JPH05142228A - Classification and identification reagent for human prostate cancer - Google Patents

Abstract

PURPOSE:To obtain the reagent, which can perform the quick, highly reliable identification without complicated procedures by chemically bonding radioactive material, fluorescence pigment, enzyme a marker for observation with an electron microscope or a group containing the structure for bonding them at the secondary step to an IgG antibodies. CONSTITUTION:Radiation active material, fluorescence pigment, enzyme a marker for observation with an electron microscope or a group containing the structure for bonding these materials in the second step are chemically bonded to IgG antibodies. The IgG antibodies are secreted from hybridoma between the antibody producing cells of anti-human prostate cancer and cells which can be subcultured for a long period in vitro. Thus, the derivative of the IgG antibodies is obtained. The derivative is contained, and the reagent for identification is obtained. The radioactive material is <125>I, the fluorescence pigment is fluorescein or rhodamine and the enzyme is peroxidase or rhodamine. The structure for bonding the radiation active material, the fluorescence pigment, the enzyme or the marker for observation with the electron microscope in the second step is biotin.

Description

Detailed Description of the Invention

The present invention comprises a derivative of an antibody secreted by a fused cell (hereinafter referred to as a hybridoma) between an antibody-producing cell for human prostate cancer and a cell capable of long-term subculture in vitro, and the derivative thereof. The present invention relates to a reagent for classification and identification of prostate cancer.

The term "antibody derivative" as used herein refers to a group containing a radioactive substance, a fluorescent dye, an enzyme, a marker for electron microscope observation, or a structure for secondarily binding them to the antibody. It is a chemically coupled product.

In recent years, cancer has become the disease with the highest mortality rate among all diseases, and has become a social problem.
Although the countermeasures have been examined from all sides, a fundamental solution has not yet been found. In Japan, gastric cancer, lung cancer, and liver cancer are more common than in the past, and account for more than 50% of all deaths due to cancer, but in recent years, different types of cancer have also increased due to changes in lifestyle. Urological malignancies including prostate cancer are typical examples.

At present, the treatment methods for these cancers are mainly surgery and are performed by combining radiation therapy, chemotherapy and immunotherapy. However, such treatment methods have limitations in terminal cancer and advanced cancer, and early diagnosis and early treatment are considered to be the best methods. To that end, a rapid and highly reliable method for identifying and diagnosing cancer cells and cancer tissues is required.

On the other hand, cancer-associated antigens present on the surface of cancer cells
A huge amount of research has been carried out on cancer-specific antigens, and its existence has been proved especially in transplantation experiments using experimental animals, but human cancer has not been sufficiently examined. Currently, the antigens present on the surface of cancer are (1) an antigen present only in the autologous, (2) an antigen commonly present in tumors of the same species,
(3) Antigens present in tumors of other organs or normal cells are considered. Although immunological techniques are very useful in the analysis of antigens, in order to produce antisera that distinguish the above three species using conventional homologous or heterologous antisera, an absorption procedure is required. Need to repeat. However, this operation is inevitably accompanied by a decrease in antibody titer and is not suitable for practical use. Moreover, even if the desired one is obtained, it is almost impossible to obtain an antibody having the same specificity with good reproducibility. From the above, cancer-related antigens on the surface of cancer cells,
It is considered necessary to introduce a new method to identify the cancer-specific antigen.

[0006] The production of a monoclonal antibody by the cell fusion method started by Milstein et al. In 1975 is one of the new methods which meet the purpose, and is a human cell surface antigen such as human lymphocyte subset, human leukemia, human melanoma. In recent years, it has been reported that a monoclonal antibody against was obtained.

[0007] However, although it is generally said that it is possible to produce a specific antibody by using the technique of producing a monoclonal antibody by the cell fusion method, the antigenicity varies depending on the cancer cell, and as a result, the purpose is It is currently unpredictable whether an antibody with excellent specificity will be obtained. As for cancer cells, there are only a few reports on the above-mentioned specific cancer cells, and particularly on prostate cancer, there is no such report, and there is no antibody that has been put to practical use.

The present inventors have conducted extensive research on a rapid and simple method for identifying human prostate cancer, and as a result, they have been secreted by hybridomas between antibody-producing cells for prostate cancer and cells capable of long-term subculture in vitro. The inventors have found that the antibody derivative of the present invention can be used for serological identification of prostate cancer, and further studied the identification method using them to complete the present invention.

[0009]

[Means and Actions] Since the antibody for producing the derivative of the present invention (hereinafter referred to as antibody A in the "Means and actions and effects" section) has a highly specific reactivity with an antigen, Compared with the conventional method, the present invention enables rapid and highly reliable identification without complicated procedures. The class of antibody A is IgG.

The sample that can be tested using the present invention may be a sample containing prostate cancer, for example, clinical materials obtained clinically from patients suspected of having prostate cancer, such as urine, lymph nodes, bone marrow, etc. Biopsy tissue can be used.

