JPH0469994B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a hybridoma cell line that produces a monoclonal antibody with antigen-antibody reaction specificity that reacts with mitochondrial aspartate aminotransferase and does not react with cytoplasmic aspartate aminotransferase. In more detail, GOT fractionation and quantification, etc.
The present invention relates to a hybridoma cell line that produces a monoclonal antibody with antigen-antibody reaction specificity that reacts with mitochondrial aspartate aminotransferase, which is extremely effective in distinguishing isoenzymes, but does not react with cytoplasmic aspartate aminotransferase.

Aspartate aminotransferase (GOT) is widely present in various animals and microorganisms, and is an important enzyme that takes part in the transfer of amino groups.
It is known to increase due to liver diseases, etc., and is an important measurement item in clinical tests.
GOT has two isozymes: S-GOT, which is present in the cytoplasmic fraction, and m-GOT, which is present in the mitochondrial fraction, and until now they have not been measured separately except for special purposes. However, recently the significance of measuring each isozyme separately has been discussed from the viewpoint of research and clinical testing, and in particular,
Since the blood concentration of GOT correlates well with the degree of liver disease and the prognosis of surgery, differential quantification of m-GOT is desired.

Currently, the method reported by Carmen et al. [J. Clin. Invest., Vol. 34, p. 131 (1955)] is often used to measure GOT. That is, GOT
This method involves measuring the amount of the coenzyme nicotinamide adenine dinucleotide that is oxidized when the oxaloacetate produced by the reaction is converted to malic acid by malate dehydrogenase based on absorbance in the ultraviolet region. In the method of measuring enzyme activity, which is typified by this method, both isozymes react with each other, making it impossible to measure them separately. for that,
When using these methods, it was necessary to perform fractionation in advance using a diethylaminoethyl ion exchanger or an electrophoresis device. These fractionation devices have drawbacks such as being expensive, requiring time for separation, and requiring a relatively large amount of sample. Furthermore, taking advantage of the fact that the two isozymes do not immunologically cross each other, S-GOT is precipitated using S-GOT antiserum, and m-GOT in the supernatant is measured using the method described above. See Special Publication No. 58-30543. ], but it still has drawbacks such as the separation method involves centrifugation and the preparation of a conjugate of antibody and sheep red blood cells is complicated. Furthermore, in the method of measuring enzyme activity, it is impossible to measure the apoenzyme that has been removed from the coenzyme (pyridoxal phosphate) unless the coenzyme is added, making the operation more complicated and expensive.

Also, direct immunoassay
A method for measuring m-GOT is also known (for example, Robert, Clinical Biochem, Vol. 12, p. 250 (1979)). Since this method takes advantage of the fact that antibodies do not cross each other, most of the drawbacks of the methods for measuring enzyme activity shown above can be overcome, but the antibodies used in these immunoassays are Because it is obtained by immunizing animals with purified antigen and separating it from serum, a large amount of purified antigen is required each time an antiserum is collected, and the amount of antibody is only small. However, it has been difficult to use in immunoassays because it has the disadvantage that its quality varies mainly due to individual differences between animals.

Therefore, in order to maximize the benefits of immunoassays, there has been a desire for antibodies that are inexpensive, available in large quantities, and of stable quality.

On the other hand, in 1975, Ko¨hler and Milstein developed fused cells (hybridoma) obtained by fusing lymphocytes from splenocytes of immunized mice with myeloma cells (myeloma). [Nature, Vol. 256, p. 495 (1975)]. Since this report, various hybridomas and monoclonal antibodies have been reported [for example, Koprowski, Proceedings
Of National Academic Sciences
USA (Proc.Natl.Acad.Sci.USA) Volume 75,
3938 pages (1978); Galfre et al. Nature vol. 266, 550 (1977); Ko¨hler et al. European Journal of Immunology (Eur. J. Immunol.) vol. 6 511
(1976)], there are no hybridoma cell lines capable of producing monoclonal antibodies with antigen-antibody reaction specificity that react with mitochondrial aspartate aminotransferase and do not react with cytoplasmic aspartate aminotransferase. Nothing has been described, nor has there been any report that its creation was successful.

