JP2000041672A - Production of ascites by using cell line producing monoclonal antibody against human hormone-beta for stimulating villi sex gland, and ascites obtained thereby - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce ascites containing a large amount of antibodies for detection of pregnancy, or the like by transplanting a cell line producing a monoclonal antibody specific to human hormone-β subunit for stimulating villi sex gland into the abdominal cavity of a laboratory animal after administration of pristane. SOLUTION: This method for producing ascites comprises administering about 1.0 ml pristane in the abdominal cavity of a laboratory animal, transplanting a cell line of FERM BP6107 producing monoclonal antibody specifically binding to β-subunit (hCG-β) of the hormone for stimulating villi sex gland derived from a human body into the abdominal cavity at three days after the administration, and gathering the ascites just before the laboratory animal is weakened by the ascites accumulated in the abdominal cavity and dies, to provide the objective ascites containing anti-hCG-β monocolonal antibody in high concentration efficiently. The anti-hCG-βmonoclonal antibody can be used for detection of hCG secreted in a humor when pregnant and is useful especially in a medical field.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing ascites for efficiently producing a large amount of a monoclonal antibody against human β-subunit of chorionic gonadotropin (hCG-β). By using this method, ascites containing a high concentration of anti-hCG-β monoclonal antibody can be obtained. Since this antibody can be used for detecting hCG secreted into body fluids during pregnancy, it is particularly effective in the medical field.

[0002]

2. Description of the Related Art Conventionally, two major methods have been used for preparing monoclonal antibodies. One is a method of culturing a monoclonal antibody producing cell line in vitro. Another method is to culture a monoclonal antibody-producing cell line in vivo.

[0003] In the first method, a monoclonal antibody-producing cell line is cultured in a culture solution. From the cell line,
The monoclonal antibody is secreted into the culture supernatant. The culture supernatant can be purified to collect the monoclonal antibody.

[0004] However, the in vitro culture method has several disadvantages. First, the concentration of the monoclonal antibody secreted into the culture supernatant is generally about 100 μ
g / ml. Therefore, in order to produce a large amount of monoclonal antibodies, it is necessary to repeatedly culture cells in a large amount of medium or to culture the medium by changing the medium little by little (that is, performing perfusion culture).

[0005] Purifying low concentrations of antibodies from culture supernatants is very laborious and costly. In particular, since the medium used for culturing the monoclonal antibody-producing cells generally contains serum, impure immunoglobulin derived from serum can be mixed into the culture supernatant. It is extremely difficult to completely remove this impure immunoglobulin by purification.

[0006] In order to prevent contamination with impure immunoglobulin, there is also a culture method using a serum-free medium or the like. But,
It is still difficult to adapt the monoclonal antibody-producing cells to such a special medium without deteriorating the antibody-producing ability. Therefore, it is not a practical method for obtaining a large amount of antibodies.

[0007] When performing perfusion culture, there is a problem that necessary culture equipment is expensive. Further, there is also a problem that it is difficult to continue culturing for a long time in an aseptic state because various bacteria are easily mixed into the culture system.

In the second method of culturing in vivo, a monoclonal antibody-producing cell line is introduced into the body of an experimental animal, for example, into the peritoneal cavity. The introduced and expanded cell line secretes the monoclonal antibody into, for example, ascites. The ascites can be purified and the monoclonal antibodies collected.

According to the method of culturing antibody-producing cells in vivo, a large amount of antibody may be produced in ascites at a concentration of about 20 mg / ml at the maximum. However, the characteristics of the cells differ for each monoclonal antibody producing cell line. Therefore, arbitrarily selecting commonly used in vivo culture conditions may not be appropriate.

[0010] For example, various agents (eg, pristane, incomplete Freund's adjuvant, etc.) are known as inducers for inducing plasmacytomas (cytomas producing antibodies) in experimental animals. However, the type and amount of suitable inducer may vary depending on the monoclonal antibody producing cell line used. Further, even if the antibody producing cells are injected into the abdominal cavity of the experimental animal treated with the inducer, the cell transplantation may not be established. Even when transplantation is established, the amount of antibody secreted into ascites may be small because the transplanted antibody-producing cells become solid cancers and proliferate.
Therefore, in order to produce a large amount of monoclonal antibodies, it is necessary to individually and extensively examine various conditions including the type of experimental animal, the number of antibody-producing cells to be transplanted, and the like for each monoclonal antibody-producing cell to be used. There is.