Upon identification, a specimen containing prostate cancer is brought into contact with a test solution containing a derivative of antibody A, and identification is carried out by immunofluorescence microscopy, immunoelectron microscopy, radioactive binding, enzyme immunoassay or the like. You can When the direct method of immunofluorescence microscopy is used, the antibody A is fluorescently labeled with fluorescein, rhodamine or the like, and when immunoelectron microscopy is used, a marker such as ferritin is labeled, When using the active binding method, ¹²⁵ I,
^It is convenient to use radioisotope-labeled with ¹³¹ I or the like, or enzyme-labeled with peroxidase, alkaline phosphatase or the like in the case of enzyme immunoassay. Of course, the indirect method is performed by using a secondary antibody or a binding substance instead of the secondary antibody (for example, an indirect method can be performed by using a biotin-labeled anti-human prostate cancer antibody and using avidin instead of the secondary antibody). You can also The above are specific examples of the derivative in the present invention. Instead of the antibody A itself, it is also possible to use a portion of the antibody A obtained by limiting decomposition of the antibody A by chemical and / or enzymatic treatment, for example, F (ab ′) ₂ . However, this is not directly related to the present invention.

The antibody A is produced, for example, as follows.

A. Step of preparing antibody-producing cells : The antibody-producing cells for human prostate cancer cells may be obtained from any animal species including human, and it is not essential to perform immunization in advance. The efficiency of collecting hybridomas can be significantly increased.

When human cells are used, those with a history of prostate cancer or those with a high antibody titer to the cells in serum can be selected. In the immunization of cancer cells, cancer cells per se or cells whose proliferative properties have been lost by glutaraldehyde treatment or mitomycin treatment may be used, and the surface antigens separated from the cells by an appropriate method and purified may be used. Good. Further, in immunization, an auxiliary such as Freund's complete or incomplete adjuvant can be mixed with the immunogen and used. The immunogen administration method for immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, etc., but subcutaneous injection or intraperitoneal injection is preferred. Immunization may be performed once or repeatedly at appropriate intervals, preferably 1 to 5 weeks. If the antibody titer against the cells in the serum of the immunized animal is measured and the animal with sufficiently high antibody titer is used as the source of the antibody-producing cells, the efficiency of the subsequent operation can be increased. For fusion, it is preferable to use animal-derived antibody-producing cells 3 to 5 days after the final immunization. The antibody-producing cells are lymphocytes that are plasma cells and their progenitor cells, which may be obtained from any part of the individual, but are generally spleen, lymph nodes,
It can be obtained from peripheral blood or any suitable combination thereof.

B. Step of cell fusion : The other parent cell, which can be subcultured for a long time in vitro, may be any cell that fuses with the antibody-producing cell to produce a hybridoma suitable for the purpose, but the probability is high. Are leukemia cells such as myeloma. The species of origin is also human,
Either rat or mouse may be used. As described below, it is preferable to use a hypoxanthine guanine phosphoribosyl transferase-deficient cell line or a thymidine kinase-deficient cell line in order to remove parent cells mixed after fusion.
For example, human-derived GM1500-6TG-A12, RPM
I8226, mouse-derived P3-X63-Ag8, P3-N
SI / 1-Ag4-1, Sp2 / 0-Ag14, X63
-Ag8.653 etc. can be used.

It is not essential that the species from which the above-mentioned antibody-producing cells are derived are the same as the species from which cells that can be subcultured for a long period of time are the same, but the efficiency of fusion, the stability of the properties of the cells after fusion, In general, it is often advantageous to use the same one in terms of easiness in culturing in the body. Particularly, as a cell capable of long-term subculture, mouse-derived P3-X
63-Ag8, P3-NSI / 1-Ag4-1, Sp2
When using / 0-Ag14 or X63-Ag8.653, it is advantageous to use syngeneic BALB / c or a hybrid mouse thereof as an animal for obtaining antibody-producing cells. At the time of fusion, it is preferable to use a fusion accelerator such as Sendai virus or polyethylene glycol, especially polyethylene glycol 1000, 1540, 2000, 4000 or
It is preferable to use 6000 or the like. About 30-55%
Allow the fusion to occur in the containing solution. Dimethyl sulfoxide may be further added as an auxiliary agent.

C. Establishment of hybridoma : In the mixture after fusion, in addition to the hybridoma, antibody-producing cells as parent cells and cells that can be subcultured for a long time in vitro remain. The former usually does not endure in vitro culture for a long period of time, so there is no problem, but the latter may be proliferated together with the target hybridoma, and therefore it is desirable to exclude this. Therefore, as the latter, after fusion using hypoxanthine guanine phosphoribosyl transferase or thymidine kinase deficient cell line, it is cultured in a medium containing hypoxanthine, aminopterin and thymidine. As a result, only the hybridoma can be selectively grown. When a hypoxanthine guanine phosphoribosyl transferase- or thymidine kinase-deficient cell line is not used as a parent cell, the hybridoma is treated by treating the cell with emetine and actinomycin D prior to the fusion to make the cell lose proliferative properties. May be selected from a mixture with parental cells.

