JPH0455200B2 - - Google Patents

Description

Detailed Description of the Invention: a. Field of Industrial Application The present invention relates to a selective adsorbent for human α ₂ -plasmin inhibitor and a method for separating human α ₂ -plasmin inhibitor from human plasma using the same. be. 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 and containing 11.7% sugar [Moroi and Aoki; The Journal of Biological
Chemistry, 251 , 5956-5965 (1976)]. In addition, its concentration in plasma is 6.13±0.88 in healthy people.
mg/100ml [N.Aoki
and T. Yamanaka, Clinica Chimica Acta
84, 99–105 (1978)]. On the other hand, human α ₂ -plasmin inhibitor has 3
It is known that there are different types of active sites. 1st
is a site that inhibits the fibrinolytic action of plasmin (hereinafter referred to as the “reactive site”)
[B. Wiman and 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 and D. Collen;
European Journal of Biochemistry, 84 , 573
-578 (1978)], and the third is the fibrin binding site at the amino group end [Y. Sakata, et al.,
See Thrombosis Research, 16 , 279-282 (1979)]. Recently, progress has been made in elucidating the physical and biological properties of human α ₂ -plasmin inhibitor, and it is known that human α ₂ -plasmin inhibitor has an important effect on the fibrinolytic mechanism [ For example, Nobuo Aoki, History of Medicine, 126, 147-155 (1983)
reference〕. In addition, when blood from a patient with α ₂ -plasmin inhibitor congenital deficiency is placed in a test tube and left at 37°C,
Coagulation occurs quickly and normally, but the coagulation completely dissolves spontaneously in 4 to 8 hours [N. Aoki, etal., Journal of Clinical
Investigation, 63, 877–884 (1979)]. In fact, in α ₂ -plasmin inhibitor deficiency, there is a very serious bleeding tendency due to early collapse of the hemostatic plug. On the contrary, this fact suggests that in severe thrombosis, reducing the amount of α ₂ -plasmin inhibitor in the blood can rapidly dissolve the causative thrombus. Conceivable. c. Structure of the _Invention The present inventors have conducted repeated research on monoclonal antibodies against human α ₂ -plasmin inhibitors, and have found that monoclonal antibodies against human α ₂ -plasmin inhibitors, We have discovered a monoclonal antibody that suppresses the fibrinolysis-inhibiting effect of human α ₂ -plasmin inhibitor, which recognizes the fibrinolysis-inhibiting site and does not recognize either the plasmin-binding site or the fibrin-binding site; We have already proposed a hybridoma cell that produces the
issue)). As a result of further research into the use of the specific action of the monoclonal antibody obtained in this way, the present inventors have developed an adsorbent that can specifically adsorb human α ₂ -plasmin inhibitor, and have used it to develop an adsorbent that can specifically adsorb human α 2 -plasmin inhibitor. The present invention was achieved by discovering that human α ₂ -plasmin inhibitor can be selectively separated. That is, the present invention 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 which inhibits both the plasmin binding site and the fibrin binding site. This is a selective adsorbent for human α ₂ -plasmin inhibitor, in which a monoclonal antibody that suppresses the fibrinolytic inhibitory effect of human α ₂ -plasmin inhibitor, which does not recognize it, is bound to an insoluble carrier as a ligand. This is a method for selectively separating human α ₂ -plasmin inhibitor from plasma. In general, chromatography that utilizes the biological affinity of adsorbents for the separation and purification of biological substances is called affinity chromatography [for example, "Experiment and Application" by Ichiro Chibata, Tetsuya Tosa, and Yuji Matsuo. Affinity Chromatography” (Kodansha Science Fix)]. In the present invention, the terms affinity, ligand, insoluble carrier, and adsorbent are each defined as follows. Affinity; Specific affinity ligand that exists between two substances; Substance that has affinity with the substance to be adsorbed or purified; Insoluble support; Support that is insoluble in water (this does not contain the ligand) Adsorbent; Ligand is immobilized on an insoluble carrier Next, the selective adsorbent for human α ₂ -plasmin inhibitor and the method for separating human α ₂ -plasmin inhibitor from human plasma according to the present invention will be described in detail. A monoclonal antibody against human α ₂ -plasmin inhibitor, which will be described later, is chemically coupled as a ligand to an appropriate insoluble carrier (e.g., Sepharose), and this is packed into a column using an appropriate buffer solution (e.g., 50mM Tris buffer PH7.4, 0.15M Equilibrate with NaCl). A specimen sample (human plasma) is added to this adsorbent to adsorb the human α ₂ -plasmin inhibitor in the specimen test. Then add a suitable wash solution (e.g. 50mM Tris buffer, PH7.4, 0.15M
Impurities are removed from the adsorbent by NaCl).
Next, the amount of human α ₂ -plasmin inhibitor in the “flow-through fraction” and “washing fraction” of plasma is measured. Thus, from that value human α ₂ from human plasma