The β subunit of human chorionic gonadotropin β subuni
t; hCG-β) as a cell line that produces a monoclonal antibody that specifically binds to it, under the accession number FERM-BP6107 of the National Institute of Bioscience and Biotechnology, Institute of Industrial Science and Technology (accession date: September 17, 1997). There are monoclonal antibody producing cell lines. For this particular cell line, it has been desired to provide an efficient method for producing ascites.

[0012]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above-mentioned problems, and uses a monoclonal antibody-producing cell line FERM BP6107 for hCG-β.
An object of the present invention is to provide an efficient method for producing ascites, which is optimized for producing a large amount of a monoclonal antibody against Ascites.

[0013]

Means for Solving the Problems The present inventors have proposed hCG-
Cell line F producing monoclonal antibodies to β
For ERM BP6107, optimization of ascites production conditions was attempted. As a result, they have found a method for producing ascites that is easy to produce, low in cost, and easy for subsequent purification operations.

The method for preparing ascites according to the present invention comprises the steps of (a) administering pristane to the intraperitoneal cavity of an experimental animal; and (b) after administering pristane, the β-subunit of human-derived chorionic gonadotropin (hCG- The cell line of Accession No. FERM BP6107, produced by the National Institute of Advanced Industrial Science and Technology, which produces a monoclonal antibody that specifically binds to β),
Transplanting into the peritoneal cavity of the experimental animal; and (c) collecting the ascites fluid immediately before the experimental animal weakens and dies due to accumulation of ascites in the peritoneal cavity.

In one embodiment, the animal is a mouse, the dose of pristane is about 0.5 to about 1.0 ml, and the cell line is 3 to 1 ml after pristane administration.
On day 0, about 1 × 10 ⁶ to about 1 × 10 ⁷ cells are transplanted.

In a preferred embodiment, the dose of pristane is about 1.0 ml and about 1 × 10 ^{7 of the} cell lines are transplanted three days after pristane administration.

In another preferred embodiment, the dose of pristane is about 1.0 ml and about 1 × 10 ^{7 of the} cell lines are transplanted 5 days after pristane administration.

In yet another preferred embodiment, the dose of pristane is about 1.0 ml and about 1 × 10 ^{7 of the} cell lines are transplanted 10 days after pristane administration.

In yet another preferred embodiment, the dose of pristane is about 0.5 ml, and the cell line is implanted about 1 × 10 ⁶ 7 days after pristane administration.

The ascites of the present invention is produced by any of the above-mentioned ascites producing methods. This ascites contains the anti-hCG-β monoclonal antibody produced by the monoclonal antibody producing cell line FERM BP6107.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

In the present invention, unless otherwise indicated,
Protein separation and analysis methods and immunological techniques known in the art may be employed. These techniques can be performed using commercially available enzymes, kits, antibodies, labeling substances and the like.

As used herein, the term "experimental animal" refers to an animal capable of establishing transplantation when the monoclonal antibody-producing cell line is appropriately injected into the peritoneal cavity and secreting the monoclonal antibody into ascites. Say. Experimental animals
Typically, it is a mammal. Examples of mammals include mice, rats, and rabbits. An example of a preferred mammal is a mouse. BALB / C as an example of a mouse
System, SJL system, NZB system, A / J system, C57B
L / 6 strain, C3H / He strain, and CBA / JNCrj strain mice. A more preferred mouse example is B
It is an ALB / C strain mouse. Although the age of the experimental animal is not particularly limited, it is typically 6 weeks or older, and preferably a large mouse of 15 weeks or older.

In the present specification, "immediately before the experimental animal weakens and dies" means that the experimental animal is weakened by ascites in the abdominal cavity, and the heart of the experimental animal is moving, but is moving on its own. Is in a difficult state.

A method for preparing ascites for efficiently obtaining a large amount of antibodies will be described below along the flow of experiments. Experimental conditions have been optimized for antibody production from cell line FERM BP6107.

1. Administration of pristane First, pristane is administered intraperitoneally to the experimental animal. Administration of pristane is performed according to methods well known in the art, but is usually performed by intraperitoneal injection.

"Pristane" is 2,6,10,14-tetramethylpentadecane. Pristane is an inducer generally used to induce plasmacytomas in mice and to promote the growth of induced plasmacytomas.