The hybridoma thus obtained generally contains two or more types of clones in many cases and is not a population of cells having completely the same properties. When it is desired to separate individual clones, it is necessary to carry out cloning. Cloning is, of course, for the production of antibodies with a single specificity, but it also means preventing changes in population that often occur during long-term culture in a system in which multiple types of clones are mixed. It is also effective and desirable to do. As a cloning method, a limiting dilution method, a soft agar method, or a fibrin gel method can be used. It is also possible to separate cells during cloning using a fluorescence activated cell sorter.
In addition, against the appearance of mutant strains that occur during long-term culture, it is possible to preserve cells having the characteristics of the original cells by performing cloning occasionally. As an example of a hybridoma that secretes an antibody against human prostate cancer, which is produced according to the above-described production method, as shown in Examples described later, P
Examples include -001, P-002, P-003 or P-004.

Hybridomas are
1-10 × 10 ⁶ cells / ml in fetal bovine serum supplemented with 5% (V / V) dimethyl sulfoxide as a frost damage protection substance
It can be stored for a long period of time by suspending in and freezing. In this case, the cooling rate during freezing is preferably -1 ° C / minute, and storage is preferably performed at -80 ° C or lower.

The thawing should be performed as soon as possible. If cells are washed with a medium immediately after thawing to remove dimethylsulfoxide, the cells can be suspended in an ordinary medium and the culture can be restarted. However, when the cell viability at the time of thawing is poor and the proliferative activity is extremely low, it is necessary to appropriately add mouse splenocytes and the like.

D. Production of antibody A: In the production of antibody A, a hybridoma producing an antibody against human prostate cancer cells is cultured in vitro or in vivo (in vivo).

In the case of in vitro culture, a nutrient medium suitable for hybridoma growth, eg 10% (V
/ V) fetal bovine serum, 5 × 10 ⁻⁵ M β-mercaptoethanol, 1 mM sodium pyruvate, and RPMI 1640 medium containing antibiotics can be used. Also, instead of RPMI1640 medium, 4.5 g / L
Dulbecco's modified Eagle's containing glucose
MEM (hereinafter abbreviated as D-MEM) may be used. A suitable initial concentration for growing cells varies depending on each hybridoma, but is generally about 10 ⁵ cells / ml, and it is desirable that the cell concentration in the culture does not exceed 2 × 10 ⁶ cells / ml.

Further, the antibody A secreted by the hybridoma can be produced by transplanting the hybridoma into a living body and allowing it to grow in a solid or ascites form, and collecting a body fluid, preferably serum or ascites, from the living body. .. The crude antibody solution obtained by this method has a drawback that it contains various substances derived from the living body as a host as impurities, but has a significantly higher concentration than the antibody solution obtained by in vitro culture. It is excellent in that it contains antibody A. When a hybridoma is transplanted into the abdominal cavity and proliferated, pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered before the transplantation, preferably 3 to 9 weeks before, Although the yield of crude antibody solution can be increased, this treatment is not essential. The living body used as a host is preferably an animal of the same strain and the same strain as the parent cell of the hybridoma to be transplanted. In this case, the hybridoma usually grows in the body without any special treatment. However, when the histocompatibility phenotypes of the hybridoma and the host do not match, administration of anti-lymphocyte antibody, X It is necessary to perform treatment such as irradiation with rays in advance. After transplantation, it usually takes 1 to 3 weeks for cells to grow.

When the hybridoma is cultured in vitro or in vivo to produce and secrete the antibody A, a radioactive substance such as leucine or lysine labeled with a radioisotope is added to the medium or administered to the host, so that the inside of the molecule is It is possible to produce an antibody A which contains a radioactive substance and has a chemical structure completely the same as that of the unlabeled substance.

As an example of antibody A against human prostate cancer,
The antibody A secreted by the hybridoma shown in Table 1 below may be mentioned. Its specificity and immunoglobulin class are as shown in Table 2 below.

Although the antibody A may be used as a crude antibody solution, it may be purified by a conventional method for immunoglobulin purification such as ammonium sulfate fractionation or ion exchange chromatography, or by affinity chromatography with Protein A or an antigen. Can be used.

If desired, these antibody A derivatives can be mixed and used. Further, the composition can be prepared by mixing with a carrier or a diluent.

[0028]

EXAMPLES Specific examples will be described below.

Example 1 Antibody against human prostate cancer cells
Preparation of A. Immune and cell fusion (1) Preparation of cells used as immunogen Long-term in vitro subcultured human prostate cancer cell 8PC93
2 x 10 ⁵ cells in a 100 mm culture dish (Falcon 3003)
10% for Eagles MEM are salt (hereinafter abbreviated as MEM)
(V / V) Fetal bovine serum and antibiotics kanamycin sulfate (final concentration 60 mg / L) at 37 ° C., 5
Culture was performed in a moist atmosphere containing% carbon dioxide. 3 days later
The cells were detached with a policeman, the culture supernatant was removed by centrifugation, further washed by centrifugation with phosphate buffered saline (pH 7.2, hereinafter abbreviated as PBS), and then suspended in PBS. About 2 × 10 ⁶ cells were obtained from one culture dish.

[0030] (2) cultured mouse of myeloma cells Sp2 / 0-Ag14 10 ⁵ cells / ml
Was cultured in D-MEM supplemented with 10% (V / V) fetal bovine serum, 1 mM pyruvate, 2 mM glutamine and 60 mg / L kanamycin sulfate, and subcultured every 3 days. The cell concentration of Sp2 / 0-Ag14 was adjusted to 2.5 × 10 ⁵ cells / ml on the day before cell fusion, and the cells were cultured in the above medium.

