JPH06141883A - Monoclonal antibody to epithelial metaplasia and hybridoma capable of producing the same - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody capable of selectively recognizing the epithelial metaplasia and a hybridoma capable of producing the monoclonal antibody. CONSTITUTION:A hybridoma [e.g. 2D11 (FERM P-13205)] capable of producing a monoclonal antibody selectively binding to the epithelial metaplasia is selected by fusing a cell capable of producing an antibody (e.g. a cell of the spleen) immunized by using a blood type active fraction of human gastric mucous membrane mucin to a cell of a myeloma. The resultant hybridoma is then subjected to cell culture or inoculated into an animal to collect the objective monoclonal antibody from the resultant ascitic fluid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a monoclonal antibody which does not react with normal tissue but only with metaplasia of epithelium and a hybridoma which produces the monoclonal antibody.

[0002]

BACKGROUND OF THE INVENTION Metaplasia is the conversion of fully differentiated normal mature tissue into related mature tissue. A classic example of this in epithelial tissue is the change of normal columnar epithelium into squamous epithelium. There are various causes of metaplasia such as mechanical stimulation and chemical stimulation to tissues, and it is known that preneoplastic changes occur depending on the expression site and species.

At present, metaplasia of epithelium has been diagnosed histologically, and it is recognized only as a morphological change of cells, and its biochemical essence is hardly known. If a monoclonal antibody that does not react with normal tissue but only with metaplasia of epithelium is obtained, it is expected to be extremely useful for diagnosing certain precancerous lesions. In addition, it is expected that it can be used for studying the relationship between metaplasia of epithelium and cancer, or biochemical study of metaplasia of epithelium.

[0004]

Therefore, the object of the present invention is to provide a monoclonal antibody which does not react with normal tissues but only with metaplasia of epithelium. Furthermore, the present invention also aims at providing a hybridoma that produces the monoclonal antibody.

[0005]

Means for Solving the Problems The present inventor has conducted diligent research to obtain a monoclonal antibody that selectively reacts with metaplasia of epithelium. As a result, the blood group active fraction of human gastric mucosa mucin was used as an antigen. It was found that a hybridoma obtained by fusing antibody-producing cells such as spleen cells of the immunized animal with myeloma cells unexpectedly produces a monoclonal antibody that selectively reacts with metaplasia of epithelium, and completed the present invention.

That is, the present invention does not react with normal tissues produced by a hybridoma obtained by cell fusion of antibody-producing cells of an animal immunized with blood group active fraction of human gastric mucosa mucin and myeloma cells. The present invention provides a monoclonal antibody that selectively reacts only with metaplasia and a hybridoma that produces the monoclonal antibody.

The present invention will be described in detail below. The blood group active fraction of human gastric mucosa mucin used for the production of the monoclonal antibody of the present invention is the method used by Yyrewicz EC et al. For the purification of cervical mucosa mucin (J. Biol. Chem., 256, 11895-
11904, 1981).

[0008] That is, cervical mucin (5g), 6M guanidine chloride, 10mM dithiothreitol (DTT), 5mM E
Solubilized with 0.3 M Tris buffer (pH 8.5) containing DTA, reacted with iodoacetamide to carboxymethylate the thiol group, and then added with 50 mM Tris buffer (pH: 0.15 M sodium chloride (pH).
Dialyze against 8.0) overnight at 4 ° C. 1% SD in mucin solution
Add both reagents to give S and 10 mM DTT (final concentration),
Bio-Gel A-50m column incubated at 0 ℃ for 15 minutes and equilibrated with 50 mM Tris buffer (pH 8.0) containing 0.1% SDS.
It is a method of charging to (2.5φ × 90 cm) and eluting with the same buffer to obtain a mucin fraction.

In order to obtain animal antibody-producing cells, the above fractions are used as an antigen to inoculate mice, rats, rabbits, sheep, horses, cows, etc. to immunize these animals, Antibody-producing cells such as splenocytes may be collected. In the immunization, Freund's complete adjuvant may be used as an adjuvant. When Freund's complete adjuvant is used, the blood group active fraction and the adjuvant are used in a volume ratio of 1: 1 to 1:10, preferably about 1: 1, and the blood group active fraction is 0.1 to 1: 1. 1 mg / ml, preferably 1
The antigen suspension may be prepared, for example, by suspending the suspension in about 0.25 to 0.5 M saline so that the concentration will be mg / ml.

