JPH0369280B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an anti-human cervical cancer antibody, a method for producing the antibody, and a method for detecting cancer cells using the antibody. [Background of the Invention] Uterine cancer has a high incidence rate among cancers in women. Among these, cervical cancer is the cancer with the highest number of deaths, and even though the number of deaths is decreasing, it can be said that it remains an important cancer. Cervical cancer has a high five-year survival rate of 87.0% if it is detected in the first stage and treated with surgery, but in the second stage it is 71.4%, in the third stage it is 50.1%, and in the fourth stage it is 18.2%.
%. Therefore, early detection is important for cervical cancer as well as for other cancers. The current method for diagnosing cervical cancer is to collect cells from the cervix, smear and fix the cells on a glass slide, and then examine them under a microscope to see if cancer cells exist. This is a method called diagnosis. In this method, cells are stained with Papanicolaou staining or Giemsa staining to detect irregular nuclear edges, enlarged and increased nucleoli, and dissimilarity between the nucleus and cytoplasm.
Based on abnormal mitotic figures, presence or absence of multinucleated cells, etc., class (normal), (abnormal but no suspicion of malignancy),
Classified into (suspicious of malignancy, (highly suspected of malignancy), and (definitely malignant). However, this method does not specifically stain only cancer cells, so diagnosis is made by cytology of early cancer. It is said that diagnosing advanced cancer is more difficult and requires considerable knowledge and training.On the other hand, as the number of people undergoing mass screening for uterine cancer is increasing year by year, it is necessary to test a large number of clinical specimens. At present, there is a need to improve staining methods in cytodiagnosis to make them more rapid and objective.In order to improve staining methods in cytodiagnosis, this inventor We considered using antigen-antibody reactions.In other words, if antibodies specific to cervical cancer cells can be obtained, cervical cancer cells can be specifically stained using the labeled antibody method. Therefore, this inventor attempted to produce an antibody against cervical cancer cells.As the antibody, he developed a monoclonal antibody that specifically reacts with only one type of antigenic determinant. Conventionally, monoclonal antibody production methods include:
An animal is immunized with malignant tumor cells, antibody-producing cells obtained from the animal are fused with myeloma cells to obtain hybridomas, hybridomas that produce antibodies are selected, and the selected hybridomas are cultured. [Nature, Vol. 256, p. 495, 1975;
-45407] is known. However, it is unknown whether cervical cancer cells possess antigenic determinants useful for the production of monoclonal antibodies. As a result of repeated research, this inventor succeeded in producing several types of monoclonal antibodies against cervical cancer cells, confirmed the location of the antigenic determinant recognized by the antibodies on the cells, and also used the labeled antibody method to develop the antibodies. They succeeded in specifically staining cervical cancer cells using fluorescent or enzyme-linked immunosorbent techniques, thereby making it possible to easily and quickly diagnose cervical cancer at an early stage. [Structure of the Invention] That is, the present invention provides: (A) an immunoglobulin subclass G _{2 a} that reacts with antigenic determinants of human cervical cancer cells but does not react with normal tissues of the human cervix; or
Monoclonal antibodies that belong to G ₂ b; (B) antibody-producing cells and myeloma cells of mammals (other than humans) immunized with human cervical cancer cells isolated from culture vessels without using proteolytic enzymes; The hybridomas produced by the fusion of the cells were cultured, and the cultures reacted with the antigenic determinants of human cervical cancer cells, but not with the normal tissue of the human cervix, and showed immunoglobulin subclass G ₂ a or
A method for producing the above-mentioned antibody, which is characterized by separating and collecting a monoclonal antibody belonging to G ₂ b, and (C) preparing a tissue sample to be examined against an antigenic determinant of human cervical cancer cells. When contacted with a labeled or unlabeled monoclonal antibody that reacts with but does not react with the normal tissue of the human cervix and belongs to immunoglobulin subclass G ₂ a or G ₂ b. , in the latter case, further contacting with a labeled second antibody,
