JPS6349076A - Hybridoma cell strain - Google Patents

Abstract

PURPOSE:To produce a monoclonal antibody effective in inhibiting anti-CK-M activity, by establishing a hybridoma bacterial strain capable of producing a monoclonal antibody inhibiting creatine kinase M-subunit activity. CONSTITUTION:An animal is immunized by using CK-MM or CK-MB as an antigen and a lymphocyte of the animal is fused with a myeloma cell to obtain a hybridoma cell strain. A large amount of monoclonal antibody capable of inhibiting anti-CK-M activity can be produced by culturing the hybridoma cell strain. The monoclonal antibody can be collected e.g. by culturing the strain in a culture vessel such as a flask and collecting the antibody from the supernatant liquid of the cultured product or by inoculating a hybridoma cell strain cultured in a culture vessel to the same kind of animal and proliferating in the animal.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention provides a method for treating creatine kinase (Creatine kinase) in biological fluids.
ine Kinase EC2, 7, 3, 2; hereafter CK
It is abbreviated as A hybridoma capable of producing a monoclonal antibody (hereinafter abbreviated as anti-CK-M activity-inhibiting monoclonal antibody) that can be used for the quantification of the MB isozyme of CK, reacts with CK, and inhibits CK-M subunit activity. It concerns cell lines.

(Prior Art) CK is an enzyme that catalyzes both the left and right reactions of formula (1).

(The abbreviations are CP: creatine phosphorus a, C: creatine,
ADP: adenosine diline fi, ATP: adenosine triphosphate. ) GK by a combination of two subunits.

It is known that three isozymes exist, and they are CK MM, CK MB, and CK-BB, respectively.
-MB is present in the heart muscle, and CK-BB is mainly present in organs such as the brain and kidneys. Among these, CK-MB exists specifically in the cardiac muscle.

Its specific measurement has been shown to be a particularly useful marker in the diagnosis of myocardial infarction.

In the field of clinical testing, measurement of CK activity is one of the important items routinely measured in the diagnosis of muscle diseases, neurological diseases, central nervous system diseases, psychosis, heart diseases, etc. CK measurement methods have been proposed. One method is to measure leftward activity; among these are:
■Method for measuring inorganic phosphoric acid produced by hydrolysis of CP,
(2) ADP is pyruvate kinase (hereinafter abbreviated as PK).

) and lactate dehydrogenase to produce reduced β-nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH).
How to measure this as a decrease in absorption.

(2) There is a method in which ADP is converted into pyruvic acid using PK and then reacted with 2,4-dinitrophenylhydrazine to measure the hydrazone produced.

In addition, methods for measuring activity in the right direction include: ■ Generated C
React with a dye and compare the colors. Alternatively, a method of measuring fluorescence, ■ a method using luciferase (Japanese Unexamined Patent Publication No. 51-4
1597, JP 55-120796, JP 56-26200, JP 57-10519
See Publication No. 9. ), ■Method using phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase (Japanese Patent Publication No. 341-1981) No. 9, JP-A-56-
See Publication No. 155000. ), (2) A method using hexokinase and glucose-6-phosphate dehydrogenase (hereinafter abbreviated as 06PDH).

■Glucokinase (hereinafter abbreviated as GlcK) and 06
There is a method using PDH.

However, since these methods cannot distinguish and measure the three isozymes, several methods have been proposed to specifically measure CK-MB, which is a useful marker for myocardial infarction. first.

(2) Adding an antibody that specifically inhibits GK-M activity.

There is a method of measuring only CK-B activity by methods ① to ② (see Japanese Patent Publications No. 56-19239 and Japanese Patent Publication No. 58-20274). In this case, although it is indistinguishable from CK-BB, the amount of CK-BB in normal serum can be ignored. In addition, ① separation of CK-MB isozyme with ion exchange resin and measurement of CK-MB activity by methods ① to ② (JP-A-54-65096, JP-A-54-
(Refer to Publication No. 163886); Furthermore, ■Method of measuring protein amount by immunoassay rather than enzyme activity [
Clinical chemistry (CIinical chem)
-istry), Vol, 29. 1232 pages (1
983)] have been reported. Among these methods, the immunoinhibition method (2) is the most useful because it is simple to operate and easy to compare with 19cK activity. In this method, polyclonal antibodies isolated from antiserum obtained by immunization with MM isozyme as an antigen are used.

