JPH0535827B2 - - Google Patents

Description

[Detailed description of the invention]

a Industrial Application Field The present invention provides human plasmin-α ₂ in a solution state.
- Plasmin inhibitor complex (plasmin-α ₂
−plasmin inhibitor complex, plasmin−α ₂ −
This invention relates to a method for immunologically measuring antiplasmin complex. More specifically, a human plasmin-α ₂ -plasmin inhibitor complex was prepared by the Sand-Deutsch method using a monoclonal antibody that specifically recognizes and binds to human α ₂ -plasmin inhibitor and an antibody that can recognize and bind to plasmin. Regarding immunological measurement methods. b. Prior Art Human α ₂ -plasmin inhibitor was first isolated and purified by Aoki and Moroi and is a potent plasmin inhibitor that instantly inhibits the esterase activity of fibrinolytic oxygen plasmin. , is known to be a single-chain glycoprotein with a molecular weight of approximately 67,000 containing 11.7% sugar [Moroi &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 a site that blocks the fibrinolytic action of plasmin (hereinafter referred to as the "reactive side") [B. Wiman & D. Collen; The Journal
of Biological Chemistry, 254 , 9291–9297
(1979)], and the second is the plasmin binding site at the carboxyl group terminal side [B. Wiman & 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., Thrombosis Research, 16
279-282 (1979)]. Human α ₂ -plasmin inhibitor inhibits plasmin activity almost instantaneously, and α ₂ -plasmin inhibitor binds to plasmin at a ratio of 1:1 to form a complex. (B. Wiman & D. Collen; The Journal of
Biological Chemistry, 254 , 9291–9297
(1979), D. Collen; Thrombosis and
Haemostasis, 43, 77-89 (1980)) In conditions where plasminogen is activated, such as in disseminated intravascular coagulation (DIC) or thrombolytic therapy using urokinase, the generated plasmin and α ₂ −
Plasmin inhibitors react and form complexes. Recently, quantifying the plasmin-α ₂ -plasmin inhibitor complex in plasma is considered to be effective for monitoring thrombolytic therapy and diagnosing DIC (e.g. NABooth & B. Bennett: British
Journal of Haematology, 50 , 537–541 (1982)
reference). Therefore, if the amount of plasmin-α ₂ -plasmin inhibitor complex in blood could be measured accurately and easily, it would be extremely useful for the prevention and diagnosis of various diseases. As conventionally known methods for measuring the plasmin-α ₂ -plasmin inhibitor complex, the first method uses two-dimensional cross-immunoelectrophoresis, and the second method involves immobilizing antibodies obtained from antiserum. This is based on the so-called Sandermansch method, which is an application of the oxygen antibody method. However, the former method had the drawback of low sensitivity and quantitative performance. Furthermore, although the latter method is extremely sensitive and effective, it has the drawback that it is extremely difficult to stably obtain an antiserum having a certain level of activity against human α ₂ -plasmin inhibitor. c. Structure of the Invention The present inventors have conducted repeated research on monoclonal antibodies against human α ₂ -plasmin inhibitor, and have found that they have been found to specifically target a site other than the site that blocks the fibrinolytic action of plasmin in human α ₂ -plasmin inhibitor. We have discovered a monoclonal antibody that recognizes this monoclonal antibody, and have created hybridoma cells that produce this monoclonal antibody, which we have already proposed. The present inventors have found that by utilizing the newly discovered specific action of the monoclonal antibody, it is possible to directly and accurately measure the amount of human plasmin-α ₂ -plasmin inhibitor complex in solution. Heading The present invention has been arrived at. That is, the present invention provides an immunoassay method using the Sanderutsch method, in which either an antibody bound to an insoluble carrier or a labeled antibody is located at a site other than the site that inhibits the fibrinolytic action of plasmin in human α ₂ -plasmin inhibitor. A human plasma sample, which is a monoclonal antibody that specifically recognizes a location, and the other is an antibody against human plasmin, characterized in that the labeled antibody and the human plasma sample are simultaneously brought into contact with the antibody bound to an insoluble carrier. This is a method for measuring the human plasmin-α ₂ -plasmin inhibitor complex in. In general, a method for measuring the presence or absence of an antigen or its amount by making antibodies that bind to two different positions on an antigen is called the Sand-Germany method.
