells to the number of myeloma cells, usually about 1: 1 to 10: 1, for about 1 to 10 minutes.

Various immunochemical methods can be used for screening anti-α-catenin antibody-producing hybridomas.
For example, an ELISA (Enzyme-linked immunosorbent assa) using a microplate coated with α-catenin
The y) method, the EIA (Enzyme immunoassay) method using a microplate coated with an anti-immunoglobulin antibody, the Western blotting method using a nitrocellulose transfer membrane after electrophoresis of a sample containing α-catenin, the tissue staining method and the like. Since the purpose of the present invention is to prepare a monoclonal antibody applicable to the tissue staining method, the ELISA method, the Western blotting method and the tissue staining method are used in combination to obtain wells producing the desired antibody. A clone is obtained from such a well by further cloning, for example, by the limiting dilution method. Hybridomas are usually selected and bred by adding HAT (hypoxanthine, aminopterin, thymidine), and using an animal cell medium (eg, RPMI1640) containing 10 to 20% fetal bovine serum.

The clones thus obtained were transplanted into the abdominal cavity of BALB / C mice preliminarily administered with blistane, and after 10 to 14 days, ascites containing a high concentration of the monoclonal antibody was collected and used as a raw material for antibody purification. can do. Alternatively, the clone can be cultured and the culture can be used as a raw material for antibody purification. The monoclonal antibody may be collected by a known method for immunoglobulin purification, for example, by ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, use of anion exchanger, and affinity chromatography. Can be easily achieved.

[0018] Qualitative and quantitative determination of α-catenin in a biological sample can be performed by an immunological method using the anti-α-catenin monoclonal antibody obtained according to the present invention. Particularly, when applied to cancer cells, diagnosis of metastasis and cancer It can be used for patient prognosis management. As an immunological method, a biological sample is appropriately treated as necessary, for example, separation of cells,
Known methods such as an immunohistological staining method, an enzyme immunoassay method, an agglutination method, a competition method, and a sandwich method can be applied to a sample obtained by extracting and purifying α-catenin. The immunohistological staining method can be performed, for example, by a direct method using a labeled antibody, an indirect method using a labeled antibody against the antibody, or the like. As the labeling agent, any known labeling substance such as a fluorescent substance, a radioactive substance, an enzyme, a metal and a dye can be used.

Specimens Biological samples include tissues, cells, plasma,
It can also be applied to body fluids such as serum, urine, ascites, sputum, stool, etc. If necessary, the sample may be pretreated appropriately. Various buffers, organic solvents and the like can be used to extract α-catenin from the sample, but a centrifugation method using a hypotonic solution and sucrose is preferable, according to the method of Tsukita et al. (Supra).

The monoclonal antibody of the present invention comprises IgG, I
Any class of gA and IgM may be used, and Fc ′ or Fab ′ or Fab fraction obtained by removing the Fc region of the antibody, or a polymer thereof may be used. It may also be a chimeric antibody thereof.

[0021]

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

(1) Preparation of Immunizing Antigen Mouse α-catenin was used as an antigen and was prepared by a gene recombination method. First, mouse rat cDNA was prepared using an anti-rat α-catenin polyclonal antibody as a screening tool.
Mouse α-catenin c from gene library by conventional method
DNA was obtained (Nagafuchi, A. et al., Cell, 65, 849-
857, 1991). In order for α-catenin to be produced in the form of a fusion protein with maltose binding protein, mouse α
Catenin cDNA was added to pMAL-cRI vector (NEW EN
GLAND Bio Labs) and introduced it into E. coli. After selecting Escherichia coli into which the vector had been introduced, induction with IPTG was performed to express a large amount of the fusion protein. Then, the cells are disrupted by ultrasonic treatment to prepare a sample for SDS-polyacrylamide electrophoresis. After electrophoresis,
A protein having a molecular weight of about 180 kd corresponding to the fusion protein was electrophoretically extracted from the gel and used as an immunizing antigen.

(2) Immunization Immunization was carried out by mixing 300 μg / dose in the hind footpads of the rat with complete Freund's adjuvant for the first time, and further twice (3 and 7 days after the first immunization) with only the antigen. Was administered. The day after the final immunization, groin lymph nodes were taken out from the immunized animals and used for cell fusion.

(3) Cell fusion and cloning Rat lymphocytes and mouse myeloma P8 were mixed at a ratio of 2.5: 1, and the method of Kohler et al. Was partially modified to give 1 g.
Polyethylene glycol (average molecular weight of 4000) was dissolved in 1 ml of RPMI medium and reacted for 2 minutes to perform cell fusion. The fused cells were seeded in a 24-well plate, cultured in HAT medium containing 10% HCF (Bokusui Brown Co.) for 9 days, transferred to HT medium, and further cultured in a flask.
The cells were cultured in I or GIT medium (Wako). The antibody titer in the culture supernatant of the well in which the proliferation was observed was measured by the enzyme antibody method, and the hybridoma to be obtained was cloned from the appropriate well by the limiting dilution method. Hybridomas were seeded in a microtiter plate at a calculated number of 2 cells / well, and several days later, clones were visually confirmed and isolated. The hybridoma α18 that produces the desired monoclonal antibody has been deposited as FERM BP-3928 at the Microbial Industry Research Institute, Industrial Technology Institute, Ministry of International Trade and Industry since July 10, 1992.

Use medium RPMI medium RPMI 1640 (manufactured by Nissui Pharmaceutical Co., Ltd.) was added with the following additives 10% fetal bovine serum, 1.2 g / l NaHCO ₃ , 60 mg / l kanamycin HAT medium RPMI medium was added with the following additives. Addition 1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine HT medium A medium obtained by removing aminopterin from HAT medium.