(3) Immunization with 8PC93 cells Female BALB / c mouse (Charles River Japan, 9
8P of the above (1) suspended in 0.5 ml of PBS
Immunizations were performed by injecting 10 ⁷ C93 cells twice, about every 3 weeks, intraperitoneally.

(4) Cell fusion Cell fusion was carried out according to the method of Kohler and Milstein (Immunoassays; Clinical Laborato).
ry Techniques for the 1980 ′s ed. by Nakamura R.
M. et al., ES Alan R. Liss, Inc. NY, 1980, p.
p301-324).

On the 4th day after the final immunization, the mouse was sacrificed, the spleen was taken out, thoroughly disentangled, passed through a 150-mesh stainless mesh and centrifuged at 400 × g, and the precipitated cells contained 0.747% ammonium chloride. Add Tris-HCl buffer (0.017M Tris (hydroxymethyl) aminomethane, pH 7.65) to remove red blood cells, and centrifuge (40
Splenocytes were collected at 0xg). The cells were washed by repeating the procedure of adding MEM and centrifuging (400 × g) and suspending the cells in fresh MEM three times, and finally suspended in MEM. Sp2 / 0-Ag14 was removed from the culture vessel by pipetting and transferred to a centrifuge tube. Centrifuge (400
After xg), MEM was added to the collected cells to suspend and centrifuge (400 × g) to remove serum, and then the cells were resuspended in MEM. 10 × 10 ⁷ splenocytes and Sp2 / 0-
After mixing 2 × 10 ⁷ pieces of Ag14 and pipetting well, centrifugation (400 × g) was performed. After removing the supernatant, the precipitate was loosened by gently tapping the centrifuge tube. MEM
30% (V / V) polyethylene glycol (PEG
The solution to which 1000) was added was kept warm at 37 ° C., and 0.6 ml thereof was added to the loosened cells. After gently stirring, the mixture was left at room temperature for 5 minutes. After centrifugation at 7 × g for 2 minutes, 5 ml of MEM was slowly added. After gently stirring, 400 ×
Centrifuge at room temperature for 5 minutes at room temperature. The supernatant was discarded, 5 ml of MEM was added to the precipitate, and the mixture was centrifuged in the same manner to remove the supernatant. The precipitated cells were added with 5 ml of the following medium and pipetted. As a medium, 10% (V / V) fetal bovine serum, 2 mM glutamine, 5 × 10 ⁻⁵ M β-mercaptoethanol and 60 mg / L kanamycin sulfate were added to D-MEM, and glucose was further added to 4. What was added so that it might become 5 g / L was used. In addition, this medium was used as D-MEM-F.
Abbreviated as BS. 40 ml D to the above 10 ml cell suspension
-MEM-FBS was added, and 25 ml of each was dispensed into a 25 cm ² culture flask (Corning Co., C-25100) and cultured at 37 ° C. overnight in a moist atmosphere containing 5% carbon dioxide. The culture was performed under the same conditions below. Next day, pipette lightly and transfer to a centrifuge tube.
Centrifugation was performed at 00 × g. 1 after removing the supernatant
× 10 ^-4 M Pipo xanthine, 4 × ^{10 -7} M aminopterin and D-ME containing 1.6 × ^{10 -5} M thymidine
Suspend in 60 ml of M-FBS (abbreviated as HAT medium hereinafter), and 0.1 ml of the suspension is added to a 96-well culture plate (Falcon).
3072) in each well and cultured for 1 week. afterwards,
25 μl of HT medium was added every 2 or 3 days. H
As T medium, HAT medium without aminopterin was used.

B. Selection of antibody-producing hybridomas and
Two weeks after the fusion of proliferating cells, the presence or absence of antibody production against 8PC93 cells in the supernatant of each well was examined by enzyme-linked solid-phase immunoassay.

Plate 8PC93 cells (Falcon 3072
) And reacted with 40 μl of the culture supernatant in each well at room temperature for 2 hours, washed well with PBS, and diluted with horse serum 1000 times to a peroxidase-conjugated anti-mouse immunoglobulin antibody (Cappel Lab. Inc. ., USA, Catalog No. 3211-0231) for 2 hours, and then washed well with PBS. Citrate buffer (0.1
M, pH 4.5) with 1 substrate (o-phenylenediamine)
200 μl of a solution containing 0.4 μl / ml of mg / ml and 31% hydrogen peroxide was added, and color reaction was carried out for 30 minutes. Cloning was performed on 2 wells containing an antibody that reacts with 8PC93 cells. Cloning was performed as follows using the limiting dilution method.

100 hybridomas of positive antibody production in 2 wells were each suspended in D-MEM-FBS, while the above A. In the same way as used for the fusion of (4),
10 ⁸ splenocytes from BALB / c mice were subjected to D-MEM-
The cells were prepared in FBS and the two cells were mixed. D-MEM-FBS was added to a cell density of 5 × 10 ⁶ cells / ml, and 0.2 ml was added to a 96-well culture plate (Falcon 3072).
) And placed in each well and cultured. Fourteen days after the start of the culture, the presence or absence of antibody production of the hybridoma was examined for each well by the above enzyme-linked solid phase immunoassay. as a result,
A total of 38 hybridomas were obtained, including the 4 strains shown in Table 1.