By inoculating the immunized animal with the above antigen suspension, an animal immunized with the blood group active fraction of human gastric mucosa as an antigen can be obtained. For example, when immunizing a mouse, the above antigen suspension is repeated 1 to 3 times, preferably
Mice can be efficiently immunized by intraperitoneal administration 2-3 times at 1-2 week intervals, for example.

Antibody-producing cells can be obtained by separating antibody-producing cells such as spleen cells from the thus obtained immunized animal. For example, when using splenocytes as antibody-producing cells, after removing the spleen, for example, washed with RPMI1640 containing 15% fetal bovine serum (manufactured by GIBCO, hereinafter abbreviated as NS-1 medium), and splenic in the same medium. Can be cut, passed through a stainless mesh, and then centrifugally washed with the same medium to obtain antibody-producing cells.

As the myeloma cells used for cell fusion, cell lines of various animals such as mouse, rat, rabbit and human can be used, but hypoxanthine guanine phosphoribosyl transferase (HGP) is preferable.
RT) -deficient myeloma cells can be mentioned.
Such myeloma cells, in the unfused state, have HGPRT
Hypoxane / Aminopterin / Thymidine (H
Nucleic acid cannot be synthesized in AT) medium, and it will die. On the other hand, hybridomas that have fused with lymphocytes and acquired HGPRT activity are
Even if aminopterin inhibits nucleic acid biosynthesis, it can grow using hypoxanthine. Since only the hybridoma grows after the fusion treatment, it is suitable for the selection of hybridoma. The myeloma cells used in the present invention are preferably non-secretory cell lines.

Examples of such myeloma cells include mouse myeloma P3 / X63-Ag8U1 (P3U1) (Current top
ics in Microbiology & Immunology 81, 1-7, 1978
Year), P3 / NS1-1-Ag4-1 (NS-1) (European J. Immun. 6 Volume, 51
1-519, 1976), P3 / X63-Ag8-6 ・ 5 ・ 3 (X63 ・ 6 ・ 5 ・ 3) (J.Imm
un.123, pp. 1548-1550, 1979), SP2 / 0-Ag14 (SP2) (Nat
ure 276, pp. 269-270, 1978), Rat Myeloma 210RC
Y3 ・ Ag1 ・ 2 ・ 3 (Y3 ・ Ag1 ・ 2 ・ 3) (Nature 277, 131-133, 1979)
), Hitomieloma SKO-007, GM1500TG-A12, etc.

Cell fusion between an animal antibody-producing cell and a myeloma cell can be performed according to a conventional method. For example, RP
It is sufficient to mix 10 ⁷ to 10 ⁸ myeloma cells with 4 to 10 times as many antibody-producing cells as described above in an animal cell culture medium such as MI1640. At that time, as a cell fusion promoting substance, an average molecular weight of 10
You can use polyethylene glycol (PEG) from 00 to 6000, polyvinyl alcohol, Sendai virus, etc.
Is preferably used. Fusion treatment is usually 5-20 at 37 ℃
Minutes, preferably at 37 ° C. for 6 minutes.

To select hybridomas from the cells after the cell fusion treatment, selective growth in a selective medium may be performed. For example, after diluting the cells in a medium such as NS-1 medium to 5 × 10 ⁶ cells / ml, add 10 ⁵ to 10 ⁶ cells / well to the image wells on the microtiter plate. Then, a selective medium such as HAT medium is added, and thereafter, the selective medium is exchanged at an appropriate interval, for example, every 3 days, and the culture may be performed. For example, P3 / X63-Ag8U1 as myeloma cells
When (P3U1) is used, only hybridomas can be selected by culturing in HAT medium for about 10 to 14 days.

In order to select a hybridoma producing a monoclonal antibody which reacts with metaplasia from the obtained hybridomas, a hybridoma producing a monoclonal antibody which binds to the blood group active fraction of gastric mucosa is selected in advance, and further, A hybridoma producing a monoclonal antibody that binds to metaplasia can be selected from them by immunohistostaining.

Hybridomas producing a monoclonal antibody that binds to the blood group active fraction of gastric mucosa can be selected, for example, by enzyme-linked immunosorbent assay (ELISA) as follows. A sample containing the blood group active fraction of gastric mucosa is dissolved in a buffer solution such as phosphate buffered saline (PBS), sodium bicarbonate buffer (pH 8.0) in advance, and polystyrene, polyvinyl, polycarbonate, poly A solid phase such as acrylamide, silicon, dextran, and cellulose such as a soft plate or glass plate, beads, sheets, wells, and tubes is added, and the mixture is left at 4 ° C. overnight to adsorb the antigen.