199-206 (1970). Thus, according to the present invention, it is possible to consistently and accurately measure the human plasmin-α ₂ -plasmin inhibitor complex in a solution state (for example, in plasma) without any quality differences in reagents. Furthermore, since the human plasmin-α ₂ -plasmin inhibitor complex is directly measured, it is not affected by other impurities and can be measured accurately and in a short time. Next, a method for measuring the content of human plasmin-α ₂ -plasmin inhibitor complex according to the present invention will be specifically explained. An antibody against human plasmin (first antibody) is immobilized on a suitable insoluble carrier (for example, a plastic container) (hereinafter referred to as "immobilized antibody").
Next, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or specimen sample to be measured. In this case, the antibody against human plasmin may be an antibody against human plasminogen. Next, a monoclonal antibody (second antibody) against human α ₂ -plasmin inhibitor labeled with a specimen sample (e.g., human plasma) and an appropriate labeling substance is used.
are added at the same time, and brought into contact with the insoluble carrier on which the first antibody is immobilized for a certain period of time and at a certain temperature to cause a reaction (one-step method). During this time, the immobilized antibody (first
antibody), the human plasmin-α ₂ -plasmin inhibitor complex in the specimen sample, and the second antibody bind. Wash this with a suitable cleaning solution. Next, the amount of the labeling substance labeled with the second antibody present on the insoluble carrier is measured. Thus, from this value, the amount of human plasmin-α ₂ -plasmin inhibitor complex in the specimen sample can be calculated. Examples of insoluble carriers used in the measurement method of the present invention include polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked textlan, polysaccharide, other paper, glass, metal, and agarose. and combinations thereof. In addition, the shape of the insoluble carrier is, for example, tray-shaped, spherical, fibrous, rod-shaped, disc-shaped, container-shaped, cell,
It can be of various shapes, such as a test tube. It is also advantageous to use radioactive substances, enzymes or fluorescent substances as labeling substances. As radioactive substances, use ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H, etc.; as enzymes, use alkaline phosphatase, peroxidase, β-D-galactosidase, etc.; as a fluorescent substance, use fluorescein isothiocyanate, etc. However, these are not limited to those exemplified, and other types can be used as long as they can be used in immunological measurement methods. A patent application was previously filed for the monoclonal antibody of the present invention and the method for producing the same (Application filed on April 17, 1982: Title of the invention: "Monoclonal antibody and method for producing the monoclonal antibody", Patent application filed in 1982)
-75778 (Unexamined Japanese Patent Publication No. 60-222426)). 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. <<Monoclonal antibody and method for producing the same>> 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 human 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 used for cell fusion are known, but in the present invention, P3-X63Ag8-U1 cells (hereinafter abbreviated as P3-U1) [Yelton, DE ct
al.，Current Topics in Microbiology and
Immunology 81, 1 (1978)] was used.
This is a cell line resistant to 8-azaguanine, and the enzyme hypoxanthine-guanine phosphoribosyltransferase (hypoxanthine-guanine phosphoribosyltransferase)
guanine phosphoribosyl transferase) and therefore HAT (hypoxanthine,