C. Production of antibody ( production by in vitro culture ) Hybridoma P-001
, P-002, P-003 or P-004, 20% fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, 4.5
D containing g / L glucose, 5 × 10 ⁻⁵ M β-mercaptoethanol and 50 mg / L kanamycin sulfate
-Suspend in MEM at 1 × 10 ⁵ cells / ml,
25 ml of this cell suspension was dispensed into a 75 cm ² tissue culture flask (Corning Co., USA) and cultured at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. The culture supernatant was collected on day 4 when the growth reached almost steady. At this time, the number of cells was about 2 × 10 ⁶ cells / ml, and the antibody contents of the supernatants were 3.1 μg / ml, 2.8 μg / ml, and
They were 2.8 μg / ml and 2.5 μg / ml.

( Production by In Vivo Culture ) 0.5 ml of pristane (2.6,10,14-tetramethylpentadecane) was intraperitoneally administered, and then in vitro was proliferated in the abdominal cavity of BALB / c mice 10 to 30 days after the administration. 5 × 1 of the hybridoma P-001, P-002, P-003 or P-004
0 ⁶ were inoculated. Ascites was collected 2 to 3 weeks after the inoculation, and the ascites supernatant was obtained by centrifugation (1000 × g, 4 ° C., 15 minutes). About 30 ml of ascites supernatant was obtained from 10 mice for each hybridoma, and their antibody contents were 2.5 mg / ml, 2.0 mg / ml, 1.5 mg / ml and 1.8 mg, respectively.
/ Ml.

The shapes, sizes and properties of these 4 strains are shown in Table-1.
Shown in.

These antibodies were administered to 10 ICR mice per group at 2 g / kg orally, 400 mg / kg intraperitoneally or 200 times.
After intravenous administration of mg / kg, and observation for 14 days, no death due to these antibodies was observed.

[0041]

[Table 1]

Example 2 Antibody against human prostate cancer cells
Characteristics A. Comparison of reactivity with other cells The following cells that had been passaged in vitro were used. Intes
tine 407 (human fetal small intestine cell line), K-562 (human leukemia cell line), Bri 7 (human peripheral blood lymphocyte line) and 8
PC93 (human prostate cancer cell line).

Using the culture supernatant obtained in Example 1, the reactivity with the above cells was examined by the enzyme-linked solid phase immunoassay. As shown in Table-2, an antibody extremely specific to 8PC93 was obtained.

B. Cell Staining by Fluorescent Antibody Method 8PC93 cells were stained by an indirect fluorescent antibody method using an antibody secreted by one of the hybridomas, P-002.

8PC93 cells were fixed on a fluorescent-free glass slide, reacted with 50 μl of culture supernatant in a humid atmosphere at 37 ° C. for 45 minutes, and then added to PBS with 1% bovine serum albumin and 10 mM HEPES (N-2). -Hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 0.
The solution added with 1% sodium azide was washed 3 times by treating it for 3 to 5 minutes. Solution 5 diluted 20-fold with fluorescein-conjugated anti-mouse IgG (Miles-Yeda Ltd., Israel, code number 65-171)
0 μl was reacted under the same conditions as above for another 45 minutes. Washing was performed in the same manner. After drying, a carbonate buffer / glycerin solution (0.05 M, pH 9.5, containing 10% glycerin) was overlaid, a cover glass was placed, and the cells were examined under a fluorescence microscope (Olympus, model AH-RFL-LB). As a result, strong fluorescence was observed on the surface of 8PC93 cells. The fluorescein-conjugated anti-mouse IgG used was previously absorbed 6 times in 8PC93 cells.

C. Identification of immunoglobulin class The immunoglobulin class of the antibody was identified by enzyme-linked solid-phase immunoassay. As a peroxidase-conjugated anti-mouse immunoglobulin antibody, anti-immunoglobulin (Ig), anti-IgG, anti-IgM (all are Cappel Lab. Inc., USA, catalog numbers 3211-021, 3211-0081, 3211-020).
1) was used. Table 4 shows the four types of antibodies examined.
It was IgG as shown in.

[0047]

[Table 2]

[0048] Using tissues including prostate cancer obtained during Example 3 Identification of prostate cancer with an antibody (1) test body surgery. After fixing 95% alcohol, embedding in paraffin by a conventional method, and
A μm section specimen was prepared.

(2) Identification method Identification was performed by the fluorescent antibody method using the antibody (ascites supernatant) secreted by the hybridoma P-002. 0.1 ml of a 100-fold diluted antibody solution was dropped onto the tissue sample,
The reaction was carried out at room temperature for 1 hour. After the reaction was completed, it was thoroughly washed with PBS. Next, fluorescein-conjugated antibody mouse IgG
0.1 ml of 100-fold diluted solution (Miles, USA) was dropped on the tissue specimen and reacted at room temperature for 1 hour. Unreacted fluorescein-conjugated anti-mouse IgG was thoroughly washed with PBS, and the presence or absence of fluorescence of the tissue specimen on the slide was observed using a fluorescence microscope (Olympus Vanox, Olympus, Japan). As a result, strong fluorescence was observed in the prostate cancer tissue site, and almost no fluorescence was observed in the normal tissue site. For the purpose of preventing non-specific binding, fluorescein-conjugated anti-mouse IgG was previously absorbed in human prostate cell line 8PC93.