(4) Screening method The obtained hybridomas were screened for clones producing the desired monoclonal antibody by the following three types of enzyme antibody methods and tissue staining methods. B) Description of method ELISA method Antigens (cell-cell adhesion device considered to contain α-catenin Adherence Junction (hereinafter referred to as AJ)) After reacting for 30 minutes at room temperature, after washing, peroxidase-labeled anti-rat immunoglobulin antibody was added and further reacted for 30 minutes at room temperature After washing unreacted labeled antibody, o-phenylenediamine solution was added, and 30 minutes at room temperature After reacting, stop the reaction by adding 2M sulfuric acid, 490nm
The absorbance of was measured.

Western Blot Method A fusion protein of α-catenin used for immunization, a fusion protein containing each part of α-catenin, and samples prepared from various cell lines and tissues were separated by SDS-polyacrylamide gel electrophoresis and then separated on a nitrocellulose membrane. Transferred to.
After blocking this membrane in 5% skim milk, a sample was added and reacted at room temperature for 30 minutes, and the antibody bound to the protein on the membrane was detected using a rat antibody blotting detection kit from Amersham.

Tissue Staining Method Frozen sections of various tissues were incubated in 95% ethanol at 4 ° C. for 15 minutes.
And further fixed in acetone at room temperature for 1 minute. Sample 1
After blocking with% BSA, a sample was added and reacted at room temperature for 30 minutes. After washing, FITC-labeled anti-rat immunoglobulin antibody was added and reacted at room temperature for 30 minutes, and unreacted antibody was washed and then examined under a fluorescence microscope.

(B) Screening flow Primary screening: Among AJ, acid-extracted fraction considered to be enriched in α-catenin, bile canalicular fraction isolated in the stage prior to AJ isolation, and purified α-catenin were selected. Positive wells were selected in at least 3 out of these 4 types of enzyme-antibody methods, including the ELISA method as an antigen and the Western blotting method using a fusion protein containing the full length of α-catenin. Secondary screening: After cloning of the positive strain for the primary screening, Western blotting was again performed using the fusion protein containing the full-length α-catenin, and clones that reacted with the fusion protein and had different reactivity to the degradation product were selected. Third screening: The cell lines selected above were cloned again, and the reactivity of the culture supernatant with various organs and cell lines of various species including human was examined by Western blotting and tissue staining. .

(5) Recovery and purification of monoclonal antibody (a) The cloned strain obtained by the above screening was intraperitoneally injected into a nude mouse (male) after 4 weeks of age, which had been pre-administered with 0.5 ml / mouse pristane 2.0- 3.0 × 10 ⁷ cell /
Ascitic fluid containing a high concentration of the monoclonal antibody was collected 10 to 14 days after the transplantation of the mouse. This ascites fluid was diluted with a 0.9% NaCl solution to be diluted 5 to 10 times, and then ammonium sulfate was added thereto so as to have a concentration of 40%, and a precipitate fraction was collected. This precipitate fraction was dissolved in a small amount of 0.9% NaCl solution and then dissolved in 0.9% Na
The Cl solution was dialyzed as an external solution. After the dialysis, high performance liquid chromatography (TSK-Gell G-3000SW) was used to obtain a purified monoclonal antibody. (B) This clone strain can be grown in a low serum medium. That is, GIT medium (Wako Pure Chemical Industries, Ltd.) was grown and the culture supernatant was collected. Ammonium sulfate was added to this supernatant
The f-precipitated fraction was collected by adding so that the concentration became 50%. This precipitate fraction was dissolved in 1 / 50-1 / 100 volume of 0.02 M phosphate buffer, and the same solution was dialyzed as an external solution. After dialysis, DEAE-cellulofine column chromatography was performed to use the first peak portion as a purified monoclonal antibody.

(6) Characteristics of Monoclonal Antibody The characteristics of the monoclonal antibody produced by the clones thus screened are as follows. Immunoglobulin class is MonoAb-ID / SP Rat Kit (ZYMED LABORA
TORIE INC). Antibody name: α18 Immunoglobulin type (L chain): IgG _2a (κ) Recognition antigen: α-catenin Species specificity, tissue specificity: human, bovine, mouse, rat,
It specifically recognizes α-catenin in newts and zebrafish. In mice, expression is found in almost all cells except those in the central nervous system.

(7) Reactivity against human cancer cells Human cancer cell lines were subjected to α using Western blotting.
When the presence or absence of catenin expression was assayed, HCT 15 (colorectal adenocarcinoma) and Colo 201 (colorectal adenocarcinoma) expressed α-catenin, but Pc 9 (lung adenocarcinoma) lacked α-catenin. Pc 9 is the first α-catenin deficient strain identified. Similar expression of cadherin was observed in these cancer cell lines. Despite expressing cadherin, Pc 9 has a very weak cell adhesion ability, suggesting a possibility that it may be correlated with the expression of α-catenin in cancer cell transfer.

[0033]

INDUSTRIAL APPLICABILITY The monoclonal antibody to α-catenin of the present invention has an extremely high binding ability, and the amount of α-catenin in tissues and cells of various animals including human can be quantified by an immunochemical assay. It can also be applied to an immunohistological staining method, and can confirm α-catenin expression in various cells and tissues.
Furthermore, since expression of α-catenin is not observed in certain cancer cell lines, it is expected as a reagent for determining cancer metastasis.

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Claims (6)

[Claims] 1. A monoclonal antibody that binds to α-catenin. 2. The monoclonal antibody according to claim 1, which binds to human α-catenin. 3. The monoclonal antibody according to claim 1, wherein the type is IgG _2a . 4. A hybridoma that produces a monoclonal antibody that binds to α-catenin according to claim 1. 5. The hybridoma according to claim 4, which is hybridoma α18. 6. An immunochemical assay reagent containing the monoclonal antibody according to claim 1.