The present invention provides a method for detecting human cervical cancer cells, which is characterized by searching for a label. In addition, this invention provides: (D) Antigenic sites of human cervical cancer cells obtained by fusing antibody-producing cells of mammals (other than humans) immunized with human cervical cancer cells with myeloma cells. A hybridoma that produces a monoclonal antibody that reacts with human cervical cancer cells, and (E) antibody-producing cells of a mammal (excluding humans) that have been immunized with human cervical cancer cells are fused with myeloma cells, and human cervical cancer is produced from the fused cells. This makes it possible to carry out a method for obtaining hybridomas, which is characterized by selecting those that produce monoclonal antibodies that react with antigenic determinants of cells. Hereinafter, the above invention will be explained in detail. (Production of Antibody) 1. Immunization To produce the monoclonal antibody of the present invention, first, a mammalian animal is immunized with human cervical cancer cells. As human cervical cancer cells, primary culture cells or established culture cells such as HeLa cells can be used. Immunization is carried out, for example, as follows. Cells were treated with fetal bovine serum (10% v/
v), Dulbecco's containing penicillin (100U/ml), streptomycin (100μg/ml)
Culture in a suitable medium such as MEM medium (hereinafter referred to as growth medium), and after growing the cells until they are spread all over the culture flask, remove the supernatant and add calcium- and magnesium-free phosphate buffer (PH7. 2,
Wash the flask with PBS (hereinafter referred to as PBS) and detach the cells from the culture flask by spraying PBS onto the cell surface with a pipette. This method causes less loss of antigens on the cell surface than detaching cells with proteolytic enzymes such as trypsin. Cervical cancer cells are collected by separation means such as centrifugation, and mammals such as mice and rats are immunized. It is preferable that the mammal be of the same lineage as the permanently proliferating cell of the partner for cell fusion. The age of the animal is preferably 5 to 10 weeks for mice, for example. The sex can be either male or female. The number of human cervical cancer cells used for immunization is preferably 5 x 10 ⁶ to 2 x 10 ⁷ per mouse, for example. The cells are preferably suspended in PBS or mixed with Freund's complete adjuvant at a ratio of 1:1 to form an emulsion and administered to animals intraperitoneally, intravenously, subcutaneously, or the like. This immunization procedure is performed 1 to 5 times at intervals of 1 to 3 weeks. The final immunization is carried out, for example, by suspending the cells in PBS and administering the suspension intravenously or intraperitoneally to the animal. Polyclonal antibodies can be obtained from the body fluids or antibody-producing cells of animals immunized in this way. 2 Cell fusion Antibody-producing cells are extracted from the animal immunized with human cervical cancer cells as described above. Antibody-producing cells can be obtained from spleen, lymph nodes, peripheral blood, etc., but spleen is preferred. For example, the spleen is removed aseptically 2 to 5 days after the final immunization, cut into small pieces with scissors in Dulbecco-MEM medium, the spleen cells are suspended, and then the spleen cells are collected and used by centrifugation. . Myeloma cells are used as permanently proliferating cells to be fused. Preferably, the myeloma cells are derived from the same species of animal as the antibody-producing cells. For example, in the case of a mouse, P3X63−Ag8.U1 (P3U1), P3/
NS1/1-Ag4-1 (NS-1), SP2/0-
Ag14 (SP2), P3X63Ag8 (X63), P3X63−
Ag8.653 (653) etc. are used. Moreover, as myeloma cells, those having characteristics that can be used as markers during selection, such as 8-azaguanine resistant cell lines and hypoxanthine guanine phosphoribosyltransferase-deficient cell lines, are preferable. These cell lines are available from, for example, American Type Culture Collection (ATCC) or Dainippon Pharmaceutical Co., Ltd. For fusion, any of these myeloma cells are cultured in a growth medium and collected by centrifugation after washing with, for example, Dulbecco's MEM medium prior to fusion. The fusion is performed, for example, as follows. Antibody-producing cells (e.g. spleen cells) and myeloma cells are mixed at a cell number ratio of 2 to 10:1, and a fusion promoter such as polyethylene glycol is gradually added while keeping the temperature at 37°C to cause cell fusion. let Fusion is stopped by adding culture medium and diluting the fusion promoter, and cells are separated by centrifugation. Cells are then, for example, suspended in HAT medium with hypoxanthine, aminopterin, and thymidine added to the growth medium and placed in a 96-well microtest plate.