Example 4 Labeling of antibody (1) Peroxida of hybridoma P-002 secretory antibody
Enzyme label : 4 mg horseradish peroxidase (Sigma
Type VI, Sigma, USA) in 1 ml distilled water,
60 μl of 0.1 M NaIO ₄ solution prepared immediately before use
In addition, the mixture was mixed at room temperature for 20 minutes. After dialyzing the reaction solution against 1 mM sodium acetate buffer (pH 4.4) overnight,
20 μl of 0.2 M sodium carbonate solution was added. right away,
Hybridoma P-002 secretory antibody dissolved in 1 ml of 0.01 M carbonate buffer (pH 9.5) (antibody obtained by purifying ascites supernatant,
The protein content (10 mg) was added, and the mixture was reacted at room temperature for 2 hours with stirring. After completion of the reaction, freshly prepared N
aBH ₄ aqueous solution (4 mg dissolved in 1 ml distilled water)
0.1 ml was added, the mixture was allowed to stand at 4 ° C. for 2 hours, and dialyzed against PBS overnight. The mixture thus obtained is used as Sephadex G
-100 [registered trademark] (Pharmacia, Sweden) column, elute with PBS and absorb at 280 nm and 40
The first fraction with a consistent 3 nm absorbance was collected. Rabbit serum albumin (10 mg) was added to 1 ml of this fraction (enzyme-antibody conjugate) to dissolve it, and the mixture was stored at -70 ° C until use.

(2) of the hybridoma P-002 secretory antibody
Alkaline phosphatase labeling : Alkaline phosphatase that has been dialyzed against PBS to remove ammonium sulfate sufficiently.
5 mg of Type VII (Sigma, USA) and 17 mg of P-002 secretory antibody were dissolved in PBS to a total volume of 1 ml. 2 to this
Add 10 μl of 0% glutaraldehyde solution, and add 2 at room temperature.
The reaction was carried out with stirring for an hour. After completion of the reaction, Sephadex G equilibrated with Tris-HCl buffer (pH 7.6)
-200 [registered trademark] (Pharmacia, Sweden) was applied to a column and eluted with the same buffer. void volume to I
Collect the high molecular weight fraction up to the gG outflow position, add bovine serum albumin to this fraction to 5% (W / V), sterilize through Millipore filter (0.22μ, Millipore), then use It was stored protected from light at 4 ° C until the time.

(3) Hybridoma P-002 secretory antibody
β-galactosidase labeling : The β-galactosidase-labeled antibody of the hybridoma P-002 secretory antibody was obtained by the same operation as in (2) above. In this case, β-galactosidase Sigma grade IV (Sigma, USA) was used.

(4) Hybridoma P-002 secretory antibody
¹²⁵ I labeling : 100 m Ci / ml Na ¹²⁵ I (carrier-free, Amersham, USA) solution 10 μl, hybridoma P-002 secretory antibody (antibody ascites supernatant purified antibody, protein content 1.0 mg / ml) solution Add 30 μl of 0.5 M phosphate buffer (pH 7.2) containing 50 μl and 0.30 mg / ml chloramine T, mix well, and mix.
After 10 seconds, 100 μl of PBS saturated with L-tyrosine was added and immediately mixed. The obtained reaction solution was applied to a column packed with Amberlite IRA 400 and eluted with PBS containing 1% bovine serum albumin. The eluted fraction was collected and stored at 4 ° C until use. The specific radioactivity of the obtained labeled product was 1.0 μCi / mg antibody protein.

(5) of the hybridoma P-002 secretory antibody
Fluorescein label : 10 mg / ml of hybridoma P
-002 To 1 ml of the secretory antibody (antibody obtained by purifying ascites supernatant) solution, 0.1 ml of 0.5 M carbonate buffer (pH 9.3) was added, and further 0.1 mg of fluorescein isothiocyanate powder was added. While stirring so as not to foam,
Reacted for hours. Immediately after the reaction, Sephadex G-25
The product was applied to a [registered trademark] (Pharmacia, Sweden) column to remove unreacted low-molecular substances, and the target fluorescein-labeled antibody in the high-molecular fraction was obtained. Until use 4
Stored in the dark at 0 ° C.

(6) Hybridoma P-002 secretory antibody
Tetramethylrhodamine labeling : 10 mg / ml hybridoma P-002 secretory antibody (antibody obtained by purifying ascites supernatant) solution (1 ml) was added with 0.5 M carbonate buffer (pH 9.3) (0.1 ml), and tetramethylrhodamine isothiocyanate was added. 0.2 mg of powder was added. The reaction was carried out at 4 ° C. for 20 hours while stirring so as not to foam. Immediately after the reaction, S
Ephadex G-25 [registered trademark] (Pharmacia, Sweden) is applied to a column to remove unreacted low-molecular substances,
The target tetramethylrhodamine-labeled antibody of the high molecular fraction was obtained. It was stored at 4 ° C protected from light until use.