On the other hand, in 1975, Köhler and Milstein proposed fused cells (hybridoma) obtained by fusing lymphocytes from lung cells of immunized mice with myeloma cells (myeloma).
We demonstrated that it is possible to produce a single, homogeneous antibody (monoclonal antibody) using
), vol. 256, p. 495 (1975)).

Since this report, various hybridomas and monoclonal antibodies have been reported [for example.

Kobrowski, Proceedings of National Academic Science (Proc, Natl, Acad, S)
ci, USA) vol. 75, p. 3938 (1978),
; Gatfre et al.

Nature-t-- (Nature), Volume 266, 55
0 page (1977); Köhler et al., European Journal of Immunology (Eur.
J, Immunol, ) + vol. 6, p. 51) (197
6)) However, nothing has been described about anti-CK-M inhibitory monoclonal antibodies, nor has there been any report that their creation has been successful.

(Problem to be solved by the invention) To obtain the polyclonal antibody described above.

It is necessary to highly purify the antigen, but MM antigen is difficult to purify to a high degree, so the crude enzyme obtained by ethanol precipitation requires chromatography with DEAE Sephacel, CM-cellulose, phenyl Sepharose, and blue Sepharose. . Particularly in immunoinhibition methods, mutual separation of 9 isozymes is important, and incomplete separation of 1 isoenzyme results in 1
for example.

Antibodies immunized with an antigen containing MB antigen mixed into MM antigen are thought to also produce antibodies that inhibit the B subunit, and if such antibodies are used as inhibitors, the MB isozyme is inhibited. It also inhibits the B subunit, giving abnormally low readings.

Correct measurements cannot be made.

Furthermore, to obtain polyclonal antibodies.

A large amount of the highly purified MM antigen is always required, and the loft varies widely due to individual differences among immunized animals.

(Means for Solving the Problems) As a result of intensive research to solve these problems, the present inventors immunized animals using CK-MM or CK-MB as antigens, and A hybridoma cell line obtained by fusing lymphocytes and myeloma cells contains a monoclonal antibody (anti-CK-M activity-inhibiting monoclonal antibody) that reacts with CK and inhibits CK-M subunit activity. Found to be produced in large quantities.

The invention has been completed.

That is, the gist of the present invention is a hybridoma cell line that is obtained by fusion with lymphocytes prepared from an animal immunized with creatine kinase and has the ability to produce a monoclonal antibody that inhibits anti-GK-M activity.

Next, the cytological properties of the 9 cell lines of the present invention will be shown.

(1) These are fused cells created by fusion of derived anti-CK-M antibody-producing lymphocytes and myeloma cells.

(2) Form Shows a morphology almost similar to myeloma cells.

For example, the size is 10 to 20 μm.

(3) Constantly produce an anti-CK-M activity-inhibiting monoclonal antibody that recognizes a single functional antigenic determinant.

(4) Proliferation Shows almost the same proliferative property as myeloma cells.

That is, it proliferates approximately 10 times in 72 hours.

(5) Preservability It can be stored very easily for a long period of time at temperatures below -120°C.

(6) Optimum growth conditions: Temperature: 37°C, pH: 7.2 (7) Growth range: Propagation is possible at temperature: 32-42°C, pH: 6.5-7.8.

In order to obtain the cell line of the present invention, a method such as the following may be employed.

That is, first, to collect the antigen CK-MM or MB, for example,
ca Chimica Acta) Volume 123, 59~
After separating CK MM and MB with DEAE Sephacel according to the method described on page 71 (1982), CK-MM
By performing chromatography on CM-cellulose, phenyl sepharose 5 blue sepharose,
Purified CKMM that can be used as an antigen can be obtained.

Further, after CK-MB is separated using DEAE Sephacel, purified CK-MB that can be used as an antigen can be obtained by chromatography using phenyl Sepharose.

Any CK-MM or MB can be used as an antigen, but in clinical chemistry, conventional antiserum is
For example, mammals that intersect with K.

Preferably, the animal is of human, monkey, pig, bovine, etc. origin and is different from the immunized animal. with these antigens.

The amount of antigen to be used, the site of administration, the usage cycle of adjuvant, which may be used to immunize a mammal, preferably a mouse or rat,
The immunization method may be similar to the conventional method for obtaining antiserum.