The dose of pristane depends on the species of the experimental animal,
It may vary depending on age, gender, and the nature of the antibody-producing cells to be implanted later. When the experimental animal is a mouse, the dose of pristane can be about 0.3 to about 2.0 ml per mouse, preferably about 0.5 to about 1.0 ml.
1 and even more preferably about 1.0 ml.

2. Implantation of antibody-producing cells After administration of pristane, monoclonal antibody-producing cells are implanted intraperitoneally into experimental animals. Cell transplantation is performed according to methods well known in the art. For example, transplantation is performed by injecting a suspension of monoclonal antibody-producing cells into the abdominal cavity of a laboratory animal.

The transplantation of the antibody-producing cells is preferably carried out on the third to tenth days after administration of pristane, more preferably
It is performed on the third, fifth, seventh, or tenth day.

The monoclonal antibody-producing cell line used in the present invention is a cell line of Accession No. FERM BP6107, which was deposited with the National Institute of Advanced Industrial Science and Technology. The cells were immunized with BAL immunized with the β-subunit of human chorionic gonadotropin (hCG-β).
Splenocytes of B / C mice were transformed with P3 × 63-Ag8.653.
Obtained by cell fusion with myeloma cells derived from a mouse. The cells produce monoclonal antibodies that specifically bind to hCG-β. The class of this monoclonal antibody belongs to IgG1.

Chorionic gonadotropin (hCG) is a glycoprotein produced from villous tissue and has a lutein-stimulating action similar to luteinizing hormone. This hormone consists of an α subunit and a β subunit. The α subunit (hCG-α) is common to the α subunit of luteinizing hormone (LH). The β subunit (hCG-β) is similar to the LH β subunit, but differs in that it has 35 more amino acids in the C-terminal part. Therefore, a part different from LH exists in the three-dimensional structure of hCG-β.

The monoclonal antibody produced by cell line FERM BP6107 does not show a cross-reaction with LH by ELISA (enzyme-linked immunosorbent assay). Therefore, it is considered that this antibody specifically binds to hCG-β by recognizing a three-dimensional structural part different from LH in hCG-β. In addition, this antibody has the hCG-α
But not hCG-β and hCG-β.

The monoclonal antibody-producing cells are usually transplanted in a state of being suspended in a solution. A cell suspension can be prepared by suspending the monoclonal antibody-producing cells in a suitable solution. Examples of the solution for suspending the cells include sterile PBS (phosphate buffer solution).

Antibody-producing cells implanted intraperitoneally in experimental animals
Is typically about 1 × 10^Five~ About 1 × 10⁸Individual
And preferably about 5 × 10^Five~ 5 × 10⁷It's an individual
More preferably about 1 × 10⁶~ About 1 × 10⁷Individual
More preferably about 1 × 10 ⁷Individual.

3. Collection of ascites 1-2 weeks after transplantation of monoclonal antibody-producing cells,
Observation is continued every day when the abdomen of the experimental animal begins to enlarge. It is preferable that the abdomen of the experimental animal is remarkably enlarged to the same degree as the degree of swelling just before the birth of the pregnant female experimental animal, and ascites is collected from the abdominal cavity when the experimental animal is significantly weakened. The amount of ascites accumulated in the abdomen is small, but the concentration of the monoclonal antibody in the ascites may be high. In such a case, the ascites is collected at an appropriate time, based on the degree of weakness of the experimental animal. Ascites fluid is typically collected about day 7 to about day 20, preferably about day 8 to about day 18, more preferably about day 10 to about day 16, following implantation of the monoclonal antibody.

When ascites is produced according to the method of the present invention,
The frequency of experimental animals that die during the experiment and those that do not have a transplant are very low. In addition, the incidence of solid cancer in experimental animals in which transplantation has been established is extremely low. Therefore, ascites containing a large amount of monoclonal antibody can be produced with good reproducibility.

[0038] Ascites is collected from a laboratory animal according to a method well known in the art. For example, the experimental animal is lightly anesthetized with ether, the anesthetized experimental animal is kept on a beaker, and a hole is made in the abdominal skin and abdominal wall using a needle. Ascites can be collected from this hole by dropping ascites into a beaker. Alternatively, the anesthetized experimental animal is placed on its back, and a needle is inserted into the abdominal cavity from the left side of the experimental animal. The ascites can be collected by slowly aspirating the ascites into the syringe with the tip of the inserted needle positioned above the side of the spleen.

