JPH052310B2 - - Google Patents

Description

[Detailed description of the invention]

a Industrial Application Field The present invention relates to human α ₂ -plasmin inhibitor (α ₂
-plasmin inhibitor; α ₂ -antiplasmin), in particular recognizes the fibrinolytic action inhibiting site (reactive site) of human α ₂ -plasmin inhibitor as an antigen, and as a result, the fibrinolytic activity of plasmin of human α ₂ -plasmin inhibitor This invention relates to hybridoma cells that produce monoclonal antibodies that inhibit lytic activity and promote fibrinolysis. b. Prior Art Human α ₂ -plasmin inhibitor was first isolated and purified by Aoki and Moroi, and is a strong plasmin inhibitor that instantly inhibits the esterase activity of the fibrinolytic enzyme plasmin. , is known to be a single-chain glycoprotein with a molecular weight of approximately 67,000 containing 11.7% sugar [Mori &aoki; The Journal of Biological
Chemistry, 251 , 5956-5965 (1976)]. On the other hand, it is known that human α ₂ -plasmin inhibitor has three types of active sites. The first is the site that inhibits the fibrinolytic action of plasmin (hereinafter this may be referred to as the “reactive side”).
[B. Wiman & D. Collen; The Journal of
Biological Chemistry, 254 , 9291−9297 (1979)
The second is the plasmin-binding site on the carboxyl terminal side [B. Wiman & D. Collen;
European Journal of Biohemistry, 84 , 573−
578 (1978)], and the third is the fibrin binding site at the amino terminal [Y. Sakata, et al.,
Thro-mbosis Research, 16 , 279-282 (1979)
reference]. If it is possible to provide a monoclonal antibody that selectively recognizes the reactive site as an antigen among these three types of active sites in human α ₂ -plasmin inhibitor, it will be possible to use this monoclonal antibody to detect fibrin in human α ₂ -plasmin inhibitor. Since it can directly suppress the dissolution inhibitory effect and promote fibrinolysis, it can be used as a thrombolytic promoter. Furthermore, by using this antibody, it is also possible to measure α ₂ -plasmin inhibitor or α ₂ -plasmin inhibitor/plasmin complex in a solution. c. Structure of the Invention According to the present invention, there is provided a hybridoma cell obtained by fusing spleen cells obtained from a mouse immunized with a human α ₂ -plasmin inhibitor and a mouse myeloma cell, and which is obtained from the hybridoma cell. The monoclonal antibody produced is a monoclonal antibody against human α ₂ -plasmin inhibitor, which recognizes a site that inhibits the fibrinolytic action of plasmin in human α ₂ -plasmin inhibitor, and recognizes both a plasmin binding site and a fibrin binding site. A hybridoma cell characterized by being a monoclonal antibody that suppresses the fibrinolysis-inhibiting effect of human α ₂ -plasmin inhibitor, which does not recognize human α 2 -plasmin inhibitor, is provided. The hybridoma cells of the present invention can be prepared by the method of Koehler and Milstein [Ko¨hler and Milstein,
Nature 256 , 495-497 (1975)]. That is, after immunizing a mouse with human α ₂ -plasmin inhibitor, the spleen cells of this mouse are fused with mouse myeloma cells, and the resulting hybridoma cells are immobilized on human α ₂ − plasmin inhibitors in microtiter plates. Systematically tested and selected for antibodies that react with plasmin inhibitors. In this way, hybridoma cells that synthesize and secrete antibodies against human α ₂ -plasmin inhibitor are selected. The obtained hybridoma cells were cultured in a serum _- free medium, and the antibody against human α ₂ -plasmin inhibitor secreted into the culture supernatant was detected on fibrin plates.
The activity of suppressing the fibrinolytic inhibitory effect of plasmin inhibitors will be tested. As a result, hybridoma cells producing monoclonal antibodies having the activity of specifically suppressing the fibrinolysis-inhibiting effect of human α ₂ -plasmin inhibitor were isolated. Next, a specific method for producing the hybridoma cells of the present invention will be explained in detail. A. Isolation and purification of antigen; Human α ₂ -plasmin inhibitor used as an antigen was isolated and purified from human plasma by the method of Aoki and Moroi. B. Immunization of mice with human α ₂ -plasmin inhibitor; male Balb/c mice are used, but mice of other strains can also be used.
The immunization regimen and the concentration of human α ₂ -plasmin inhibitor should then be chosen so that a sufficient number of antigen-stimulated lymphocytes are formed. For example, mice were immunized with a small amount of α ₂ -plasmin inhibitor several times intraperitoneally at certain intervals, and then intravenously administered several times. Remove splenocytes for fusion several days after the final immunization. C Cell fusion; The spleen is removed aseptically and a single cell suspension is prepared from it. The spleen cells are fused with mouse myeloma cells from an appropriate line by use of an appropriate fusion promoter. A preferred ratio of spleen cells to myeloma cells is about 20:1 to about 2:1.