For example, when using a mouse.

o, o o i~10■ per mouse per time,
Preferably 0.01 to 1 CK-MM or MB.

For the first time, mix well with an adjuvant (e.g. Freund's complete adjuvant) and administer subcutaneously, intraperitoneally, etc.
After a week or more has elapsed, the adjuvant (for example, Freund's incomplete adjuvant) is mixed well and administered subcutaneously, intraperitoneally, etc. Furthermore, after more than two weeks, CK-M
Administering only M or MB intravenously, subcutaneously, intraperitoneally, etc.,
Be fully immunized. Animals thus immunized are sacrificed, preferably 2 to 4 days after the final immunization, and lymphocytes are harvested. For lymphocyte preparation, lung and lymph nodes.

Peripheral blood etc. are used. 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.

This is hypoxantine-guanine-phosphoribosyltransferase (Hypoxantine guanine-phosphoribosyltransferase).
inephosphoribosyl transfe
race) is missing.

It cannot grow on hypoxanthine-aminopterin-thymidine (1-IAT) 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, for example, commercially available mouse myeloma P3, X63, Ag8, 6, 5, 3 (X63, 6, 5,
3), P3・X63・Ag8・Ul (P2O3),
Lantmyeloma 210・RCY3・Agl・2・
It is preferable to use 3 or the like.

This myeloma is cultured in a medium such as Eagle's minimal medium (MEM), RPM1)640 medium (RPM1)640) containing serum, preferably fetal bovine serum.

Next, the lymphocytes and myeloma obtained above were each suspended in a medium such as MEM or RPM I 1640,
Mix. The mixing ratio at this time can be selected arbitrarily, but preferably the ratio of lymphocytes to myeloma is 1:1 to zo
:i, preferably a ratio of 5:1 to 10:1. The mixed cells are fused using a fusion promoter.

As for the fusion method.

For example, Immunological Methods Vol. 2, p. 285 (I
Mmmunological Methods Vol.
II, 1981. Aca-demic Pres.
s). As the fusion promoter, various polymeric substances, viruses, etc. can be used, but polyethylene glycol (PEG) and Sendai virus are preferably used. PEG has an average molecular weight of 400~
20,000 can be used, but preferably 1.0
00 to 7,500 may be used. The concentration used is preferably 40 to 60%.

The fused cells were washed to remove the fusion promoter.

5-15 vol, MEM or RPM1 containing % serum
) 640 medium and 0.5-5X in a 96-well culture dish etc.
Dispense at the rate of lOb/well. Furthermore, if a selective medium (for example, HAT medium) is added to each hole and the selective medium is replaced as appropriate, unfused myelomas will die after 10 to 14 days, and only hybridomas will grow. Incidentally, lymphocytes cannot grow in vitro for a long time and die after 10 to 14 days.

A method for searching for hybridomas producing antibodies can be carried out by knowing the inhibitory activity of the culture supernatant. For example, use the supernatant as a reagent system for measuring CK activity (
5 fixed amounts of CK-MM added to the first reagent of Reference Example 2)
Measure activity. It is sufficient to select a strain whose supernatant shows a lower activity than the control when no supernatant is added.

By the above method, a hybridoma cell line that produces a monoclonal antibody that inhibits anti-CKM activity can be created.

A type of hybridoma cell line was created by fusing mouse myeloma cells with mouse lung lymphocytes that had been previously immunized with CK-MM according to this method.

The hybridoma was named CKH-1. This stock.

On January 23, 1985, procedures for deposit were carried out with the Fermentation Research Institute, and the deposit was accepted as IFO-50088.

This CKH-1 can be frozen and stored almost permanently at -120°C or lower, and is always available for distribution.

This hybridoma CKH-1 can be grown in 9 commonly used media. For example, using RPM1640 or MEM containing 5 to 20% fetal bovine serum as a medium,
37°C1 carbon dioxide concentration 5vol.

It grows well in air containing %. It also has myeloma tumorigenic properties, so it can be used in vivo (for example).

syngeneic animals, nude mice, etc.), and anti-C-M
Activity-inhibiting monoclonal antibodies can be produced.