Therefore, the present inventors conducted extensive research in search of a hybridoma cell line that can efficiently produce antibodies that can be applied to immunoassays that can easily and sensitively measure m-GOT, and found that mitochondrial aspartic acid A hybridoma cell line obtained by fusing myeloma cells with splenic lymphocytes from mice immunized with aminotransferase reacts with large amounts of mitochondrial aspartate aminotransferase but not with cytosolic aspartate aminotransferase. The present invention was completed based on the discovery that a monoclonal antibody can be produced that has specificity for antigen-antibody reactions.

That is, the present invention provides a specific antigen-antibody reaction in which the parent cells are mouse splenocytes and mouse myeloma cells, and which reacts with mitondria aspartate aminotransferase and does not react with cytoplasmic aspartate aminotransferase. This is a hybridoma cell line that produces monoclonal antibodies with specific characteristics.

Next, the cytological properties of the cell line of the present invention will be shown.

(1) Origin: These are fused cells created by fusion of myeloma cells and spleen lymphocytes of mice immunized with mitochondrial aspartate aminotransferase.

(2) Morphology: Shows a morphology almost similar to myeloma cells. For example, the size is 10-20 μm.

(3) Function: anti-m- that recognizes a single antigenic determinant
Constantly produce GOT monoclonal antibodies.

(4) Proliferative property: Shows a proliferative property almost similar to that of myeloma cells. That is, it proliferates approximately 10 times in 72 hours.

(5) Preservability: It can be stored extremely easily for a long period of time at -70℃ or below.

(6) Optimal growth conditions: temperature 37℃, pH 7.2.

(7) Growth range: Can grow at a temperature of 32°C to 42°C and a pH of 6.5 to 7.8.

To obtain the cell line of the present invention, for example, the following method may be employed. That is, first, a mammal, preferably a mouse or a rat, is immunized with m-GOT. All m-GOTs can be used as antigens, but in terms of clinical chemistry, conventional antisera are derived from mammals that cross human m-GOTs, such as humans, pigs, cows, rabbits, etc. , and is preferably derived from an animal different from the immunized animal. This m-
To collect GOT, for example The Journal
Of Biological Chemistry Volume 253 8842
Page, Journal of Biochemistry, Volume 82
It can be collected according to the method described on page 847. Amount of antigen used, site of administration, use of adjuvant, etc.
The immunization method may be similar to the conventional method for obtaining antiserum. For example, when using mice, 0.001 to 10 mg per mouse, preferably 0.01 mg per dose.
For the first time, ~1 mg of m-GOT is thoroughly mixed with an adjuband (e.g., Freund's complete adjuvant) and administered subcutaneously, intraperitoneally, etc., and after 3 weeks or more, an adjuband (e.g., Freund's incomplete adjuvant) is administered again. Mix well and administer subcutaneously, intraperitoneally, etc. Furthermore, after more than 3 weeks, m-
GOT alone is administered intravenously, intraperitoneally, subcutaneously, etc.
Be fully immunized. The animals thus immunized are killed, preferably 2 to 4 days after the final immunization,
Collect lymphocytes. Spleen, lymph nodes, peripheral blood, etc. are used to prepare lymphocytes. The lymphocytes are suspended in a culture medium.

