JPH0424039B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monoclonal antibody having antigen-antibody reaction specificity that reacts with mitochondrial aspartate aminotransferase and does not react with cytoplasmic aspartate aminotransferase, and a method for producing the same. , more specifically, a monoclonal antibody with antigen-antibody reaction specificity that reacts with mitochondrial aspartate aminotransferase and does not oppose cytoplasmic aspartate aminotransferase, which is extremely effective for isoenzyme differentiation, such as the differential quantification of GOT. and its manufacturing method.

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 measures 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 by measuring the absorbance under ultraviolet light. In the method of measuring enzyme activity represented by this method, it also reacts with two isozymes,
It is impossible to measure them separately. Therefore, when using these methods, it was necessary to perform fractionation in advance using a diethylaminoethyl ion exchanger or an electrophoresis device. These sorting devices are
The disadvantages are that the equipment is expensive, the separation takes time, and a relatively large amount of sample is required. Furthermore, by 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 these methods utilize the fact that antibodies do not cross each other, they can almost overcome the drawbacks of the methods for measuring enzyme activity shown above. However, 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 only the smallest amount of antibody can be obtained. It has been difficult to use it in immunoassays because it has the disadvantage that the antibody is not single and homogeneous, and 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 [e.g.
Koprowski, Proceedings of National Academic Science USA, Vol. 75, p. 3938 (1978); Galfre et al., Nature, Vol. 266, p. 550 (1977) 2005); European Journal of Koehler et al.
Immunology (Eur. J. Immunol.) Vol. 6, p. 511 (1976)] is a monoclonal antibody with antigen-reactive specificity that reacts with mitochondrial aspartate aminotransferase and does not react with cytoplasmic aspartate aminotransferase. Nothing has been described about antibodies, nor has there been any report that their creation has been successful.

Therefore, the present inventors aimed to provide an antibody that can be applied to an immunoassay that can easily and sensitively measure m-GOT, and a manufacturing method that can efficiently produce the antibody.
As a result of extensive research, we found that a hybridoma cell line obtained by fusing myeloma cells with spleen lymphocytes from mice immunized with mitochondrial aspartate aminotransferase reacted with a large amount of mitochondrial aspartate aminotransferase, resulting in the production of cytoplasmic asparagine. The present invention was completed by discovering that a monoclonal antibody having antigen-antibody reaction specificity that does not react with acid aminotransferase can be produced, and that the antibody can achieve the above-mentioned objects.

That is, the present invention reacts with m-GOT and produces S-
A monoclonal antibody with antigen-antibody reaction specificity that does not react with GOT (hereinafter referred to as anti-m-GOT monoclonal antibody) and a hybridoma cell line capable of producing anti-m-GOT monoclonal antibody are cultured, and anti-m -Anti-m- characterized by collecting GOT monoclonal antibody
This is a method for producing GOTO monoclonal antibodies.

Next, examples of cell lines used in the present invention include cell lines exhibiting the following cytological properties.

(1) Origin: A fused cell created by fusion of anti-m-GOT antibody-producing lymphocytes and emyloma cells.

(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 used in the present invention, the following method may be employed, for example. That is, first, m-
Immunize with GOT. As antigens, all m-
GOT can be used, but in terms of clinical chemistry, conventional antisera are derived from mammals that cross human m-GOT, such as humans, pigs, cows, rabbits, etc., and are different from the immunized animal. Animal origin is preferred. This m-GOT may be collected, for example, according to the method described in The Journal of Biological Chemistry, Vol. 253, p. 8842, and Journal of Biochemistry, Vol. 82, p. 847. The immunization method, such as the amount of antigen used, the site of administration, and the use of adjuvant, may be in accordance with conventional methods for obtaining antiserum. For example, when using mice, 0.001 to 10 mg per mouse,
Preferably, 0.01 to 1 mg of m-GOT is mixed well with an adjuband (for example, Freund's complete adjuband) and administered subcutaneously, intraperitoneally, etc. for the first time, and then administered again after three weeks or more. ) For example, Freund's incomplete adjuvant) is mixed well and administered subcutaneously, intraperitoneally, etc. Furthermore, after 3 weeks or more, m-GOT alone is administered intravenously, intraperitoneally, subcutaneously, etc. to sufficiently immunize the animal. Animals thus immunized are sacrificed, preferably 2 to 4 days after the final immunization, and lymphocytes are harvested. 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), Rattomyeloma 210・RCY3・
It is preferable to use Ag1, 2, 3, etc.

