JPS6024196A - Anti-m-got monoclonal antibody and its preparation - Google Patents

Abstract

PURPOSE:To provide the titled antibody reactive to mitocondrial aspartate aminotransferase (m-GOT), non-reactive to cytosolic aspartate aminotransferase (S-GOT), and having an antigen-antibody reaction specificity effective extremely for the discrimination of isozymes e.g. fractional determination of GOT, etc. CONSTITUTION:The present invention provides a monoclonal antibody (anti-m- GOT monoclonal antibody) having antigen-antibody reaction specificity, i.e. reactive to m-GOT and non-reactive to S-GOT. The antibody can be prepared by culturing a hybridoma cell strain capable of producing anti-m-GOT monoclonal antibody, and separating the produced anti-m-GOT monoclonal antibody from the cultured product. The cell strain to the used in the present invention is prepared as follows. A mammal such as mouse is immunized with m-GOT. The antigen is preferably the one originated from mammals having antiserum crossing over the human m-GOT from the viewpoint of clinical chemistry, e.g. originated from human, pig, etc. and other than the immunized animal. The m-GOT can be separated by conventional process.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anti-m-GOT monoclonal antibody and a method for producing the same, and more particularly.

The present invention relates to an anti-m-GOT monoclonal antibody that is extremely effective in differentiating isoenzymes, such as in the differential quantification of GOT, and a method for producing the same.

Aspartate amine transferase (GOT)
It is an important enzyme that is widely present in various animals and microorganisms and takes part in the transfer of amino groups, but it is especially important for GO in serum.
It is known that T content increases due to myocardial infarction, muscle disease, liver disease, etc., and is an important measurement item in clinical tests. GOT has 5-G present in both cytoplasmic parts.
There are two isozymes: OT and m-GOT, which are present in the mitochondrial fraction.

Previously, they were not 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, it is desired to separately quantify m-GOT.

Currently, GOT measurements have been reported to Carmen et al. [Journal of Clinical Investigation (J,
Cl1n, Invest,) Volume 34, Page 131 (1
955)) is often used. That is, this is a method in which the amount of the coenzyme nicotinamide adenine dinucleotide oxidized when oxaloacetate produced by the GOT reaction is converted to malic acid by malate dehydrogenase is measured by absorbance in ultraviolet light. 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 five separations, and requiring a relatively large amount of sample. Furthermore, we take advantage of the fact that the two isozymes do not immunologically cross each other.

5-GOT was precipitated using 5-GOT antiserum.

A method in which m-GOT in the supernatant is measured by the method described above [see Japanese Patent Application Laid-open No. 19918-1983. ]Although there is.

Again, there are drawbacks such as the means of separation involves centrifugation and the preparation of a conjugate of antibodies and sheep red blood cells is complicated.

Furthermore, the method of measuring enzyme activity is that the apoenzyme is removed from the coenzyme (pyridoxal phosphate).

Measurement is impossible unless the coenzyme is added, making the operation more complicated and expensive.

In addition, m-G
Methods of measuring OT [e.g. Robert
+Clinical Biochemistry (C1in Bi
ochem) vol. 12, p. 250 (1979)] is also known. This takes advantage of the fact that antibodies do not cross each other.

Although most of the drawbacks of the methods for measuring enzyme activity listed above can be overcome, the antibodies used in these immunoassays cannot be obtained using conventional methods, i.e., by immunizing animals such as rabbits with purified antigens and separating them from the serum. Because it is obtained by a method, a large amount of purified antigen is required each time antiserum is collected, and only the smallest amount of antibody can be obtained, and the antibody is not single or homogeneous. However, its quality also varied mainly due to individual differences between animals, making it difficult to use in immunoassays.

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

On the other hand, in 1975, Köhler and Milstein used fused cells (hybridoma) obtained by fusing lymphocytes from the lung cells of immunized mice with myeloma cells. Single, homogeneous antibody (monoclonal antibody)
[Natur +- (Natur
e) Vol. 256, p. 495 (1975)), and since this report, various hybridomas and monoclonal antibodies have been reported [for example, Kobrowski (Kobrowski et al.
prowski) + Proceedings of the National Academy of Sciences (Pro
c, Natl, Acad, Sci, USA)
Volume 75, page 393B (, 1978); Garfur (G
alfre) et al. + - (Nature) 266
Volume, 550 pages (1977);
) and European Journal of Immunology (E
ur, J. Immunol, ) + 6, p. 511 (1976)], but nothing is described about anti-m-GOT monoclonal antibodies.