That is, by culturing this hybridoma cell line, a large amount of anti-CK-M activity-inhibiting monoclonal antibody can be collected. There are two main methods for collecting monoclonal antibodies. One is
This method uses a culture medium to culture in a culture container such as a flask, and collects antibodies from the supernatant. For example, 5~10ν
o1. in MEM or RPMr1640 medium containing % serum.

When 0.5 to 5 x 10' hybridoma cell lines are planted, they grow 10 to 20 times in 2 to 4 days, and antibodies are collected from the supernatant after culturing.

Another method is to inoculate the hybridoma cell line thus cultured in a culture vessel into a syngeneic animal. That is, hybridoma cell lines 105-107
Administer subcutaneously or intraperitoneally to syngeneic animals for 7 to 20 days.
This method collects serum and ascites fluid when the hybridoma cell line proliferates and the tumor grows. When administering intraperitoneally, administer 2.6.1 in advance (3 to 7 days).
When mineral oils such as 0°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 commonly applicable to proteins, such as pentasulfate fractionation, ion exchangers, and affinity chromatography with immobilized CK-M.

The physicochemical properties of the anti-CK-M activity-inhibiting monoclonal antibody thus obtained are shown.

(1) Action Acts on CK to generate an antigen-antibody reaction and inhibits CK-M subunit enzymatic activity.

(2) Antigen-antibody reaction specificity Reacts with CK subunit.

(3) Optimum pH 6-9 (4) Stable pH range 3-1) (5) Method for measuring titer Measure by dilution rate that can be detected by immunoassay.

(6) Range of suitable temperature for action 20~40°C (7) Conditions for inactivation It is deactivated by heating at 100°C for 10 minutes.

(8) Molecular weight 140,000~180,000 (Example) Next, one embodiment of the present invention will be specifically explained using examples.

Reference example 1 Clinica Kimi Chim Acta (C1inica Chim
icaActa) Volume 123, pp. 59-71 (198
2) According to the method described, pig hearts were first minced, homogenized, centrifuged at 30,000×g, and 70%
Crude enzyme was obtained by ethanol precipitation.

Next, the crude enzyme was dissolved and dialyzed, and then separated into CK-M which passes through and CK-MB which adsorbs using a DEAE-Sephacel column (pH 7,5).The CK-MB fraction is separated by increasing the salt concentration. It eluted. Both aliquots were dialyzed against Tris buffer containing 20% ammonium sulfate, adsorbed onto a phenyl Sepharose column, and eluted by lowering the ammonium sulfate concentration. Through the above operations, purified antigens CK-MM and MB were obtained.

Example 1 8 week old mouse Ba1b/c (obtained from Nippon Talea)
Then, 50 μg of pig CK-MM obtained in Reference Example 1 was mixed with complete Freund's adjuvant (obtained from Gyudon Kagaku) 1:1.
The mixture was mixed and emulsified and administered intraperitoneally. Three weeks later, 50 μg of porcine CK-MM was intravenously injected for boosting. Three days later, the lungs were taken out and suspended in 2MEM medium (manually produced by Gyudon Kagaku). .

Washed. On the other hand, mouse myeloma X63.6.5.
3 (obtained from Kyoto University) was cultured for 2 days prior, and cells in the logarithmic growth phase were collected by centrifugation.

Myeloma X63, 6, 5, 310 for 10' lung cells
7 and pelleted by centrifugation, then 50% PEG4000-RPM1)640 (
Obtained from Gipco. ) 1 m2 was gradually added over 1 minute, and after stirring gently for 1 minute.

9mj! RPM1) Gradually add 640 medium.

PEG4000 was diluted. Remove the PEG solution by centrifugation and pellet the HA containing 109/6 live fetal serum.
10 ml of T medium was added and 0.1 ml was dispensed into each hole of a 96-well culture dish (obtained from Nunc). 4,8
．． 1) Half of the culture solution was discarded three times a day, and fresh HAT medium was added.

After 14 days, hybridoma growth was observed in 56 of the 96 wells, and the supernatant was treated with a reagent system for measuring CK activity (
Added to the first reagent of Reference Example 2).

A fixed amount of CK-MM activity was measured. The strain in the well of the supernatant that showed a lower activity than the control when no supernatant was added was selected and cloned using the limiting dilution method to obtain the monoclonal hybridoma CKH-1. . This CKH
-1 was completely defeated by the cytological properties described above.