(7) Hybridoma P-002 secretory antibody
Fluorescein labeling : 244 mg (1 mM) d-biotin (Wako Pure Chemical Industries) and 173 mg (1.5 mM) N-hydroxysuccinimide (Eastman Kodak, USA) were added to 8 ml of dimethyl sulfoxide (Wako Pure Chemical Industries) and 1,2. -Dissolved in a mixed solution of 5 ml of dimethoxyethane (Hanai Chemical), and 206 mg (1 mM) of N, N'-dicyclohexylcarbodiimide (Kanto Chemical) dissolved in 0.5 ml of 1,2-dimethoxyethane. Was added and reacted overnight at 4 ° C. The generated precipitate was removed by filtration to obtain a filtrate.
The solvent in the filtrate was removed by concentration under reduced pressure, and the remaining oily substance was dissolved in 10 ml of dichloromethane (Wako Pure Chemical Industries, Ltd.) and cooled to 4 ° C. 0.1M NaHCO ₃ solution 1 cooled to 4 ° C
0 ml was added to this and mixed well by shaking. The formed dichloromethane layer was removed, and 10 ml of 0.1M NaHCO ₃ was removed.
Then, 10 ml of distilled water cooled to 4 ° C. was added, the same operation was repeated, and anhydrous sodium sulfate powder (Koizumi Kagaku) was added to the produced dichloromethane layer for dehydration treatment.
After the powder was filtered off, n-hexane was gradually added until cloudiness occurred. This solution was cooled to −20 ° C., the precipitated crystals were placed in a desiccator, the solvent was removed and dried,
Biotin-N-hydroxysuccinimide ester was obtained.

This biotin-N-hydroxysuccinimide was dissolved in dimethylsulfoxide and adjusted to a concentration of 1 mg / ml, then 60 μl of this solution and Hybridoma P-00.
2 1 ml of a solution of secretory antibody (antibody ascites supernatant was purified, protein content 1 mg / ml) was mixed and reacted at room temperature for 4 hours. After the reaction was completed, it was dialyzed against PBS at 4 ° C. for 3 days. The external dialysis solution was exchanged 3 times. The solution in the dialysis tube after dialysis was stored as a biotin-labeled antibody at 4 ° C. until use.

[0058] Using Example 5 Identification of prostate cancer with labeled antibodies (1) tissues, including prostate cancer obtained during test body surgery. That is, a living tissue having a size of 5 × 5 × 2 mm or less was treated with a PLP fixing solution (0.01M NaIO ₄ , 0.075 M lysine, 0.0375 M sodium phosphate buffer containing 3% paraformaldehyde,
It was fixed in pH 6.2) at 4 ° C. for 6 hours with shaking. Then, in PBS containing 10% sucrose at 4 ° C. overnight, 1
Washed with PBS containing 5% sucrose at 4 ° C for 6 hours, PBS containing 20% sucrose at 4 ° C for 6 hours, and PBS containing 20% sucrose and 10% glycerin at 4 ° C for 1 hour. I went. After washing, treat, freeze in dry ice / ethanol, and use cryostat for 10μ
m frozen sections were prepared.

(2) Identification method The frozen section was placed on a slide glass coated with bovine serum albumin and dried. Next, PB kept at 4 ℃
It was washed with S for 3 times for 5 minutes and reacted with 0.005 M periodic acid for 10 minutes at room temperature. After washing 3 times with PBS kept at 4 ° C, the plate was treated with PBS containing 10% horse serum for 10 minutes at room temperature. A 10-fold diluted solution of the hybridoma P-002 secretory antibody labeled with peroxidase, alkaline phosphatase, fluorescein, rhodamine or biotin obtained in Example 4 was placed on the section with a capillary pipette, and
The reaction was carried out at room temperature for 1 minute. After completion of the reaction, P kept at 4 ° C
Washed 5 times with BS.

Hereinafter, identification was performed by the following method.

(A) Fluorescent antibody method In the case of fluorescein and rhodamine-conjugated antibody,
The above sample was sealed with glycerin and the fluorescence microscope (Olympus
Vanox, Olympus) was used to observe the presence or absence of fluorescence.

In the case of the biotin-conjugated antibody, 5 μg / ml of fluorescein-conjugated avidin (Funakoshi Chemical Co., Ltd.) was dropped on the above sample and reacted at 37 ° C. for 1 hour. After completion of the reaction, the plate was washed 5 times with PBS, encapsulated with glycerin, and the presence or absence of fluorescence in the sample was observed using a fluorescence microscope (Olympus Vanox, Olympus).

The results are shown in Table 3.

(B) Enzyme immunoassay In the case of a peroxidase-conjugated antibody, 1 is used as a substrate solution.
% H ₂ O ₂ 0.02 ml / ml and 3,3′-diaminobenzidine-HCl 0.5 mg / ml in 0.1 M Tris / HCl buffer (pH 7.6) was used, and the mixture was reacted at room temperature for 10 minutes and then 4 The cells were washed 3 times with PBS kept at 0 ° C and once with distilled water. After post-staining with veronal-acetate buffer (pH 4.0) containing 1% methyl green, dehydration with ethanol and clearing with xylol were carried out by a conventional method, and sealed with balsam, and observed with a microscope for the presence or absence of coloring (brown). ..