Nor has there been any report that its creation has been successful.

Therefore, the present inventors have conducted extensive studies with the aim of providing 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, anti-m-G
We discovered that a hybridoma cell line obtained by fusing OT antibody-producing lymphocytes with myeloma cells produces a large amount of anti-m-GOT monoclonal antibodies, and that the antibodies achieve the above objects9. completed.

That is, 1 the present invention reacts with m-GOT, and 5-GOT
A monoclonal antibody (anti-m-GOT monoclonal antibody) having an antigen-antibody reaction specificity that does not react with Anti-m-GOT characterized by collecting
This is a method for producing O monoclonal antibody.

Cell lines to be used in the present invention include, for example, 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 almost the same morphology as myeloma cells. For example, the size is 10 to 20 μm.

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

(4) Proliferative property: Shows substantially the same proliferative property as 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°C or lower.

(6) Optimal growth conditions: temperature 37°C, pH 7.2° (7) Growth range: temperature 32°C to 42°C, pH 6,
It can grow between 5 and 7.8.

For example, the following method may be employed to obtain the cell line used in the present invention. 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 clinical chemistry, conventional antiserums cross-reactive with human m-GOTs from mammals (e.g., humans, pigs, cows, rabbits, etc.). and is preferably derived from an animal different from the immunized animal. To collect this m-GOT 2For example, The Journal of Biological Chemistry Vol. 253 88
It may be collected according to the method described in p. 42, Journal of Biochemistry, Vol. 82, p. 847. Immunization methods such as the amount of antigen to be used, the site of administration, and the use of adjuvants may be in accordance with conventional methods for obtaining antiserum. For example, when using mice, 0.0 per mouse per time.
01-10n+g, preferably 0.01-1mg m-
GOT is initially mixed well with an adjuvant (eg, Freund's complete adjuvant) and administered subcutaneously.

The drug is administered intraperitoneally, etc., and after 3 weeks or more, an adjuvant (for example, Freund's incomplete adjuvant) is mixed well and administered subcutaneously intraperitoneally, etc.

Furthermore, after 3 weeks or more, m-GOT alone was administered intravenously.

Administer intraperitoneally or subcutaneously to sufficiently immunize. Animals thus immunized are preferably immunized 2 to 4 days after the final immunization.
After a day, kill the animals and collect the lymphocytes.

Lungs, lymph nodes, immature 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 preferably does not grow in a hybridoma selection medium. Most commonly, 8-azaguanine resistant cell lines are used. This is hyboxanthin-guanine-phosphoribosyltransferase (Hypoxanthin-guanine-phosphoribosyltransferase).
tin guaninephosphoribosyl
transferase) is deficient.

It cannot grow on hypoxanthine-aminobuterine-thymidine (HAT) medium, which is a type of selective medium. Also.

It is desirable that the myeloma used does not secrete antibodies itself. From the above points, 2. For example, commercially available mouse myeloma P
3.X63.8g8.6.5.3 (X63.6.5.3
), P3.X63.Ag8.Ul (P2O3), rat myeloma 210.RCY3.Agl.2.3, etc. are preferably used.

The myeloma was cultured in Eagle's minimal medium (MEM) containing serum, preferably fetal bovine serum, RPM1164.
Culture in a medium such as 0 medium (RP old 1640).

Next, the lymphocytes and myeloma obtained above are suspended and mixed in a medium such as MEM or RP Old 1640. The mixing ratio at this time can be selected arbitrarily.

Preferably, the ratio of lymphocytes to myeloma cells is 1:1 to 20:1. Preferably, a ratio of 5:1 to 10:1 may be used. The mixed cells are fused using a fusion promoter. Examples of fusion methods include 1, for example, Immunolo-gical Methods, Vol. 2, p. 285.
thods, VolI [1981+ Academy
c Press). As the fusion promoter, various polymeric substances, viruses, etc. can be used.