The concentration of the monoclonal antibody contained in the collected ascites fluid can be measured by a method known to those skilled in the art, usually after purifying the antibody. Examples of the method for measuring the concentration of the monoclonal antibody include a spectrophotometric method, an enzyme immunoassay, and a fluorescent antibody method. In the absorptiometry, for example, the absorbance at 280 nm of a solution containing a monoclonal antibody is measured, and the concentration is measured by conversion from the extinction coefficient.
From the antibody concentration and ascites volume, the total amount of monoclonal antibody in the ascites is calculated. According to the method of the present invention, typically about 20 mg or more, preferably about 30 to about 50 mg or more, more preferably about 40 to 50 mg per experimental animal.
An ascites fluid containing about 50 mg of antibody may be made.

From the ascites fluid produced according to the method of the present invention, monoclonal antibodies can be purified by methods well known to those skilled in the art. For purification, for example, a purification method using protein A, a purification method by DEAE anion exchange chromatography, a purification method by affinity chromatography, an ammonium sulfate fractionation method, a PEG fractionation method, and an ethanol fractionation method are appropriately combined. Used.

[0041]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

Each embodiment will be described with reference to Table 1.

[Table 1]

The antibody-producing cells used were all from the monoclonal antibody-producing cell line under the accession number FERM BP6107. The mice used were all female BALB / C strains (15 weeks old).

The antibody specific to hCG-β contained in the collected ascites was purified by the following method, and then its concentration was determined.

First, Protein A (Protein A Sepharos)
e 4 Fast Flow (Pharmacia))
Binding buffer (1.5M glycine / 3M NaCl, p
H8.9). Ascites obtained from mice,
After diluting 3-fold with binding buffer, apply to equilibrated column. While monitoring the eluate from the column at 280 nm, wash the column with binding buffer until elution of the impurities is complete. After washing, elution buffer 1 (100 m
M citric acid, pH 6), elution buffer 2 (100 mM citric acid, pH 5), and elution buffer 3 (100 mM citric acid, pH 4) were sequentially applied to the column (flow rate: about 1).
ml / min), and collect the IgG-containing eluate.

The absorbance at 280 nm of the collected IgG-containing eluate was measured with an absorptiometer. The concentration of the antibody was determined by converting the measured absorbance by an extinction coefficient. When the purity of the IgG-containing eluate recovered by this method was confirmed by electrophoresis according to a conventional method, contamination was found to be below the detection limit.

Example 1 Intraperitoneal injection of 5 mice
ml of pristane was injected.

Three days after pristane administration, the cells were placed in sterile PBS.
The antibody-producing cells were suspended and 1 × 10 ⁸Cells / ml of cell suspension
A play solution was prepared. Cell suspension 100 μl (0.1 ml)
Was injected intraperitoneally. Thereby, 1 × 10⁷Individual details
The vesicles were implanted intraperitoneally in mice.

On the eleventh day after the cell transplantation, the abdomen of the mouse was significantly enlarged. Therefore, ascites was collected from the abdominal cavity of the mouse 11 days after the transplantation. As shown in Table 1, the amount of ascites collected was an average of 2.2 ml per mouse.

The transplantation was established in all of the mice into which the antibody-producing cells were transplanted. None of the mice died in the middle of the experiment, no transplant was established, and no mouse developed solid cancer.

As shown in Table 1, the concentration of the antibody contained in the ascites was 18.1 mg / ml on average. Therefore, the obtained antibody averaged 39.8 mg per mouse.

Example 2 Intraperitoneal injection of 5 mice
ml of pristane was injected.

Five days after pristane administration, the cells were placed in sterile PBS.
The antibody-producing cells were suspended and 1 × 10 ⁸Cells / ml of cell suspension
A play solution was prepared. Cell suspension 100 μl (0.1 ml)
Was injected intraperitoneally. Thereby, 1 × 10⁷Individual details
The vesicles were implanted intraperitoneally in mice.

Ten days after the cell transplantation, the abdomen of the mouse was significantly enlarged. Therefore, on day 10 after the transplantation, ascites was collected from the abdominal cavity of the mouse. As shown in Table 1, the amount of ascites collected was an average of 2.2 ml per mouse.

In all of the mice transplanted with the antibody-producing cells, the transplant was established. None of the mice died in the middle of the experiment, no transplant was established, and no mouse developed solid cancer.

As shown in Table 1, the concentration of the antibody contained in the ascites was 18.5 mg / ml on average. Therefore, the obtained antibody averaged 40.7 mg per mouse.

Example 3 Intraperitoneal injection of 5 mice
ml of pristane was injected.