Next, the antigen solution was discarded, washed with PBS, and then 1
Add PBS containing% bovine serum albumin (BSA),
After allowing it to stand overnight, block the site where no antigen is bound with BSA. After that, 50 μl of each hybridoma supernatant is added, and the mixture is allowed to stand at room temperature for 1 hour, for example, and then washed 3 times with PBS. Next, biotinylated anti-mouse immunoglobulin antiserum (second antibody) is added, and the mixture is allowed to stand at room temperature for 1 hour. PB
After washing 3 times with S, an avidin-bound enzyme is added, and the mixture is allowed to stand at room temperature for 15 minutes. After washing 4 times with PBS, add an enzyme substrate to develop the color, and measure with an absorptiometer, a fluorometer, etc. to select a hybridoma producing an antibody that binds to the blood group active fraction of gastric mucosa mucin. can do.

From the hybridomas obtained as described above, in order to select a hybridoma which produces a monoclonal antibody that binds to metaplasia of epithelium, screening by immunohistological staining may be performed as follows.
First, a formalin-fixed paraffin-embedded pathological tissue section is subjected to deparaffinization, hydrophilic treatment, and treatment with hydrogen peroxide-methanol to eliminate endogenous peroxidase, and a specimen that has been blocked with normal serum is reacted with the culture supernatant. Then biotinylated anti-mouse IgG antibody, avidin
React with biotin peroxidase complex,
3-amino-9-ethylcarba
Zole, AEC) is used as a substrate to develop color. Hematoxylin staining can be used as a counterstain to select hybridomas that produce antibodies that react with metaplasia of the epithelium.

Most of the hybridomas producing antibodies that react with the blood group active fraction of gastric mucosa, most of the antibodies have been reported by Bara et al. (Br. J. Cancer, Vol. 41, Vol. 209-221, 1980, Cancer Res., 4
Volume 6, Pages 3983-3989, 1986, Int.J.Cancer, Volume 47, 304-310
Page, 1991), but produces several% of hybridomas, but the produced antibody does not stain normal tissues by immunohistochemical staining, but only epithelial metaplasia. , And had properties different from any of the anti-gastric mucosal mucin antibodies already reported.

In this way, cells that produce antibodies that specifically react with metaplasia of the epithelium are selected and then cloned by the limiting dilution method or the like to obtain antibody-producing cells originating from a single hybridoma. be able to. An example of the hybridoma obtained as described above is hybridoma 2D11. Monoclonal antibody obtained from hybridoma 2D11 reacts with intestinal metaplasia of gastric mucosa and squamous metaplasia of cervix, gallbladder, and lung, but normal duodenal mucosa or normal stratified squamous epithelium Does not react with the tissue at all.

The above hybridoma 2D11 was used on October 2, 1992.
It has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology on the 0th day as Microindustrial Research Institute No. 13205 (FERM P-13205). Antibody production by the above-mentioned monoclonal antibody-producing hybridoma is performed by culturing in an animal cell culture medium such as RPMI1640 medium containing 10 to 20% fetal bovine serum or serum-free medium using a culture flask or a culture bottle, and the culture supernatant solution. Can be carried out by separating. The culturing method and conditions can be easily selected by those skilled in the art according to the usual animal cell culturing method.

As a method for producing a larger amount of antibody, for example, a mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered to an animal of the same origin as the parent myeloma cell of the hybridoma. After that, a method in which the hybridoma is intraperitoneally administered to allow the hybridoma to proliferate in a large amount can be adopted. According to this method, the hybridoma forms an ascites tumor in about 10 to 18 days, and a high concentration (about 1 to 20 mg / ml) of antibody is produced in serum and ascites.

If further purification is required, the ascites fluid is fractionated with ammonium sulfate, and then DEAE cellulose ion exchange chromatography, Sepharose 4B to which protein A or a blood group active fraction of gastric mucosa is bound is used. It can be purified by affinity column chromatography, molecular sieve column chromatography and the like.

[0025]

INDUSTRIAL APPLICABILITY This monoclonal antibody is useful for differentiating the site of metaplasia of epithelium from the site of other changes, and for diagnosing certain precancerous lesions. It is also a useful tool for studying the relationship between metaplasia and cancer, or for biochemical analysis of metaplasia.