Dispense 200 μl/well and culture in a CO ₂ incubator at 37°C, 5% CO ₂ , and 95% humidity (hereinafter, all culture conditions in the CO ₂ incubator are the same as below). Replace half of the culture medium with fresh HAT medium every 2 days. After culturing for about one week, the medium is changed to HT medium, which is a growth medium supplemented with hypoxanthine and thymidine. 3 Screening and cloning of hybridomas Next, culture in HT medium for several days, and when the hybridoma colonies have spread to about half of the wells of the microtest plate, determine which wells have hybridomas containing monoclonal antibodies against cervical cancer cells. Screen to see if it is producing. Screening is performed, for example, as follows. A portion of the culture supernatant from a well in which hybridomas are growing is taken, and whether or not it reacts with cultured cervical cancer cells is examined using a known labeled antibody method such as an enzyme antibody method or a fluorescent antibody method. Next, hybridomas that produce monoclonal antibodies that react with cervical cancer cells are cloned using known techniques such as limiting dilution and soft agar to create hybridomas that produce a single monoclonal antibody. Select a group. It is desirable to repeat cloning and screening two or more times. 4 Production of Monoclonal Antibodies Monoclonal antibodies are produced by culturing the hybridomas obtained as described above in vitro (in a culture device or in a nutrient medium) and in vivo (in a living body or in an animal tissue). Cultivation is performed, for example, as follows. For in vitro cultivation, hybridomas are cultured in a suitable medium such as a growth medium, for example in a CO ₂ incubator. When the hybridomas have grown to their growth limit, the culture solution is collected, and the hybridomas and the culture supernatant are separated using a solid-liquid separation means such as centrifugation. Depending on the purpose, the monoclonal antibody in the culture supernatant can be used without purification, but if it is to be separated, it can be salted out with ammonium sulfate and then dialyzed with 0.02M phosphate buffer (PH7.2). Thereafter, it is purified by passing it through a diethylaminoethyl cellulose column or the like. Hybridomas isolated from the culture supernatant can be treated with dimethyl sulfoxide (5-10% v/v), for example.
and in a suitable medium such as Dulbecco-MEM supplemented with fetal bovine serum (10-20% v/v).
By suspending the cells at a density of 10×10 ⁶ cells/ml, placing them in a suitable ampoule, and gradually freezing them at -80°C or lower, it is possible to preserve them in a living state for a long period of time. In particular, hybridomas can be preserved semi-permanently under ultra-low temperatures such as liquid nitrogen. When culturing hybridomas in vivo, the hybridomas are transplanted into any animal, but it is preferable to use the same species as the animal from which the spleen cells used for cell fusion were collected. for example
In the case of BALB/c mice, 0.5 ml of 2,6,10,14-tetramethylpentadecane is injected intraperitoneally 1 to 3 weeks before hybridoma transplantation, and 2 to 10 × 10 ^Six hybridomas are injected intraperitoneally. After 1 to 2 weeks, ascites fluid containing a high concentration of monoclonal antibodies accumulates in the mouse's peritoneal cavity and the abdomen becomes enlarged, so the ascites fluid is collected and purified in the same manner as the culture supernatant. 5 Characteristics of Monoclonal Antibodies The characteristics of the monoclonal antibodies obtained as described above are investigated, for example, as follows. First, in order to examine which site of cultured cervical cancer cells the monoclonal antibody reacts with, a known labeled antibody method, such as a fluorescent antibody method or an enzyme antibody method, is performed. Next, we examined the specificity of monoclonal antibodies.
The reactivity with cultured human normal cells and cultured human cancer cells and with existing cancer-related antigens is determined by a known label immunoassay (eg, enzyme immunoassay). Further, the immunoglobulin class and subclass of a monoclonal antibody can be determined by a labeled (eg, enzyme) immunoassay using an anti-immunoglobulin class/subclass antibody. The monoclonal antibody of this invention belongs to the immunoglobulin subclass G ₂ a or G ₂ b and reacts with antigenic determinants of human cervical cancer cells but does not react with normal tissues of the human cervix. . (Applications) The monoclonal antibody of the present invention is used, for example, in a method for detecting human cervical cancer cells in cytodiagnosis. This detection method involves contacting a tissue sample to be examined with the labeled monoclonal antibody of the present invention, and then using a label detection means to search for a label attached to the cell, or by searching for a label attached to the cell using a label detection means. After contacting the cell with the monoclonal antibody of the present invention, the cell is brought into contact with a labeled second antibody (an antibody that can bind to the monoclonal antibody of the present invention), and the label attached to the cell is searched for using a label detection means. It is done by twisting. Labels include radioactivity,
Enzymes, fluorescent compounds (eg, fluorescein isothiocyanate, tetramethylhodamine isothiocyanate), and the like are used. Production of second antibody,
Attachment of the label to the antibody is carried out by a conventional method. The above detection method includes (a) the labeled or unlabeled antibody of the invention, (b) optionally a labeled second antibody, and (c) other necessary reagents and Equipment (e.g. tissue collection equipment, buffer solutions,
It is convenient to make and use a diagnostic kit that includes a chamber slide (e.g. chamber slide). Since the monoclonal antibody of this invention specifically reacts with human cervical cancer cells, it greatly contributes to speeding up and objectifying the diagnosis of cervical cancer. [Examples] The present invention will be explained in more detail below using Examples. Example 1 Production of monoclonal antibodies against human cervical cancer (1) Immunization HeLa cells (purchased from Dainippon Pharmaceutical Co., Ltd.), which are human cervical cancer-derived cells, were incubated in a growth medium.