Example 2 8-week-old mice Ba1b/c (manufactured by Claire Japan).

), 50 μg of pig CK-MB obtained in Reference Example 1 was mixed with complete Freund's adjuvant (manual from Gyudon Kagaku) and 1:
Mix emulsified with 1 and administer intraperitoneally, 83 weeks later, 50μg
Booster immunization was carried out by intravenously injecting pig CK-MB, and 3 days later, the lungs were taken out and suspended in 2MEM medium (obtained from Gyudon Kagaku).

Washed. Thereafter, hybridomas were produced and screened in the same manner as in Example 1, and further cloning was performed to obtain monoclonal hybridoma CKH-2. This CKH-2 closely matched the cytological properties described above.

Reference example 2 Hybridoma CKH-1 obtained in Example 1.

300 m of culture cultured in RPM1) 640 medium containing 10% tallow serum to reach a m cell concentration of 2 x 10' cells/ml.
The supernatant was collected by centrifugation, and the crude antibody fraction was separated by 50% saturated ammonium sulfate fractionation, followed by dialysis.

Adsorbed on protein A Sepharose (pH 8,0),
The anti-CK-M activity-inhibiting monoclonal antibody 3.2■ was obtained by elution with a citrate buffer at pH 4.0.

This antibody was called CKA-1, and was added to the reagent system for measuring CK activity shown below to measure the degree of inhibition of CKMM activity.

First, Glc derived from Bacillus stearothermophilus
Commercially available from KC Seikagaku Kogyo. ) 1.4 units/mi! ,
o6PDH from Iconostoc mesenteroides
(Purchased from Oriental Yeast Industry Co., Ltd.) 1.2 units/ml, AD Pl, 2 mM, NADP
o, 75mM, glucose 25mM, AMP6.25m
A first reagent consisting of M, Ap5'A 12.5 μM, N-acetylcysteine 12.5 mM, magnesium acetate 12.5 mM, sodium azide 10 mM, E DTA 2.5 mM, and imidazole-acetic acid buffer (pH 6,7) 150 mM. Prepared first.

A second reagent was prepared consisting of CPloomM, 10 mM sodium azide, and 25 mM Tris-acetate buffer (pH 8.5).

Add the anti-CK-M activity-inhibiting monoclonal antibody CKA-10.5 to the above first reagent, add CK-MM, MB or BB to 0.5 ml of the first reagent, place in a cell with an optical path length of 1Ω, Incubate for 3 min then reagent 0
．． The remaining CK activity was measured from the change in absorbance at 340 nm using a spectrophotometer in which 125 mff was added and the cell chamber was kept at a constant temperature of 30°C. As a control, add the same amount of CK-MM, MB or BB to the first reagent without antibody,
The following measurements were made in the same manner.

The results are shown in Table 1.

Table I Inhibitory activity of CKA-1 As shown in Table 1, CKA-1 inhibits the CKM subunit well, but hardly inhibits the B subunit, and is an antibody that has been defeated by the above-mentioned physical and chemical properties. there were.

Reference Example 3 The hybridoma cell line CKH-2 obtained in Example 2 was cultured in the same manner as in Reference Example 2, and from 500 ml of the supernatant, anti-CK
-M activity-inhibiting monoclonal antibody 7.8■ was obtained. This antibody was called CKA-2 and added to the reagent system for measuring CK activity in the same manner as in Reference Example 2.

The inhibition rate for each CK activity was measured.

The results are shown in Table 2.

Table 2 Inhibitory activity of CKA-2 As shown in Table 2, CKA-2 inhibits the GK-M subunit well, but hardly inhibits the B subunit, and is an antibody that closely matches the physicochemical properties described above. Met.

(Effects of the Invention) Since the hybridoma cell line of the present invention has the ability to produce a monoclonal antibody that inhibits anti-CK-M activity, this antibody can be obtained in large quantities easily and at low cost. Therefore, using this antibody, CK-MB isozyme can be measured in feces with high sensitivity by immunoinhibition method.

Claims (1)

[Claims] (1) A hybridoma cell line that is obtained by fusion with lymphocytes prepared from an animal immunized with creatine kinase and has the ability to produce a monoclonal antibody that reacts with creatine kinase and inhibits creatine kinase M subunit activity.