[0026]

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

Example 1 Preparation of Antigen The mucous membrane of human stomach (O type, non-secretory type) was scraped off with a slide glass, boiled as PBS suspension for 10 minutes, homogenized and centrifuged to give a mucin-containing supernatant. 20 ml was obtained. The supernatant was dialyzed against distilled water overnight and then freeze-dried to produce crude stomach mucin.
40 mg was prepared. Crude gastric mucin was equilibrated with 20 mM phosphate buffer (pH 7.4) containing 1% SDS Sepharose 4B column (25φ ×
Gel filtration (400 mm) gave 11 mg of a sugar-containing fraction (antigen) that eluted with a void volume.

Example 2 Immunization 50 μg of the antigen obtained in Example 1 was intraperitoneally administered to BALB / c mice (female, 6 weeks old) as an emulsion of Freund's complete adjuvant for sensitization. Two weeks later, the same operation was performed, and further two weeks later, 50 μg of the antigen was intraperitoneally administered as a physiological saline solution for the final sensitization. Three days after the final antigen sensitization, the cervical spine of the mouse was dislocated and killed, and the spleen was aseptically removed. The spleen was placed in a Petri dish containing NS-1 medium, cut into small pieces with scissors, the tissue was loosened by pipetting, and a splenocyte suspension was obtained through a stainless mesh. Next, this suspension was centrifuged at 500 g for 7 minutes and the splenocytes collected were suspended in 30 ml of fresh NS-1 medium. This washing operation was repeated 3 times, and finally the splenocytes were stained with trypan blue to count the number of viable cells.

Example 3 Cell fusion 5 × 10 ⁷ mouse myeloma cells P3U1 were washed with 30 ml of NS-1 medium, P3U1 and 1.5 × 10 ⁸ splenocytes obtained in Example 2 were mixed, and centrifuged. Then, the supernatant was removed to obtain a cell pellet. Add 1 ml of PEG1500 to this pellet, then RPMI164
2 ml of 0 solution was gradually added to the final volume of 10 ml with RPMI1640, and the pellet obtained by centrifugation again contained 15% fetal bovine serum.
The spleen cells were suspended in HAT medium at 5 × 10 ⁸ cells / ml, and 0.1 ml of each was dispensed into a 96-well microplate.
The culture was performed in a 5% CO ₂ -incubator at 37 ° C., and 50 μl of HAT medium was added to each of the 4th and 6th days of the culture.

Example 4 Selection of Antibody-Producing Hybridoma (1) After cell fusion, the growth of the hybridoma was observed from day 9 and the antibody production in the culture supernatant was measured by the following method. The presence or absence of antibody production in the culture supernatant of the hybridoma obtained in Example 3 was examined by enzyme immunoassay. 50 μl of supernatant from wells in which hybridomas have grown
Each of them was added to a 96-well soft plate previously coated with the antigen. PBS for 1 hour at room temperature
After washing 3 times with 50 μl of peroxidase-labeled anti-mouse IgG antiserum (second antibody, CALTAG), the mixture was allowed to stand at room temperature for 1 hour. After washing 3 times with PBS, 0.01% hydrogen peroxide,
Add 50 μl of citrate-phosphate buffer (pH 5.0) containing 3.5 mM O-phenylenediamine and develop color for 30 minutes, then add 2N-H ₂ SO ₄
After stopping the reaction with 50 μl, the absorbance at a wavelength of 490 nm was measured to obtain 20 strains of hybridomas that produce antibodies that react with the blood group active fraction of gastric mucosa.

Example 5 Selection of Antibody-Producing Hybridoma (2) The 20 strains obtained in Example 4 were screened by immunohistostaining. Formalin-fixed paraffin-embedded pathological tissue sections in deparaffinizing xylene for 15 minutes 2
Soaked twice. Then, through the ethanol series of 99%, 90%, 80%, and 70%, soaking in 0.3% hydrogen peroxide-added methanol (30% hydrogen peroxide aqueous solution: methanol = 1: 100) for 20 minutes, the endogenous peroxidase activity After removing the water, it was washed with PBS. After 5% normal horse serum-PBS was dropped on the section and reacted for 20 minutes to block non-specific binding, it was lightly rinsed with PBS. 200-500 μl of culture supernatant is added to the specimens treated in this way.
After mounting and reacting for 30 minutes, the cells were washed 5 times with PBS for 3 minutes each. next. Biotinylated anti-mouse IgG antibody (horse, Vector
(Manufactured by the company), load 200-500 μl, react for 30 minutes, then with PBS
Washed 5 times for 3 minutes each.