is within the range of 0.5 for approximately 10 ⁸ splenocytes
The use of ~1.5 ml of fusion medium is appropriate. Many myeloma cells used for cell fusion are known, but in the present invention, P3-X63-Ag8-U1 cells (hereinafter abbreviated as P3-U1) [Yelton, DEet
al., CurrentTopics in Microbiology and
Immu-nology, 81 , 1 (1978)] was used. This is an 8-azaguanine resistant cell line that is resistant to the enzyme hypoxanthine-guanine phosphoribosyltransferase (hypoxanthine-guanine phosphoribosyltransferase).
transferase) is deleted and therefore HAT
(hypoxanthine, aminopterin, thymidine)
It does not survive in the culture medium. Moreover, this cell line itself does not secrete antibodies, and is a so-called non-secreting type. A preferred fusion promoter is, for example, polyethylene glycol having an average molecular weight of 1000 to 4000, although other fusion promoters known in the art may also be used. In the examples of the present invention, polyethylene glycol with an average molecular weight of 1540 was used. D. Selection of fused cells; in a separate container (e.g., a microtiter plate), a mixture of unfused spleen cells, unfused myeloma cells, and fused cells is grown in a selective medium that does not support unfused myeloma cells. Dilute and culture for sufficient time (about 1 week) to kill unfused cells. The medium should be drug resistant (e.g. 8-
A medium (such as the HAT medium described above) that is resistant to azaguanine and does not support unfused myeloma cells is used. Unfused myeloma cells die in this selective medium. These unfused spleen cells are non-neoplastic cells, so after a certain period of time (about 1 week)
die out. Cells fused to these cells can survive in selective media because they have both the neoplastic properties of myeloma parent cells and the properties of parent spleen cells. E. Confirmation of antibodies against human α ₂ -plasmin inhibitor in each container; after hybridoma cells are thus detected, their culture supernatants are collected and tested for antibodies against human α ₂ -plasmin inhibitor by enzyme linked immunoassay (Enzyme Linked Immunoassay). Sorbent
Assay). F. Selection of hybridoma cells producing antibodies with activity against human α ₂ -plasmin inhibitor; hybridoma cells producing antibodies against human α ₂ -plasmin inhibitor were cultured in a serum-free medium. The containing culture supernatant was concentrated and incubated with human α ₂ -plasmin inhibitor for a certain period of time. Further, plasmin was added to this human α ₂ -plasmin inhibitor mixture, and the mixture was placed on a fibrin plate to measure the area of fibrin dissolution. In this way, hybridoma cells producing antibodies with activity against human α ₂ -plasmin inhibitor are selected. Specifically, FERM BP-1781 and FERM BP-
1782 hybridoma cells are listed. The present invention will be described in detail below with reference to Examples. Example 1 (1) Preparation of human α ₂ -plasmin inhibitor Human α ₂ -plasmin inhibitor was prepared from 2360 ml of human plasma according to the method of Aoki and Moroi described above.
Obtained 7.7mg. (2) Immunization of mice Male Balb/c mice were immunized with 100 μg of human α ₂ -plasmin inhibitor and Complete Freund's adjuvant.
The patient was immunized intraperitoneally twice with an interval of 21 days using an emulsion of . Further, 7 days later and 88 days later, 30 μg of human α ₂ -plasmin inhibitor was additionally administered intravenously together with physiological saline. Four days after the final immunization, the spleen cells were used for cell fusion. (3) Preparation of spleen cell suspension The spleen was aseptically removed and passed through a stainless steel mesh to obtain a single cell suspension. Cells were treated with L-glutamine 0.39 g/, kanamycin sulfate 0.2 g/and NaHCO ₃ 2.0
RPMI-1640 medium (GIBCO
(manufactured by). Proliferated cells were washed three times with RPMI-1640 and resuspended in RPMI-1640 medium. (4) Preparation of myeloma cells Mouse myeloma culture P3-U1 contains L-glutamine 0.39g/, kanamycin sulfate 0.2g/,
RPMI- ₁₆₄₀ medium (10% FCS-
The cells were cultured in RPMI-1640 (abbreviated as RPMI-1640). Myeloma cells were in the log phase of cell division at the time of cell fusion. (5) Cell fusion Suspend spleen cells and myeloma cells in serum-free RPMI-1640 medium at a ratio of 10:1, and
Centrifuged at 200g. After removing the supernatant medium, the sediment was incubated with 1 ml of a 50% polyethylene glycol solution (PH 8.2) with an average molecular weight of 1540 for 2 minutes.