(aminobuterin, thymidine) does not survive in culture medium. Furthermore, this cell line itself does not secrete antibodies, and is a so-called non-secreting type. As a preferred fusion promoter, for example polyethylene glycol having an average molecular weight of 1000 to 4000 can be advantageously used, but other fusion promoters known in the art can also be used. d Selection of fused cells; in a separate container (e.g., a microtiter plate), the 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. In addition, 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 α ₂ -plasmin inhibitor in each container; thus, after hybridoma cells are detected, the culture supernatant is collected and analyzed for antibodies against human α ₂ -plasmin inhibitor by enzyme linked immunoassay (Enzyme Linked Immunoassay). Sorbent
Assay). f. Cloning of hybridoma cells producing the antibody of interest; When hybridoma cells producing the antibody 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. In the measurement reagent of the present invention, such a monoclonal antibody is used as either the first antibody or the second antibody. That is, the monoclonal antibody can be bound to an insoluble carrier and used as an immobilized antibody, or can be attached with a labeling substance and used as a labeled antibody. Other antibodies used in conjunction with the monoclonal antibody may be any antibody that can recognize and bind to human plasmin. As described above, according to the present invention, human plasmin-α ₂ −
It is possible to accurately and easily measure the amount of plasmin inhibitor complex in a sample (eg, human plasma) containing the complex. The present invention will be described in detail below with reference to Examples. Example 1 The first and second antibodies used in this example were described in the specification previously filed by the present inventors (April 17, 1982).
Filed in Japan: Title of the invention: “Monoclonal antibody and method for producing monoclonal antibody”, Patent application No. 59-75778
The following materials obtained by the method described in JP-A-60-222426) were used. First Antibody An antibody component was purified from an antiserum against human plasminogen obtained by immunizing a rabbit with human plasminogen, and used by immobilizing it on an insoluble carrier (microtiter plate) as described below. Second antibody Antibody name “1B10G11” obtained in Example 3 of the application specification (FERM
BP-1782) was used and labeled with alkaline phosphatase. An antibody against human plasminogen at a concentration of 50 μg/ml was left on a microtiter plate at 4° C. overnight to immobilize it. Add to this phosphate buffered saline containing 0.5% bovine serum albumin (hereinafter referred to as 0.5% BSA).
-PBS) was added thereto, and the mixture was allowed to stand at room temperature for 2 hours, and then washed three times with 0.5% BSA-PBS. Next, a mixed solution of a human plasma sample diluted with phosphate buffered saline (PBS) and an alkaline phosphatase-labeled monoclonal antibody (IB10G11) at a concentration of 329 ng/ml was added, and the mixture was allowed to contact for 2 hours at room temperature to react.
After washing with 0.5% BSA-PBS, add alkaline phosphatase substrate solution at a concentration of 1.0 mg/ml and incubate for 30 min.
After reaction at room temperature for minutes, MICROPLATE
Absorbance at a wavelength of 405 nm was measured using a PHOTOMETER. The results are shown in Figure 1 of the attached drawing.
From this figure, it can be understood that the relationship between the concentration of the plasmin-α ₂ -plasmin inhibitor complex and the absorbance is a linear relationship. From this calibration curve, the amount of plasmin-α ₂ -plasmin inhibitor complex (μg/ml) in the patient's plasma sample was calculated and shown in Table 1.

【table】

【table】 [Brief explanation of the drawing]

The attached drawing shows the relationship between the concentration of human plasmin-α ₂ -plasmin inhibitor complex in a sample and the absorbance.

Claims (1)

[Scope of Claims] 1. In an immunoassay method using the Sand-Deutsch method, either the antibody bound to an insoluble carrier or the labeled antibody inhibits the fibrinolytic action of plasmin in human α ₂ -plasmin inhibitor. A monoclonal antibody that specifically recognizes a site other than human plasmin, the other being an antibody against human plasmin, characterized in that the labeled antibody and the human plasma sample are brought into contact with the antibody bound to an insoluble carrier at the same time. Method for measuring human plasmin-α ₂ -plasmin inhibitor complex in plasma specimen. 2. The measuring method according to claim 1, wherein the labeled antibody is an antibody labeled with oxygen, a radioactive isotope, or a fluorescent substance.