Cultured in a CO ₂ incubator. Once the cells have formed a monolayer, discard the culture medium and peel off the cells by spraying PBS onto the bottom of the culture container with a pipette.
Cells were collected by centrifugation (400 xg, 5 minutes). The HeLa cells were suspended in PBS, thoroughly mixed with Freund's complete adjuvant at a ratio of 1:1, and 1× It was injected intraperitoneally in 10 ⁷ pieces (0.4 ml). Two weeks later, HeLa cells collected in the same manner as above were suspended in PBS and administered into the tail vein at 1×10 ⁷ cells (0.4 ml) per animal. (2) Cell fusion Three days after the second immunization, the spleens were aseptically removed from the two immunized mice, and the finely chopped spleen cells were suspended in Dulbecco-MEM medium. Cells were collected by centrifugation for minutes at room temperature. After washing the cells twice with Dulbetsko-MEM medium, they were left at room temperature until fusion. Mouse myeloma cells NS-1 (purchased from Dainippon Pharmaceutical Co., Ltd.) were cultured in a growth medium in a CO ₂ incubator. Immediately before cell fusion, NS-1 cells were transferred to a centrifuge tube and centrifuged at 200 xg for 5 minutes at room temperature to collect the cells.
Cells were washed two more times with Dulbecco-MEM medium and left at room temperature until confluence. 1.5 x 10 ⁸ spleen cells and 3 x 10 ⁷ NS-1 cells
The cells were transferred to a centrifuge tube, mixed, and centrifuged at 400×g for 5 minutes at room temperature. The supernatant was removed and the cells formed into a compact pellet. Centrifuge tube at 37°C
While keeping this, Darbetzko-MEM 37
1 ml of 50% polyethylene glycol (PEG4000, Merck) warmed to °C was gradually added over 1 minute and mixed with the cells. The cell pellet was continued to be stirred with the pipette tip for an additional minute. Next, 10 ml of Dulbecco's MEM medium warmed to 37°C was added over 5 minutes while stirring, centrifuged at 400 xg for 5 minutes at room temperature, and the supernatant was removed. Fetal bovine serum (10% v/v), penicillin (100 U/ml), streptomycin (100 μg/ml), hypoxanthine (1x
10 ^-4 M), aminopterin (4 x 10 ^-7 M), and thymidine (1.6 x 10 ^-5 M).
MEM medium (hereinafter referred to as HAT medium) at 60%
ml to make a cell suspension and transfer it to three 96-well micro test plates (Falcon 3072).
200 μl/well was dispensed and cultured in a CO ₂ incubator. Add half of the culture solution to a new HAT every 2 days.
Replaced with medium. Seven days after the start of culture, hybridoma colonies became visible to the naked eye, but the medium was changed from HAT medium to HT medium, which does not contain only aminopterin, and culture was continued. (3) Screening and cloning of hybridomas On the 11th day after the start of culture, hybridoma colonies had grown to about half of the bottom area of the wells in most of the wells of the 96-well microtest plate. We screened to see if they produced antibodies against. HeLa in a 96-well microtest plate
Cells were cultured until they formed a monolayer, and after removing the culture supernatant, the wells were filled with a 1% w/v bovine serum albumin solution in PBS (hereinafter referred to as 1% BSA) and left at room temperature for 1 hour. .
After washing the wells three times with phosphate buffer containing 0.05% v/v Tween 20 (hereinafter referred to as Tween-PBS), remove the hybridoma culture supernatant.