Meanwhile, myeloma cells (myeloma) are prepared.
It is preferable to use myeloma derived from the same species as the immunized animal. Furthermore, the myeloma is preferably a drug-resistant mutant strain, and unfused myeloma is preferably one that does not grow in a hybridoma selection medium. Most commonly, 8-azaguanine resistant cell lines are used. It is a hypoxanthine-guanine-phosphoribosyltransferase (Hypoxantin-guanine-phosphoribosyltransferase).
guanine phosphoribosyl transferase) and cannot grow on hypoxanthine-aminopterin-thymidine (HAT) medium, a type of selective medium. In addition, it is desirable that the myeloma used itself does not secrete antibodies. From the above points, for example, commercially available mouse myeloma P3, X63, Ag8, 6, 5,
3 (X63・6・5・3), P3・X63・Ag8・U1
(P3U1), rat myeloma 210・RCY3・Ag1・
It is preferable to use 2, 3, etc.

The myeloma is then incubated with Eagle's minimal medium (MEM) containing serum, preferably fetal bovine serum.
Culture in a medium such as RPMI1640 medium (RPMI1640).

Next, the lymphocytes and myeloma obtained above are suspended and mixed in a medium such as ME or RPMI1640. The mixing ratio at this time can be selected arbitrarily, but preferably lymphocytes to myeloma cells are 1:1 to 20:1, preferably 5:1 to 10:1.
It is sufficient to use the ratio of The mixed cells are fused using a fusion promoter. As a fusion method, for example, Immunological Methods Vol. 2, p. 285 (Immunological Methods Vol. 1981,
Academic Press). As the fusion promoter, various polymeric substances, viruses, etc. can be used, but polyethylene glycol (PEG) and Sendai virus are preferably used. PEG having an average molecular weight of 400 to 20,000 can be used, preferably 1,000 to 7,500. The concentration used is preferably 40 to 60 vol.%.

The fused cells are washed to remove the fusion promoter and placed in MEN or MEN containing 5-15 vol.% serum.
Suspend in RPMI1640 medium and place in a 96-well culture dish, etc.
Dispense at a ratio of 5×10 ⁶ /well. Furthermore, if a selective medium (for example, HAT medium) is added to each well and the selective medium is exchanged as appropriate, unfused myelomas will die and only hybridomas will grow after 10 to 14 days. By the way, lymphocytes are grown in vitro (in vitro) for a long time.
It cannot grow in vitro) and dies after 10 to 14 days.

The search for hybridomas producing the desired antibody can be carried out by knowing the antibody titer of the culture supernatant. For example, the target m-
GOT is adsorbed onto a carrier such as a 96-well microtiter plate, the non-adsorbed surface is protected with bovine serum albumin, and the supernatant is collected and added. After completing the antigen-antibody reaction for 1 to 24 hours, a washed and labeled second antibody (for example, rabbit anti-mouse immunoglobulin if the lymphocytes are mouse-derived) is added and reacted in the same manner. As a label, a radioisotope, an enzyme, a fluorescent substance, etc. can be used. Similarly, after washing, detection is performed using a detection method depending on the labeling method. For example, in the case of an isotope, its radioactivity is measured, in the case of an enzyme, its enzymatic activity is measured by adding a substrate, and in the case of a fluorescent substance, its fluorescence intensity is measured with a fluorophotometer, and the detection is stronger than in the target experiment. Select a hole in the culture dish. Since there is a possibility that two or more types of hybridoma clones exist in the holes selected by this method, each clone is made into one clone (monoclone) by cloning. This cloning method can be carried out, for example, by the limiting dilution method. That is, dilute the solution to one per well, dispense it into a 96-well culture dish, and culture. 5～
When colonies appear after 10 days, search for hybridomas in holes where there seems to be only one colony per hole using the same procedure as above, dilute the antibody-producing strain again as above, and perform the same procedure as above. All you have to do is repeat it and get a monoclone.

By the above method, a hybridoma cell line that produces an anti-m-GOT monoclonal antibody can be created.

According to this method, a type of hybridoma cell line was created by fusing mouse myeloma cells with mouse spleen lymphocytes that had been previously immunized with m-GOT, and was named hybridoma MATH-1.
On March 15, 1981, this strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
This stock was not deposited. This MATH-1 can be stored frozen almost permanently at -80°C or lower, and is always available for distribution.