Incubation was performed at 37°C. then serum free
9 ml of RPMI-1640 medium was added and the cells were carefully resuspended for 5 minutes. The suspension was then centrifuged at approximately 200 g for 5 min, followed by 8 x 10 ⁶ cells/ml.
10% FCS−RPMI− to obtain a concentration of
1640 medium and then distributed onto 96 microwell plates (approx.
100μ). This fused cell is 5% at 37°C.
Cultured using _CO2 . (6) Selection and culture of hybridoma cells producing antibodies against human α ₂ -plasmin inhibitor One day after cell fusion, 100μ of HAT medium was added to each well. Thereafter, half of the medium was replaced with a new HAT medium at 2-day intervals and cultured. Eight days later, the culture supernatant of the hybridoma cells was screened for antibodies against human α ₂ -plasmin inhibitor by enzyme immunoassay. The antigen used in the screening was human α ₂ -plasmin inhibitor,
The antibody was an alkaliphosphatase-labeled rabbit anti-mouse antibody. All of the 349 wells in total were positive by enzyme immunoassay, indicating that they produced antibodies against α ₂ -plasmin inhibitor. When active cell proliferation was observed, HT medium was added. HT a total of 4 times at 1-day intervals
The medium was exchanged using the same medium, and then culture was performed using a normal 10% FCS-RPMI-1640 medium. Example 2 (Selection of hybridoma cells producing antibodies against human α ₂ -plasmin inhibitor) Fibrinolytic inhibitory activity of human α ₂ -plasmin inhibitor from among the hybridoma cells capable of producing antibodies against human α ₂ -plasmin inhibitor. The following method was used to screen hybridoma cells that produce antibodies that have the effect of suppressing cancer. 10% FCS-RPMI for hybridomas in each well
-1640 times cultured in the ground, and the number of cells was approximately 2×10 ⁷ . The cells were centrifuged at approximately 200g for 5 minutes,
After removing the culture supernatant, cells were placed in serum-free RPMI−
Washed with 10 ml of 1640 medium. Approximately 200g for another 5 minutes
Centrifuge the cells at
ml/, transferrin 5.0ml/, ethanolamine 5.0ml/, sodium selenite 5.0
ml/, L-glutamine 0.39g/, kanamycin sulfate 0.2g/, Hepes 2.38g/, and
PRMI-1640 supplemented with NaHCO ₃ 1.5g/:
Dulbecco's MEM: Ham's F-12 (2:1:1)
Mixed serum-free medium (hereinafter referred to as “MITES medium”)
) was suspended in 10 ml and cultured for 3 days. The culture supernatant was collected and concentrated 25 times.
0.4 μg of human α ₂ -plasmin inhibitor was added to 25 μg of this concentrated solution and incubated at 37° C. for 30 minutes. Next, add 0.025 units of plasminogen and 0.031 units of urokinase and adjust the volume.
It was set to 40μ. 10μ of this was placed on a fibrin plate. The fibrin plate was left to stand for 18 hours at 37°C and a humidity of 95% or higher, and the dissolved area was measured. As a result, it was found that the effect of completely suppressing the fibrinolytic inhibitory activity of human α ₂ -plasmin inhibitor added to the antibody produced by 1D10 hybridoma cells (FERM International Deposit No. FERM BP-1781) was found. Reference Example 1 Cloning of Hybridoma Cells A hybridoma cell (1D10) that showed a positive result in an antibody activity test against human α ₂ -plasmin inhibitor was cloned by the following method. Dilute 1D10 cells to 0.9 cells per well of a 96-well microtiter plate.
Add Balb/c mouse thymocytes as feeder cells and distribute into plates using 10% FCS-RPMI-1640.
Cultured in medium. Observation under a microscope confirmed that it was definitely a single cell colony. Antibodies against human α ₂ -plasmin inhibitor in the culture supernatant of hybridoma cells were screened by enzyme immunoassay. A total of 26 wells were positive by enzyme immunoassay and produced monoclonal antibodies against human α ₂ -plasmin inhibitor. Purification of monoclonal antibodies: In order to produce large quantities of monoclonal antibodies against human α ₂ -plasmin inhibitor, approximately
10 ⁷ hybridoma cells were injected intraperitoneally into pristane-pretreated Balb/c mice. Approximately 1
Using the ascitic fluid collected after a week, the method of Ey et al.
Ey, SJProwse and CRJenkin,
Immunochemistry, 15 , 429-436 (1978)]
According to Protein A-Sepharose 4B (protein
The antibody was purified using a Sepharose 4B) column. 20 mg of a monoclonal antibody against human α ₂ -plasmin inhibitor was obtained from 2.5 ml of ascites fluid. Characteristics of the purified monoclonal antibodies; The specific class of the purified monoclonal antibodies was determined by Ochterlony gel diffusion test using class-specific anti-mouse antisera. The results are shown in Table 1 below. Most of the antibodies against human α ₂ -plasmin inhibitor were H chain r ₁ and L chain K.