50 μl/well was dispensed and left at room temperature for 2 hours. After washing the wells 4 times with Tween-PBS, peroxidase-labeled anti-mouse IgG+
IgM rabbit antibody (Zymet Laboratories)
A 1/1000 diluted solution was dispensed at 100 μl/well and left at room temperature for 2 hours. Tween
After washing the wells four times with PBS, 100 μg/ml of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (hereinafter referred to as ABTS) and 0.1 M citric acid containing 0.03% hydrogen peroxide were added. A buffer solution (PH4.2) was added at 100 μl/well and allowed to react at room temperature for 30 minutes. The reaction was stopped by dispensing 2 mM sodium azide solution at 100 μl/well, and the development of blue-green color in the wells was observed, and it was determined that antibodies against cervical cancer were present in the colored wells. The same procedure was carried out by adding 100 μl/well of a solution of normal human serum diluted to 1/10 with PBS.
A 96-well microplate coated after being left overnight at 4°C was also tested. Wells containing hybridomas producing monoclonal antibodies that react with HeLa cells are
There were 7 wells out of 288 wells, and 0 out of 288 wells reacted with normal human serum. Next, the hybridomas in these 7 wells were cloned by the limiting dilution method. The thymus of a mouse (BALB/c, 3 weeks old) was removed aseptically, cut into small pieces with scissors, and the thymocytes were suspended in a growth medium to make a cell suspension of 5 × 10 ⁶ cells/ml. A diluted solution of 5 hybridomas/ml was dispensed into a 96-well microtest plate at 200 μl/well, and CO ₂
Cultured in an incubator. 12 days after starting culture
On day 1, the culture supernatants of wells containing only one hybridoma colony per well were screened for hybridomas producing the above-mentioned anti-cervical cancer cell monoclonal antibody. This cloning and screening procedure was repeated twice, and two types of hybridomas, 1C2 and 2G12, were established. (4) Production of monoclonal antibodies Each established hybridoma was suspended in a growth medium at an initial cell density of 3 x ¹⁰ cells/ml, cultured in a CO ₂ incubator, and after 4 days, the culture solution was centrifuged (400 x g, 10 minutes) and
Culture supernatant containing monoclonal antibodies was collected. In culturing hybridomas in vivo,
2 weeks before transplanting the hybridoma
Intraperitoneally administered to BALB/c mice (8 weeks old)
Each animal was injected with 0.5 ml of 2,6,10,14-tetramethylpentadecane. These mice were injected 1x with two types of hybridomas.
Dulbecco MEM at a cell density of ¹⁰⁷ cells/ml
Cell suspension suspended in culture medium per animal
0.5 ml was injected intraperitoneally. After 15 days, the ascites fluid (5 to 8 ml) accumulated in the abdominal cavity of the mouse was collected by suction using a syringe, and after centrifugation (400 x g, 10 minutes), the ascites supernatant containing the monoclonal antibody was collected. In this way, two types of hybridomas,
1C2 and 2G12 were cultured, and the monoclonal antibodies produced by each hybridoma were isolated as follows. An equal amount of saturated ammonium sulfate was gradually added to the culture supernatant or ascites supernatant containing the monoclonal antibody at 4°C for salting out, and the mixture was dialyzed against 0.02M phosphate buffer (PH7.2) for 24 hours. This dialyzed fraction was passed through a diethylaminoethyl cellulose column (Watmann) equilibrated with 0.02M phosphate buffer (PH7.8), and the eluate was ultrafiltered, and the protein concentration was approximately 10 mg/ml as determined by absorbance measurement at 280 nm. Concentrate under reduced pressure until
Approximately 3 ml of a concentrated solution containing the monoclonal antibody was obtained. Each of the obtained monoclonal antibodies was
They were named -1 and M-2. All of these are
The molecular weight determined by electrophoresis was approximately 150,000. Example 2 Characteristics of monoclonal antibodies (1) Fluorescent antibody method HeLa cells were grown in a laboratory culture chamber/slide (4804, Miles Laboratories) in growth medium at an initial cell density of 1 x ¹⁰⁴ cells/ml. After culturing for 3 days, the culture supernatant was removed and the cells were filled with 1% BSA. After being left at room temperature for 1 hour, the cells were washed three times with Tween-PBS, 200 μl of culture supernatant containing monoclonal antibodies were added, and the mixture was allowed to react at room temperature for 2 hours. Next, remove the supernatant, wash 4 times with Tween-PBS, and add 100 μl of FITC-labeled anti-mouse IgG + IgM goat antibody solution (1/10 dilution in PBS, KPL).