This hybridoma MATH-1 can be grown in a commonly used medium. For example, using RPMI or MEM containing 5 to 20 vol.% fetal bovine serum as a medium, it grows well at 37° C. in air containing 5 vol.% carbon dioxide concentration. It also has myeloma tumorigenic properties, so it proliferates in vivo, such as in syngeneic animals, nude mice, etc., and anti-m-
GOT monoclonal antibodies can be produced.

By culturing this hybridoma cell line, anti-m-GOT monoclonal antibodies can be collected in large quantities. There are two main methods for collecting monoclonal antibodies. One method is to culture in a culture container such as a flask using a medium, and collect the antibody from the supernatant. For example, if 0.5 to ⁵ x 105 hybridoma cell lines are planted in MEM or RPMI1640 medium containing 5 to 10 vol.% serum, they will grow 10 to 20 times in 2 to 4 days, and the supernatant after the culture will This is a method of collecting antibodies from fluid. Another method is to inoculate the hybridoma cell line thus cultured in a culture vessel into a syngeneic animal. That is, 10 ⁵ to 10 ⁷ hybridoma cell lines are administered intraperitoneally or subcutaneously to a syngeneic animal, and after 7 to 20 days, when the hybridoma cell line proliferates and the tumor grows, serum and ascites are collected. It's a method. When administering intraperitoneally, administer 2,6,10,14-
When mineral oils such as tetramethylbentadecane are administered, more ascites is produced.

The antibody thus obtained can be purified and used if necessary. That is, it can be purified using means commonly applicable to proteins, such as ammonium sulfate fractionation, ion exchangers, and affinity chromatography with immobilized m-GOT.

The physicochemical properties of the anti-m-GOT monoclonal antibody thus obtained are shown.

(1) Action: Acts on m-GOT to cause an antigen-antibody reaction.

(2) Antigen-antibody reaction specificity...does not react with S-GOT, but reacts with m-GOT.

(3) Optimal PH...6-9 (4) Stable PH range...3-11 (5) Method for measuring titer...Measure by dilution rate that can be detected by immunoassay.

(6) Suitable temperature range for action…20~40℃ (7) Conditions for inactivation...Deactivation occurs by heating at 100℃ for 10 minutes.

(8) Molecular weight...150000 The hybridoma cell line of the present invention has anti-m-
Since GOT monoclonal antibodies can be obtained in large quantities easily and inexpensively, m-GOT can be measured easily and with high sensitivity by immunoassay.

Next, the present invention will be specifically explained using examples.

Reference example 1 Journal of Biochemistry Volume 82
According to the method described on page 847, pig hearts were first minced, freeze-thawed, homogenized, heat treated at 65°C, and then centrifuged at 6000 rpm to obtain a crude extract.

Next, a GOT fraction obtained by fractionating 80% saturated ammonium sulfate from the crude extract was desalted by dialysis, and S-GOT and m-GOT were separated using a hydroxyapatite column. Furthermore, the m-GOT fraction
Pass through DEAE cellulose column (PH7.3),
The fraction was adsorbed on a CM cellulose column (PH6.3), eluted by increasing the salt concentration, and purified to obtain the antigen m-GOT.

Reference Example 2 The antigen m-GOT was obtained in exactly the same manner as in Reference Example 1 except that bovine heart was used.