Preferably polyethylene glycol (PEG)'+
Sendai virus may be used. PEG having an average molecular weight of 400 to 20,000 can be used, but preferably PEG has an average molecular weight of 1,000 to 7,500.The concentration used is preferably 40 to 60% by volume.

The fused cells were washed to remove the fusion promoter.

MEM or RPM11 containing 5-15vo10% serum
640 medium and 0.5 to 5XI in a 96-well culture dish etc.
Dispense at the ratio of OG/hole. In addition, each hole was filled with selective medium (
For example, add HAT medium) and replace the selective medium as appropriate.

After 10-14 days, unfused myelomas die.

Only hybridomas grow. By the way, lymphocytes cannot grow for long periods in vitro.

It will die 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, and the non-adsorbed surface is protected with bovine serum albumin.

Collect and add the supernatant. After 1 to 24 hours of antigen-antibody reaction, wash and label the second antibody (e.g., rabbit anti-mouse immunoglobulin if the lymphocytes are mouse-derived).
Add and react in the same way.

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 using a fluorophotometer.

Select the hole in the culture dish that was detected more strongly than in the target experiment. 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 by, for example, the limiting dilution method. That is, the solution is diluted to one per well, dispensed into a 96-well culture dish, and cultured. 5-10
When colonies appear after a day, 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 repeat the same procedure as above. , all you need to do is 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 created by fusing mouse myeloma cells with mouse lung lymphocytes previously immunized with m-GOT was named bilidoma MATH-1.
The procedure for depositing this strain with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry was carried out in Japan, but this strain was not deposited. This M
ATH-1 can be stored frozen almost permanently at -80''C or below, and is always available for distribution.

The anti-m-GOT monoclonal antibodies of the present invention include, for example,
By culturing the hybridoma cell line obtained above, a large amount can be collected. For example, there are broadly two methods for collecting monoclonal antibodies. One method is to culture in culture containers such as two flasks using a medium, and collect antibodies from the supernatant. For example, ti'MEM or R
PM11640 medium, 0.5-5 X 105 +I
If you plant 1 hybridoma cell line, it will grow in 2 to 4 days.
In this method, antibodies are collected from the supernatant after the culture is grown 20 times. Another method is to inoculate the hybridoma cell line thus cultured in a culture vessel into a syngeneic animal.

In other words, 105 to 107 hybridoma cell lines 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. be. When administering intraperitoneally, administer 2. in advance (3 to 7 days before). 6. 10.
When mineral oils such as 14-tetramethylpentadecane are administered, larger amounts of ascites are obtained.

The antibody thus obtained can be purified and used if necessary. That is, it can be purified using means applicable to common 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 of the present invention thus obtained are shown, and these physicochemical properties are those of the antibody obtained from hybridoma MAT11-1.

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

(2) Antigen-antibody reaction specificity: No reaction with 5-GOT.

Reacts with m-GOT.

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

(6) Suitable temperature range for action: 20 to 40°C (7) Conditions for deactivation: Deactivation occurs by heating at 100°C for 10 minutes.

(8) Molecular weight...150,000 The anti-m-GOT monoclonal antibody of the present invention can be used in large quantities
Moreover, since it can be obtained easily and inexpensively, m-GOT can be measured easily and with high sensitivity by immunoassay.

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

Reference Example 1 According to the method described in Journal of Biochemistry, Vol. 82, p. 847, pig hearts were first minced, freeze-thawed, homogenized, and heat-treated at 65°C.
A crude extract was obtained by Orpm centrifugation.

Next, the GOT fraction obtained by fractionating 80% saturated ammonium sulfate from the crude extract was dialyzed and desalted, and 5-GOT (!: m-GOT) was separated using a hydroxyapatite column.Furthermore, the m- The GQT fraction was passed through a DEAE cellulose column (pH 7, 3), and both fractions were adsorbed on a CM Nacellu column (pH 6, 3), eluted by increasing the salt concentration, and purified to obtain the antigen m-GOT. I got it.

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 50.17 g of pig-made mice obtained in Reference Example 1 were given to 8-week-old mouse BALB/C (obtained from Shizuoka Laboratory Animal Cooperative Association).
-GOT was mixed and emulsified with complete front adjuvant (obtained from Gyudon Kagaku) in a ratio of 1=1, administered intraperitoneally, and 4 weeks later, 50 μg of incomplete Freund's adjuvant (obtained from Gyudon Kagaku) was mixed and emulsified.
Obtained from Gyudon Kagaku. ) and administered. Furthermore, 4 weeks later, 50 μg of porcine m-GOT was intravenously injected for boosting.
Two days later, the lungs were removed.