The reaction was allowed to take place at ℃ for 30 minutes. Remove supernatant and PBS
After washing four times with , it was examined using a fluorescence microscope (Nikon Diaphoto TMD-EF). Of the two types of monoclonal antibodies, strong fluorescence was observed on the cell surface of HeLa cells with M-1, and strong fluorescence was observed on the intercellular space of HeLa cells with M-2. In this way, the location on cells of the antigenic determinant recognized by each monoclonal antibody was confirmed. (2) Determination of class and subclass of monoclonal antibodies HeLa cells were cultured in a 96-well microtest plate, and when a monolayer was formed, the culture supernatant was removed, 1% BSA was added, and the mixture was left at room temperature for 1 hour. did. Next, remove the supernatant and add Tween-
After washing four times with PBS, culture supernatant containing monoclonal antibodies was dispensed at 100 μl/well and left at room temperature for 2 hours. After washing four times with Tween-PBS, the classes and subclasses of the two monoclonal antibodies were determined using MonoAb-ID EIA KIT (Zymets Laboratories) (1st
table).

[Table] (3) Cross-reactivity with antibodies to known cancer-related antigens Whether the monoclonal antibody of the present invention reacts with known cancer-related antigens is determined by inhibition of monoclonal antibody binding by antibodies to known cancer-related antigens. I investigated it as. CEA, ferritin, AFP, and β ₂ -microglobulin were investigated as known cancer-related antigens. HeLa cells were cultured in a 96-well microtest plate until they formed a monolayer, and after removing the culture supernatant, the wells were filled with 1% BSA and incubated at room temperature.
I left it for a while. After washing 3 times with Tween-PBS,
Anti-CEA rabbit antibody (Dako), anti-ferritin rabbit antibody (Dako), anti-AFP rabbit antibody (Hoechst), and anti-β ₂ -microglobulin rabbit antibody (Seikagaku) were each used with bovine serum albumin (0.1% w/v), It was diluted to 1/50 with 10 mM Tris-HCl buffer containing Tween 20 (0.05% v/v), dispensed at 100 μl/well, and left at room temperature for 3 hours. After washing four times with Tween-PBS, culture supernatant containing monoclonal antibodies was dispensed at 100 μl/well and left at room temperature for 2 hours. After washing the wells four times with Tween-PBS, a 1/1000 dilution of peroxidase-labeled anti-mouse IgG + IgM rabbit antibody (Zymets Laboratories) was dispensed at 100 μl/well and left at room temperature for 2 hours. After washing the wells four times with Tween-PBS, add 0.1M citrate buffer (PH4.2) containing 100 μg/ml ABTS and 0.03% hydrogen peroxide.
100 μl/well was added and allowed to react at room temperature for 30 minutes. 2mM sodium azide solution
Dispense 100μ1/well to stop the reaction,
Absorbance at 414 nm was measured (Titertech Multiscan, Flow Laboratories). As a result, although the antibodies against known cancer-related antigens are reacted in large excess, the binding between the monoclonal antibodies and HeLa cells, which are the antigens, is hardly inhibited. It is thought that it does not react with antigens (Table 2).

[Table] [Numbers in the table indicate (absorbance when adding antibody to cancer-related antigen/absorbance when not adding antibody to cancer-related antigen.] (4) Cultured human normal cells and cultured human cells Reactivity with cancer cells Flow4000 (Fetoken) as cultured human normal cells
Cultured human cancer cells such as C32 (melanin-deficient melanoma), G402 (Ken leiomyoblastoma), and MIA PaCa−
2 (pancreatic cancer), PA-1 (ovarian teratoma), HT-
1197 (bladder cancer), COLO-201 (colon adenocarcinoma)
ZR-75-1 (breast cancer), IMR-32 (neuroblastoma), MOLT-3 (acute lymphoblastic leukemia) [all purchased from Dainippon Pharmaceutical Co., Ltd.] were isolated on a 96-well microtest plate. The cells were cultured until a layer was formed. After removing the culture supernatant, the wells were filled with 1% BSA and left at room temperature for 1 hour. After washing three times with Tween-PBS, the culture supernatant containing the monoclonal antibody was dispensed at 100 μl/well and left at room temperature for 2 hours. After washing the wells four times with Tween-PBS, a 1/1000 dilution of peroxidase-labeled anti-mouse IgG + IgM rabbit antibody (Zymets Laboratories) was dispensed at 100 μl/well and left at room temperature for 2 hours. After washing 4 times with Tween-PBS, add 100 μg/ml ABTS.