The myeloma is washed with serum, preferably Eagle's minimal medium (MEM) containing fetal bovine serum.
Culture in a medium such as PRMI1640 medium (RPMI1640).

Next, the lymphocytes and myeloma obtained above are suspended and mixed in a medium such as MEM 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. Examples of fusion methods include, for example, Immunolo-gical Methods Vol 1981, p. 285.
Academic Press). As the fusion promoter, various polymeric substances, urinary tract, etc. can be used, but preferably polyethylene glycol (PEG) and Sendai virus may be 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 MEM or MEM 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.

Search for hybridomas producing the desired antibody can be performed 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 clones of two or more types of hybridomas exist in the holes selected by this method, they are made into individual clones (monoclones) 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 the 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 repeat 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.

The anti-m-GOT monoclonal antibody of the present invention is
For example, by culturing the hybridoma cell line obtained above, a large amount can be collected.
There are, for example, 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,
MEM or RPMI1640 containing 5-10 vol.% serum
When 0.5 to ⁵ x 105 hybridoma cell lines are planted in a medium, they grow 10 to 20 times in 2 to 4 days, and antibodies are collected from the supernatant after the culture. Another method is to inoculate the hybridoma cell line thus cultured in a culture vessel into a syngeneic animal. That is, hybridoma cell lines
In this method, 10 ⁵ to ^{10 7} cells are administered intraperitoneally or subcutaneously to a syngeneic animal, and after 7 to 20 days, the hybridoma cell line proliferates and when the tumor becomes large, serum and ascites are collected. When administering intraperitoneally, a larger amount of ascites can be obtained by administering mineral oil such as 2,6,10,14-tetramethylpentadecane in advance (3 to 7 days).

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 anti-m-GOT of the present invention thus obtained
The monoclonal antibody exhibits physicochemical properties, and these physicochemical properties are those of the antibody obtained from the hybridoma MATH-1.

(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) Optimum 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) Range of suitable temperature for action…20~40℃ (7) Conditions for inactivation...Deactivation is achieved by heating at 100°C for 10 minutes.

(8) Molecular weight...150000 The anti-m-GOT monoclonal antibody of the present invention is
Since it can be obtained easily and inexpensively in large quantities, 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.

Reference Example 3 50 μg of porcine m-GOT obtained in Reference Example 1 was added to an 8-week-old mouse BALB/C (obtained from Shizuoka Experimental Animal Cooperative Association) with a complete front adjuvant band (obtained from Hani 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-
A booster immunization was given by intravenously injecting GOT, and 2 days later, the spleen was taken out, suspended in NANKS medium (obtained from Osaka University Institute of Technology), and washed. On the other hand, mouse myeloma X63-6-5-3 (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, in a 37℃ water bath.
50vol.% PEG4000−RPMI11640) obtained from Kivco. ) was gradually added over 1 minute, and after stirring gently for another 1 minute, 9 ml of
PEG4000 (obtained from Sigma) was diluted by gradually adding it to RPMI1640 medium. PEG by centrifugation
The solution was removed, 10 ml of RPMI640 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 dose (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 on a 96-well microtiter plate and detected using a second antibody labeled with peroxidase (obtained from Vector). 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.

Example 1 Hybridoma MATH-1 obtained in Reference Example 3 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, 60.5 ml of 5 x 10 ⁶ hybridoma MATH-1 was intraperitoneally administered to BALB/C mice to form tumors, and 12 days later, serum serum 0.7
ml/mouse and ascites 2.0ml/mouse were collected, and anti-m-GOT
Monoclonal antibodies were 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 COT in clinical tests.

Reference example 4 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 conducted in the same manner as in Reference Example 3, 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 Reference Example 3 to obtain a hybridoma cell line.

Example 2 The cell line obtained in Reference Example 4 was cultured in exactly the same manner as in Example 1, 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)

[Scope of Claims] 1. A monoclonal antibody having antigen-antibody reaction specificity that reacts with mitochondrial aspartate aminotransferase and does not react with cytoplasmic aspartate aminotransferase. 2. Cultivate a hybridoma cell line created in advance by fusing mouse myeloma cells with spleen lymphocytes of a mouse immunized with mitochondrial aspartate aminotransferase, and react with mitochondrial aspartate aminotransferase from the culture, Characterized by collecting a monoclonal antibody having antigen-antibody reaction specificity that does not react with cytoplasmic aspartate aminotransferase.Reacts with mitochondrial aspartate aminotransferase but does not react with cytoplasmic aspartate aminotransferase. A method for producing a monoclonal antibody having antigen-antibody reaction specificity.