- The degree of separation of plasmin inhibitors can be calculated. Various types of insoluble carriers can be used for the selective adsorbent in the present invention, and for example, cepharose, polyacrylamide, cellulose, dextran, maleic acid polymer, or mixtures thereof are preferably used. The shapes of these inert carriers include powder, granules, pellets, beads, films,
It can be in various forms such as fibrous. As mentioned above, the antibody used in the adsorbent of the present invention is a monoclonal antibody against human α ₂ -plasmin inhibitor, which recognizes the site that inhibits the fibrinolytic action of plasmin in human α ₂ -plasmin inhibitor. A monoclonal antibody that suppresses the fibrinolysis-inhibiting effect of human α ₂ -plasmin inhibitor that does not recognize either the plasmin binding site or the fibrin binding site.
was first discovered by the present inventors and a patent application was filed earlier (Application filed on April 17, 1982; title of the invention: "Monoclonal antibody, hybridoma cells, and method for producing monoclonal antibody" Patent application 1982-
No. 75778). The monoclonal antibody of the present invention and the method for producing the same are explained in detail in the patent application specification, and the contents thereof will be briefly explained below. 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 intraperitoneally several times at certain intervals with a small amount of α ₂ -plasmin inhibitor, and then intravenously administered several times. Spleen cells are removed 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 are known for use in cell fusion, but in the present invention P3-X63-Ag8-U1 cells (hereinafter abbreviated as P3-U1) [Yelton, DEetal., Current Topicsin]
Microbiology and Immunology, 81 , 1
(1978)] was used. This is an 8-azaguanine resistant cell line that is resistant to the enzyme hypoxanthineguanine phosphoribosyltransferase (hypoxanthineguanine phosporibosy).
transferase) is deleted and therefore
HAT (hypoxanthine, aluminopterin,
thymidine) does not survive in 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. D. Selection of fused cells; a mixture of unfused spleen cells, unfused myeloma cells, and fused cells in a separate container (e.g., a microtiter plate) using a selective medium that does not support unfused myeloma cells. and culture for a 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. G. Cloning of hybridoma cells producing antibodies of interest; When hybridoma cells producing antibodies of interest are cloned by an appropriate method (e.g. limited dilution method), antibodies are produced in two different ways. According to the first method, monoclonal antibodies produced by hybridoma cells can be obtained from the culture supernatant by culturing hybridoma cells in an appropriate medium for a certain period of time. According to a second method, hybridoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. Monoclonal antibodies produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time. The monoclonal antibody obtained as described above does not recognize either the plasmin-binding site or the fibrin-binding site in human α ₂ -plasmin inhibitor, but recognizes the site that inhibits the fibrinolytic action of plasmin. selectively binds to. In the adsorbent used for separating α ₂ -plasmin inhibitor in human plasma according to the present invention, such a monoclonal antibody is used as a ligand by chemically bonding it to an insoluble carrier. Generally speaking, such a binding method may be any method in which a monoclonal antibody is chemically bound to an insoluble carrier. As described above, according to the present invention, it is possible to easily and selectively separate α ₂ -plasmin inhibitor from human plasma. The present invention will be described in detail with reference to Examples. Example Separation of α ₂ -plasmin inhibitor from human plasma An adsorbent (0.5 ml) chemically bound to the monoclonal antibody 1D10C1 against α ₂ -plasmin inhibitor as a ligand was packed into a column, and a washing solution (50 mM Tris buffer pH 7.4) was packed into a column. , 0.15 M NaCl), and then 1.0 ml of human plasma was added. Furthermore, the inner wall of the column was washed with 1.0 ml of the above washing solution, and the eluted fraction was designated as the "pass-through fraction." Next, the unadsorbed substances were eluted with 2.0 ml of the above washing solution and designated as a "washing fraction." All the above operations were performed at 4°C. The separated “pass-through fraction” and “washing fraction” are immediately divided into 4