0.1M citrate buffer (PH4.2) containing 0.03% hydrogen peroxide was added at 100 μl/well and allowed to react at room temperature for 30 minutes. The reaction was stopped by dispensing 2 mM sodium azide solution at 100 μl/well, and the absorbance at 414 nm was measured (Titertech Multiscan, Flow Laboratories). Comparing the absorbance when reacting the monoclonal antibody with HeLa cells and the absorbance when reacting with other cultured cells, M-1 and HT-1197
It was observed that there was almost no reaction except for strong reaction with MIA PaCa-2 and weak reaction with C-32. From this result, it was confirmed that these monoclonal antibodies reacted quite specifically with cervical cancer cells (Table 3).

[The symbols in the table are Absorbance of cultured cells/Absorbance of HeLa cells <0.25: -, 0.26-0.50: ±, 0.51-0.75: +, 0.76-1.00: ++. ]

Test Example 1 Cervical Cancer Cytology A cotton swab or a wooden or plastic spatula was used to scrape the uterine vagina, and then the inside of the cervix was rubbed while rotating, and cells were evenly smeared onto a glass slide. It was fixed in 95% ethanol solution for 20 minutes. After washing three times with PBS, the cells were left in PBS containing 0.3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity. After washing 3 times with PBS, 2%
It was left in horse serum for 30 minutes. 1 ml of the culture supernatant containing the monoclonal antibody was dropped onto the cell smear on a slide glass and allowed to react at room temperature for 1 hour. After washing 3 times with PBS, peroxidase-labeled anti-mouse IgG
Rabbit antibody (Zymets Laboratories) 1000
Drop 1 ml of the 1:1 dilution onto the cell smear and let it cool at room temperature.
The reaction was allowed to proceed for 30 minutes. After washing 3 times with PBS, 250μ
The mixture was left in PBS containing g/ml of 3,3'-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide at room temperature for 7 minutes. After washing three times with PBS, nuclear staining was performed with hematoxylin for 5 minutes at room temperature. After washing under running water for 10 minutes, it was left in a 95% ethanol solution for 10 minutes, and then in xylene for 15 minutes. Excess xylol was wiped off, the sample was sealed before drying, and observed under a microscope. Neither of the two types of monoclonal antibodies reacted with normal cases at all, and all cases with cervical cancer showed positive reactions (Table 4).

【table】

Claims (1)

[Scope of Claims] 1. Reacts with antigenic determinants of human cervical cancer cells, but does not react with normal tissues of the human cervix, and is an immunoglobulin subclass G ₂ a or G ₂ b
A monoclonal antibody that belongs to 2. The antibody according to claim 1, which is produced by a hybridoma obtained by fusion of antibody-producing cells of a mammal (other than humans) immunized with human cervical cancer cells and myeloma cells. 3. Hybridomas produced by fusion of antibody-producing cells of mammals (excluding humans) immunized with human cervical cancer cells isolated from an incubator and myeloma cells without the use of proteases are cultured, and one offspring is generated from the culture. Isolation and collection of monoclonal antibodies that react with antigenic determinants of cervical cancer cells but do not react with normal tissues of the human cervix and belong to immunoglobulin subclass G ₂ a or G ₂ b. A method for producing the above antibody, characterized by: 4. The manufacturing method according to claim 3, wherein the culturing is carried out in a nutrient medium. 5. The manufacturing method according to claim 3, wherein the culture is carried out in animal tissue. 6. The sample of tissue to be examined is tested for immunoglobulin subclass G ₂ a or G ₂ b that reacts with antigenic determinants of human cervical cancer cells but not with normal tissue of the human cervix. A method of searching for a label by contacting a monoclonal antibody belonging to the human body with or without a label, and in the case of the latter, further contacting with a labeled second antibody. How to detect cervical cancer cells. 7. The method according to claim 6, wherein the label is a fluorescent compound, an enzyme, or a radioisotope.