Ten days after pristane administration, antibody-producing cells were suspended in sterile PBS to prepare a cell suspension of 1 × 10 ⁸ cells / ml. 100 μl of cell suspension (0.1 m
l) was injected intraperitoneally. As a result, 1 × 10 ⁷ cells were transplanted into the peritoneal cavity of the mouse.

On the thirteenth day after the cell transplantation, the abdomen of the mouse was significantly enlarged. Therefore, on day 13 after transplantation, ascites was collected from the abdominal cavity of the mouse. As shown in Table 1, the amount of ascites collected was an average of 3.7 ml per mouse.

In one mouse transplanted with the antibody-producing cells, the transplant was not established. One mouse died during the experiment. None of the mice developed solid cancer.

As shown in Table 1, the concentration of the antibody contained in the ascites was 11.0 mg / ml on average. Therefore, the obtained antibody averaged 40.7 mg per mouse.

Example 4 Intraperitoneally of 5 mice
0.5 ml of pristane was injected.

Seven days after pristane administration, the cells were placed in sterile PBS.
The antibody-producing cells were suspended and 1 × 10 ⁷Cells / ml of cell suspension
A play solution was prepared. Cell suspension 100 μl (0.1 ml)
Was injected intraperitoneally. Thereby, 1 × 10⁶Individual details
The vesicles were implanted intraperitoneally in mice.

Sixteen days after the cell transplantation, the abdomen of the mouse became significantly enlarged. Therefore, on the 16th day after the transplantation, ascites was collected from the abdominal cavity of the mouse. As shown in Table 1, the amount of ascites collected was an average of 2.0 ml per mouse.
One mouse died during the experiment. None of the mice failed to transplant and did not develop solid cancer.

As shown in Table 1, the concentration of the antibody contained in the ascites was 11.8 mg / ml on average. Therefore, the obtained antibody averaged 23.6 mg per mouse.

As described above, according to the method of this embodiment,
At least 20 mg per mouse, up to about 40 mg
Ascites containing some monoclonal antibodies was obtained.

[0067]

According to the present invention, a very efficient method for producing ascites using the monoclonal antibody producing cell line FERM BP6107 is provided. In the method for producing ascites of the present invention, the conditions for producing ascites for a specific cell line FERM BP6107 are optimized. Therefore,
It is easy to produce ascites, low in cost, and easy to purify antibodies from ascites.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) (C12P 21/08 C12R 1:91) (72) Inventor Miya ▲ Saki ▼ Hitoshi Makoto Kazuma, Kadoma City, Osaka Prefecture 1006 Matsushita Electric Industrial Co., Ltd. F-term (reference) 4B024 AA01 BA01 BA44 DA02 GA03 HA01 HA15 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA92X AB05 BA08 BD29 CA25 CA44 CA46

Claims (7)

[Claims] 1. A method for preparing ascites, comprising: (a) a step of administering pristane to an intraperitoneal cavity of an experimental animal; (b) after administration of pristane, a β-subunit of human-derived chorionic gonadotropin (hCG) Transplanting a cell line of Accession No. FERM BP6107 of the National Institute of Bioscience and Biotechnology, which produces a monoclonal antibody that specifically binds to β) into the peritoneal cavity of the experimental animal; and (c) the peritoneal cavity Collecting the ascites just before the experimental animal weakens and dies due to accumulation of ascites therein. 2. The experimental animal is a mouse, the dose of pristane is about 0.5 to about 1.0 ml, and the cell line is about 1 × on day 3 to 10 after pristane administration.
The method for producing ascites according to claim 1, wherein 10 ⁶ to about 1 × 10 ⁷ cells are transplanted. 3. The method of claim 2, wherein the dose of pristane is about 1.0 ml, and about 1 × 10 ^{7 of the} cell lines are transplanted three days after pristane administration. 4. The method of claim 2, wherein the dose of pristane is about 1.0 ml, and about 1 × 10 ^{7 of the} cell lines are transplanted 5 days after pristane administration. 5. The method of claim 2, wherein the dose of pristane is about 1.0 ml, and about 1 × 10 ^{7 of the} cell lines are transplanted 10 days after pristane administration. 6. The method of claim 2, wherein the dose of pristane is about 0.5 ml, and about 1 × 10 ^{6 of the} cell lines are transplanted 7 days after pristane administration. 7. Ascites produced by the method for producing ascites according to any one of claims 1 to 6.