Example 1 50 μg of porcine m-GOT obtained in Reference Example 1 was applied to an 8-week-old BALB/C mouse (obtained from Shizuoka Experimental Animal Cooperative Association) with a complete front ajium band (obtained from Hanui Chemical). :1 was mixed and emulsified and administered intraperitoneally, and 4 weeks later, 50 μg was mixed with incomplete Freund's adjuvant (obtained from Hanui Chemical Co., Ltd.) and administered in the same manner. Furthermore, after 4 weeks, 50 μg of pig m
-GOT was intravenously injected for booster immunization, and 2 days later, the spleen was taken out and NANKS medium (obtained from Osaka University Institute of Technology).
The mixture was loosened, suspended, and washed. On the other hand, mouse myeloma X636.5.3 (obtained from Flow Laboratory) was cultured for 2 days, and cells in the logarithmic growth phase were collected by centrifugation. Mix 10 ⁸ splenocytes with 10 ⁷ myeloma X63, 6, 5, 3,
After pelleting by centrifugation, dissolve in water at 37°C.
Gradually add 1 ml of 50 vol.% PEG4000-RPMII1640 (obtained from Kibuco Co., Ltd.) over 1 minute, and then
After stirring gently for 6 minutes, 9ml of
PEG4000 (obtained from Sigma) was diluted by gradually adding RPMI1640 medium. PEG by centrifugation
The solution was removed, and 10 ml of RPMI1640 medium containing 10 vol.% fetal bovine serum was added to the pellet, and 0.1 ml was dispensed into each hole in a 96-well culture dish (obtained from Nunc).
The next day, add 0.1 ml of HAT medium, 2, 3,
Half the amount (0.1ml) for 5 times on days 5, 8, and 11.
The culture medium was discarded and fresh HAT medium was added. 14
After a few days, hybridoma growth was observed in 78 out of 96 wells, and the supernatant was collected using the antigen pig m-GOT.
was adsorbed onto a 96-well microtiter plate and detected using a second antibody labeled with peroxidase (obtained from Vector Co., Ltd.). As a result, about 10 or so were found to be colored. Select 3 strains with the highest cell density and clone them using the limiting dilution method, which involves diluting and culturing the cells to a density of 1 cell/well in a 96-well culture dish.
Hybridoma MATH-1 was obtained.

Reference Example 3 Hybridoma MATH-1 obtained in Example 1 was
The cells were cultured in RPMI1640 medium containing 10 vol.% fetal bovine serum, and the supernatant at a cell concentration of 10 ⁶ cells/ml was collected to obtain an anti-m-GOT monoclonal antibody.
When this antibody was assayed using the method used for the search in Example 1, sufficient color was developed to allow measurement at 256-fold dilution.

Next, 5 × 10 ⁶ hybridoma MATH-1 were intraperitoneally administered to BALB/C mice that had been administered 0.5 ml of 2,6,10,14 tetramethylpentadecane 6 days earlier to form tumors. later serum
0.7ml/mouse and ascites 2.0ml/mouse were collected, and anti-m-
GOT monoclonal antibody was obtained. When this antibody was assayed in the same manner as the culture supernatant, it was measurable at a magnification of approximately 500,000 times.

In addition, these two antibodies are human m-
Since it reacted with GOT and did not react with any S-GOT, it was fully applicable to the differential quantification of GOT in clinical tests.

Example 2 Using the bovine m-GOT obtained in Reference Example 2,
Using a 96-well microtiter plate that had been immunized in the same manner as in Example 1, fused in the same manner as in Example 1, and adsorbed with bovine m-GOT, a search was performed in the same manner as in Example 1, and 10 strains of bovine m-GOT were detected. Since antibody-producing strains were obtained, one of them was cloned in the same manner as in Example 1 to obtain a hybridoma cell line.

Reference Example 4 The cell line obtained in Example 2 was cultured in exactly the same manner as in Reference Example 3, and an anti-m-GOT monoclonal antibody was obtained from the supernatant.

When this antibody was assayed in the same manner as in Reference Example 3, the color was measurably developed up to 512 times.

This antibody reacted with human m-GOT, but did not react with any S-GOT.

Claims (1)

[Claims] 1. A monoclonal antibody whose parent cells are mouse splenocytes and mouse myeloma cells and which has antigen-antibody reaction specificity that reacts with mitochondrial aspartate aminotransferase but does not react with cytoplasmic aspartate aminotransferase. The hybridoma cell line that produces it.