The preparation of the avidin biotin peroxidase complex was carried out using Vectastain ABCkit (Vector).
Avidin DH: biotinylated peroxidase = 1:
The mixture was mixed at a molar ratio of 3 and reacted at room temperature for 30 minutes to form a complex. A solution containing this complex was dropped on a tissue section and reacted for 30 minutes. Then, it was washed with PBS 5 times for 3 minutes each. Next, the substrate solution prepared using AEC Substrate Kit (Vector) was dropped on the tissue section, and 20
Let react for minutes. It was washed with tap water for 5 minutes.

Hematoxylin staining was used as a counterstain. Tissue sections were stained in Mayer's hematoxylin solution for 3 minutes and washed with tap water for 20 minutes. Among the 20 strains, only the antibody produced by one hybridoma stained only the intestinal metaplasia of the gastric pylorus in the immunohistological staining. This hybridoma was cloned by the limiting dilution method to obtain hybridoma 2D11.

Example 6 Production of Monoclonal Antibody (1) The hybridoma 2D11 obtained in Example 5 was treated with NS-1 medium,
The culture supernatant was collected by culturing at 37 ° C. and 5% carbon dioxide incubator while scaling up the 96-well plate, 25 dl flask, and 75 dl flask.

Example 7 Production of Monoclonal Antibody (2) Hybridoma 2D1 was hybridized with 6-week-old BALB / c mice by intraperitoneal injection of 0.5 ml of pristane (manufactured by Aldrich) and 3 weeks later.
1 was intraperitoneally injected at a rate of 1 × 10 ⁷ cells / mouse.
One to two weeks after the hybridoma transplantation, after confirming the swelling of the abdomen, the laparotomy was performed and the ascites was collected. About 5 from 1 mouse
Ascites fluid of 1 ml was collected, and 1 ml of ascites contained about 10 mg of monoclonal antibody protein.

Example 8 Characteristics of Monoclonal Antibody (1) The reactivity of the monoclonal antibody produced by hybridoma 2D11 with each tissue was examined by the same method as in the screening of Example 5. The results are shown in Table 1.

[0037]

[Table 1]

As a result, it was shown that the monoclonal antibody produced by hybridoma 2D11 selectively reacts with intestinal metaplasia of gastric mucosa and squamous metaplasia of cervix, gallbladder and lung.

Example 9. Characteristics of monoclonal antibody (2) The antigen recognition site of the monoclonal antibody produced by hybridoma 2D11 was examined by the following method. Hybridoma 2D11
Affinity chromatography of gastric mucin was carried out using a gel bound with a monoclonal antibody produced by Saccharomyces cerevisiae, and only high-molecular-weight (molecular weight: several million) mucin was bound.

The gel bound with the monoclonal antibody was prepared as follows. Purified 2D11 5 mg (1.2 ml
0.2 M phosphate buffer (pH 7.5) and 0.8 g of formyl cellofine (Seikagaku Corporation) were shaken at 4 ° C for 2 hours, and then 6 mg of NaCNBH ₃
Was added and shaken at 4 ° C. for 8 hours. After washing the gel with distilled water, 1.5 ml and 6 mg of phosphate buffer containing 1 M ethanolamine
NaCNBH ₃ was added and shaken at room temperature for 2 hours, the gel was washed with distilled water, and then suspended in PBS to prepare. The reaction of this antibody with gastric mucin and metaplastic tissue of the stomach disappeared by the periodate treatment of gastric mucin and tissue. Therefore, this antibody is considered to be an antibody that recognizes the sugar chain of mucin.

Example 10 Characteristics of Monoclonal Antibody (3) The subclass of the monoclonal antibody produced by hybridoma 2D11 was determined by the ELISA method and the immunoblot method using the Subclass Kit of Binding Site. As a result, this antibody was found to be an antibody of the IgG2b subclass.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/574 Z 9015-2J 33/577 B 9015-2J (C12P 21/08 C12R 1:91)

Claims (3)

[Claims] 1. A monoclonal antibody that reacts with metaplasia of epithelium 2. A monoclonal antibody produced by a hybridoma of an antibody-producing cell of an animal immunized with a blood group active fraction of human gastric mucosa mucin and a myeloma cell of the animal, and reacting to metaplasia of epithelium. 3. A hybridoma producing a monoclonal antibody that reacts with metaplasia.