[Table] Here, antibody 1B10G11 is designated as FIKEN International Deposit No.
Regenerated by FERM BP-1782 hybridoma cells. Reference Example 2 Suppression of human α ₂ -plasmin inhibitor activity by antibody against human α ₂ -plasmin inhibitor 1 μg of human α ₂ -plasmin inhibitor and 5 μg of each monoclonal antibody were added to 50 μg of 0.05M phosphate buffered saline (hereinafter abbreviated as PBS). Dissolve at 37℃
Incubation was carried out for 30 minutes. Next, plasminogen 0.025 units and urokinase 0.031 units.
Unit was added to make the liquid volume 60μ. this house
10μ was placed on a fibrin plate. The fibrin plate was allowed to stand for 18 hours at 37°C and a humidity of 95% or higher, and the dissolved area was measured. The results are shown in Table 2 below. Note that the values in the table below are relative values when the area dissolved by 0.025 units of plasminogen and 0.031 units of urokinase is taken as 100%.

[Table] Reference Example 3 Effect of monoclonal antibody against human α ₂ -plasmin inhibitor on binding between human α ₂ -plasmin inhibitor and fibrin I ¹²⁵ -labeled human α ₂ -plasmin inhibitor
0.01 µM and 0.05 µM monoclonal antibody against _α2 -plasmin inhibitor in 2% bovine serum albumin - 0.05 M Tris buffer (PH7.4) - 0.15 M
After adding NaCl and incubating at 37°C for 30 minutes, the mixture was left at 4°C overnight. To this antigen-antibody reaction mixture, 2.5mM CaCl ₂ , 7μM fibrinogen fraction, and 2 units/ml thrombin were added.
Incubation was carried out at 37°C for 30 minutes at 100μ. Formation of a coagulum (fibrin clot) was observed. After 30 minutes, 100μ of 200mM EDTA was added to remove calcium ions, and the coagulated product was wound up with a bamboo skewer. The coagulated material is wound on a bamboo skewer for 5 minutes.
Washing solution 3 times [2% BSA, 0.05M Tris buffer (PH
7.4), 0.15M NaCl, 2mM EDTA]. Finally, the coagulated material was collected from the bamboo skewer into a test tube, and the radioactivity (cpm) of the coagulated material was measured. Table 3 shows the ratio of radioactivity of the coagulum to radioactivity in the original reaction mixture. Table 3 also shows the results of using a common commercially available mouse IgG as a comparative antibody.