Desalting and concentration were performed at °C. Preparation of human α ₂ -plasmin inhibitor calibration curve The α ₂ -plasmin inhibitor amount was determined according to the specification previously filed by the present inventors (filed on May 1, 1980; title of the invention “Human α ₂ -plasmin inhibitor”). The measurement was carried out using an immunoassay test using a monoclonal antibody against the antibody and the Sand-Deutsch method described in Kitt's Japanese Patent Application No. 59-86101 (Japanese Unexamined Patent Publication No. 60-231168)). The first and second antibodies used in this example were described in the specification previously filed by the present inventors (filed on April 17, 1982; title of the invention: "Monoclonal antibodies, hybridoma cells, and method for producing monoclonal antibodies. The following product obtained by the method described in Japanese Patent Application No. 75778/1983 was used. First antibody The antibody named "1D10C1" obtained in Example 3 of the above application specification (Japanese Patent Application No. 59-75778) was used, and it was immobilized on an insoluble carrier (microtiter plate) as shown below. Using. This antibody is a monoclonal antibody that does not recognize either the plasmin binding site or the fibrin binding site in human α ₂ -plasmin inhibitor, but also recognizes the site that inhibits the fibrinolytic action of plasmin and binds selectively to that site. be. Second Antibody The antibody name "1B10G11" obtained in Example 3 of the application specification (Japanese Patent Application No. 59-75778) was used. This antibody is a monoclonal antibody that specifically recognizes a site other than the reactive site in α ₂ -plasmin inhibitor, and was used after being labeled with alkaline phosphatase. A monoclonal antibody (1D10C1) that specifically recognizes the reactive site in human α ₂ -plasmin inhibitor at a concentration of 20 μg/ml was immobilized on a microtiter plate at 4° C. overnight. Add to this a buffer containing 1% bovine serum albumin (15mM Na ₂ CO ₃ , 35mM NaHCO ₃ , 3mM
After adding NaN ₃ ) and leaving it at room temperature for 4 hours, 1%
Washing solution containing bovine serum albumin (20mM phosphate buffer, 0.135M NaCl, 2mM _NaN3 , 0.05%
Tween 20) was washed 5 times. Next, α ₂ -plasmin inhibitor diluted to various concentrations with a dilution solution (20mM phosphate buffer PH7.4, 0.135M NaCl) was added and left at room temperature for 4 hours. Thereafter, the monoclonal antibody (1B10G11) that recognizes a site other than the reactive site in α ₂ -plasmin inhibitor was washed 5 times with the washing solution and further labeled with alkaline phosphatase.
was added and left overnight at 4°C. After washing with the above washing solution, add 1 mg/ml of alkaline phosphatase substrate solution.
Add at a concentration of MICROPLATE
PHOTOMETER (MTP-20, manufactured by Corona Electric Co., Ltd.)
After 20 minutes, the absorbance at a wavelength of 405 nm was measured. The results are shown in the attached drawings. From this figure, it can be understood that the relationship between the concentration of α ₂ -plasmin inhibitor and the absorbance is a linear relationship. Therefore, by using a monoclonal antibody that specifically recognizes the reactive site in α ₂ -plasmin inhibitor as one antibody in the Sand-Deutsch method, the amount of α ₂ -plasmin inhibitor can be easily measured. Using the attached drawing as a calibration curve, the amount of α ₂ -plasmin inhibitor in human plasma “flow-through fraction” and “washing fraction” was measured. Measurement of the amount of α ₂ -plasmin inhibitor in the sample A monoclonal antibody (1D10C1) that specifically recognizes the reactive site in human α ₂ -plasmin inhibitor was left at a concentration of 20 μg/ml on a microtiter plate at 4°C overnight. Fixed. Add to this a buffer containing 1% bovine serum albumin (15mM Na ₂ CO ₃ , 35mM NaHCO ₃ , 3mM
After adding NaN ₃ ) and leaving it at room temperature for 4 hours, 1%
Washing solution containing bovine serum albumin (20mM phosphate buffer, 0.135M NaCl, 2mM _NaN3 , 0.05%
Tween 20) was washed 5 times. Next, samples (the above-mentioned "pass-through fraction", "washing fraction" and human plasma) diluted to various concentrations with a dilution solution (20mM phosphate buffer, 0.135M NaCl) were added, and the samples were incubated at room temperature for 4 hours.
I left it for a while. Thereafter, the monoclonal antibody (1B10G11) that recognizes a site other than the reactive site in human α ₂ -plasmin inhibitor that was washed 5 times with the washing solution and labeled with alkaline phosphatase.
was added and left overnight at 4°C. After washing with the washing solution, add 1 mg of alkaline phosphatase substrate solution/
Add at a concentration of ml and MICROPLATE
PHOTOMETER (manufactured by Corona Electric Co., Ltd., MTP-12)
After 20 minutes, the absorbance at a wavelength of 405 nm was measured. The results are shown in the table below. α ₂ -plasmin inhibitor was not detected in the “pass-through fraction” and “washing fraction”, and α ₂ -plasmin inhibitor was completely separated from human plasma by adding human plasma to the adsorbent. This was confirmed. 【table】

[Brief explanation of the drawing]

The attached document shows the relationship between concentration and absorbance of human α ₂ -plasmin inhibitor.

Claims (1)

[Scope of Claims] 1. 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 which recognizes both the plasmin binding site and the fibrin binding site. A selective adsorbent for human α ₂ -plasmin inhibitor, in which a monoclonal antibody that suppresses the fibrinolysis-inhibiting effect of human α 2 -plasmin inhibitor, which does not even recognize α ₂ -plasmin inhibitor, is bound to an insoluble carrier. 2. The selective adsorbent according to claim 1, wherein the insoluble carrier is at least one selected from the group consisting of cepharose, polyacrylamide, cellulose, dextran, and maleic acid polymer.