The mixture was loosened, suspended, and washed in NANKS medium (obtained from Osaka University Institute of Technology). On the other hand, mouse myeloma, X636.5
・3 (obtained from Flow Laboratories) was cultured for 2 days prior, and cells in the logarithmic growth phase were collected by centrifugation. Myeloma X63, 6, 5, 31 with 108 tile cells
After mixing with 07 and pelletizing by centrifugation, 37
50vol. in a water bath at ℃. %PEG4000-RPM
1 ml of I11640 (obtained from Kibuco) was gradually added over 1 minute, and after stirring gently for another 1 minute, 9 ml of RPM11640 medium was gradually added over 6 minutes to PE.
G4000 (obtained from Sigma) was diluted.

Remove the PEG solution by centrifugation and add 1°v to the pellet.
ol, 10ml of RPM11640 medium containing % fetal bovine serum
1 was added and 0.1 ml was dispensed into each hole in a 96-well culture dish (obtained from Nunc Co.). The next day, the HAT medium was diluted with 0.
Add 1 ml at a time for 5 days (2, 3, 5, 8, and 11 days)
Half of the culture solution (0.1 ml) was discarded twice and fresh HAT medium was added. After 14 days, hybridoma growth was observed in 78 of the 96 wells, so the supernatant was used to adsorb the antigen, porcine m-GOT, on a 96-well microtiter plate, and a second antibody labeled with peroxidase (Vector Co., Ltd. As a result of a search using the detection method obtained from
Among them, three strains with particularly high color development were selected and cloned using the limiting dilution method, in which the cells were diluted and cultured at a density of one cell/well in a 96-well culture dish, and hybridoma MATH
-1 was obtained.

Example 1 The hybridoma MATI (-1 obtained in Reference Example 3) was
ol.

The cells were cultured in RPM11640 medium containing % fetal bovine serum.

The supernatant with a cell concentration of 106 cells/ml was collected and
-GOT monoclonal antibody was obtained. This antibody was assayed using the method used for the search in Example 1.

A 256-fold dilution produced enough color to be measurable.

Next, hybridoma MATI (-15x 106) was intraperitoneally administered to BALB/C mice that had been administered 0.5 ml of 2.6.io, 14 tetramethylpentadecane 6 days earlier to form tumors. Afterwards, 0.7 ml of serum and 2.0 ml of ascites were collected from each animal, and anti-m
-GOT monoclonal antibody was obtained. When this antibody was assayed in the same manner as the culture supernatant, it was measurable even at a magnification of about 500,000 times.

In addition, these two antibodies reacted with human m-GOT and did not react with any 5-GOT, so
It was sufficiently applicable to the differential quantification of GOT in clinical tests.

Reference Example 4 Using the bovine m-GOT obtained in Reference Example 2, Example 1
and fused in the same manner as in Example 1.

A search was conducted in the same manner as in Reference Example 3 using a 96-well microtiter plate to which bovine m-GOT was adsorbed.

Since 10 bovine m-GOT 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, 51
The color was measurably developed up to twice as much.

This antibody reacted with human m-GOT, but none of the 5
- It also did not react with GOT.

Patent applicant: Unikorikiki Co., Ltd.

Claims (2)

[Claims] (1) Mitochondrial aspartate aminotransferase (Mitochondrial aspartate aminotransferase)
teaminotransferase, also known as Mitc
ondrial glutamicoxaloacet
ic transaminase; hereinafter abbreviated as m-G
It's called OT. ) and reacts with cytosolic aspartate aminotransferase (Cytosolic GOT or 5olable GOT; hereinafter referred to as 5-GOT).
A monoclonal antibody having antigen-antibody reaction specificity that does not react with m- (hereinafter referred to as OT monoclonal antibody for anti-m-). (2) Cultivate a hybridoma cell line capable of producing anti-m-GOT monoclonal antibodies, and use anti-m-GOT from the culture.
Anti-m characterized by collecting T monoclonal antibody
- Method for producing GOT monoclonal antibody.