[Table] This result shows that the monoclonal antibodies against each human α ₂ -plasmin inhibitor are monoclonal antibodies that do not recognize the fibrin binding site of human α ₂ -plasmin inhibitor. Reference Example 4 (Search for a monoclonal antibody that recognizes the reactive site of human α ₂ -plasmin inhibitor) In this example, a monoclonal antibody against human α ₂ -plasmin inhibitor that recognizes the reactive site of human α ₂ -plasmin inhibitor was used. This study investigated the effects of α ₂ -plasmin inhibitor 0.15 μM and monoclonal antibody against α ₂ -plasmin inhibitor 0.75 μM were added to 2% bovine serum albumin solution [0.05 M
Tris buffer (PH7.4), 0.15M NaCl] at 37°C;
Incubation was carried out for 30 minutes and the mixture was left at 4°C overnight. Mix 60μ of this reaction mixture with 20μ of plasmin solution (0.47μM), add 0.05M Tris buffer (PH7.4) and 0.15M NaCl to make a total volume of 500μ, and prepare two bottles for each sample.
Incubation was performed at 37°C for 2 or 20 minutes.
Next, 3.5mM synthetic substrate S-2251 (HD-valyl-
Add 200μ of L-leucyl-L-lysyl-P-nitroanilide dihydrochloride) and measure the amount per unit time using a spectrophotometer (Beckman.DU-8).
Changes in absorbance at a wavelength of 405 nm were measured.
As a control, changes in absorbance were similarly examined for a sample in which only plasmin was reacted and a sample in which human α ₂ -plasmin inhibitor and plasmin were reacted without adding a monoclonal antibody. The results are shown in Table 4 below.

[Table] From the results of Reference Examples 3 and 4 above, the monoclonal antibody obtained from the hybridoma cells of the present invention specifically recognizes the reactive side of human α ₂ -plasmin inhibitor, and the monoclonal antibody obtained from the hybridoma cells of the present invention specifically recognizes the reactive side of human α 2 -plasmin inhibitor. It turns out that I don't recognize any of them.

Claims (1)

[Claims] 1 Hybridoma cells obtained by fusing spleen cells obtained from mice immunized with human α ₂ -plasmin inhibitor and mouse myeloma cells, and monoclonal antibodies produced from the hybridoma cells are human α 2 -plasmin inhibitors. ₂
- A monoclonal antibody against a plasmin inhibitor, which recognizes the site that inhibits the fibrinolytic action of plasmin in human _{α 2} -plasmin inhibitor and does not recognize either the plasmin binding site or the fibrin binding site. A hybridoma cell characterized by being a monoclonal antibody that suppresses the fibrinolysis-inhibiting effect of.