In the case of an alkaline phosphatase-conjugated antibody,
0.2M Tris-HCl buffer (pH 8.5) containing 0.39M magnesium sulfate, 0.2% lead citrate, 0.6% β-glycerophosphate and 4% sucrose as a substrate solution [filtered before use] And react at room temperature for 10 minutes, then at 4 ℃
It was washed with PBS kept at 3 times and once with distilled water. next,
After reacting in a 1% yellow ammonium sulfide solution at room temperature for 5 minutes, it was thoroughly washed with PBS, encapsulated with glycerin, and observed with a microscope for the presence or absence of coloring (black brown).

The results are shown in Table 3.

[0067]

[Table 3]

Example 6 Imaging of Cancer Cells Using Antibody 1 × 10 ⁷ human prostate cancer cell line 8PC93, 1 group 3
Female, 8-week-old BALB / cnu / nu mice were subcutaneously transplanted into the right thigh. 30 days after transplant, tumor size is 1
At the time of reaching cm ² or more, the ¹²⁵ I-labeled hybridoma P-002 secreting antibody prepared in Example 4 or ¹²⁵ I-labeled mouse IgG as a control was intravenously injected. The dose of the former is 35 μCi / animal and the dose of the latter is 10 μCi / animal. Whole body scintigraphy was performed using a gamma camera 96 hours after the intravenous injection.

As a result, in the ¹²⁵ I-labeled antibody administration group, marked accumulation of radioactivity at the tumor site was observed in all cases as compared with the control group.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location G01N 33/532 B 8310-2J 33/534 8310-2J 33/535 8310-2J 33/574 D 9015 -2J 33/577 B 9015-2J // A61K 39/395 X 8413-4C C12N 5/20 15/06 C12P 21/08 8214-4B (C12P 21/08 C12R 1:91) (72) Inventor Yasuhiko Kobayashi 3-26-1-102, Hyakunincho, Shinjuku-ku, Tokyo (72) Tomoko Chiku 1410-2, Naga, Nagareyama-shi, Chiba (72) Inventor Kenichi Matsunaga 3-26-1-303 (72-1) Hyakunin-cho, Shinjuku-ku, Tokyo ) Inventor Takami Fujii 5-11-21 Towa, Adachi-ku, Tokyo (72) Inventor Chikio Yoshipu 2-19-46 Higashi, Kunitachi, Tokyo

Claims (18)

[Claims] 1. An IgG antibody secreted by a hybridoma between an anti-human prostate cancer antibody-producing cell and a cell capable of long-term subculturing in vitro, a radioactive substance, a fluorescent dye, an enzyme, for electron microscope observation. Of the above-mentioned marker or a group of IgG antibodies chemically bound to a group containing a structure for secondarily binding them. 2. The derivative according to claim 1, wherein the radioactive substance is ¹²⁵ I. 3. The derivative according to claim 1, wherein the fluorescent dye is fluorescein or rhodamine. 4. The derivative according to claim 1, wherein the enzyme is peroxidase or alkaline phosphatase. 5. The derivative according to claim 1, wherein the structure for secondarily binding a radioactive substance, a fluorescent dye, an enzyme or a marker for electron microscope observation is biotin. 6. The human prostate cancer cell line 8 is human prostate cancer cell line.
The derivative according to any one of claims 1 to 5, which is PC93. 7. The antibody is the human prostate cancer cell line 8PC93.
7. The derivative according to any one of claims 1 to 6, which is an antibody against 8. The antibody is hybridoma P-001, P-
The derivative according to claim 7, which is an antibody secreted by 002, P-003 or P-004. 9. An IgG antibody secreted by a hybridoma between an anti-human prostate cancer antibody-producing cell and a cell capable of long-term subculturing in vitro, a radioactive substance, a fluorescent dye, an enzyme, for electron microscope observation. Reagent for classification and identification of human prostate cancer containing a derivative of an IgG antibody chemically bound with a marker or a group containing a structure for secondary binding thereof. 10. The reagent according to claim 9, which contains a carrier or a diluent. 11. The reagent according to claim 9, wherein the radioactive substance is ¹²⁵ I. 12. The reagent according to claim 9, wherein the fluorescent dye is fluorescein or rhodamine. 13. The reagent according to claim 9, wherein the enzyme is peroxidase or alkaline phosphatase. 14. The structure for secondarily binding a radioactive substance, a fluorescent dye, an enzyme or a marker for electron microscope observation is biotin.
The reagent according to. 15. The reagent according to any one of claims 9 to 14, wherein the human prostate cancer is human prostate cancer cell line 8PC93. 16. The antibody is the human prostate cancer cell line 8PC9.
3. An antibody to 3, which is characterized in that
5. The reagent according to any one of 5. 17. The antibody is hybridoma P-001 or P-001.
The reagent according to claim 16, which is an antibody secreted by -002, P-003 or P-004. 18. The reagent according to any one of claims 1 to 17, which is used for identifying human or animal prostate cancer.