JPS61286753A - Immunoassay using monoclonal antibody and kit therefor - Google Patents

Abstract

PURPOSE:To enable accurate immunoassay within a short time, by setting one kind of the antibody bonded to an insoluble carrier particle as the monocronal antibody capable of specifically recognizing the reactive site in a human alpha-2-plasmin inhibitor. CONSTITUTION:Two or more kinds of the antibodies bonded to insoluble carrier particles, for example, comprising polystyrene are selected from ones specifically recognizing the respectively different antigenic determinant regions of a human alpha2-plasmin inhibitor and, as at least one kind of said antibodies, a monoclonal antibody capable of specifically recognizing the fibrinolytic action inhibiting region of the plasmin in the human alpha2-plasmin inhibitor is used. Next, the carrier particles are washed with a washing solution and dispersed in a reaction liquid to obtain an insoluble carrier specimen which is, in turn, mixed with a alpha2-plasmin inhibitor solution to perform reaction for a predetermined time at predetermined temp. while the coagulated reaction liquid is diluted to measure absorbancy and the quantity of the human alpha2-plasmin inhibitor in a specimen can be calculated from the measured value.

Description

DETAILED DESCRIPTION OF THE INVENTION a. Industrial Application Field The present invention relates to human α2-plasmin inhibitor (al-plasmin inhibitor) in solution.
.

The present invention relates to a method and kit for immunologically measuring α, -antlplasmin>. For more details, please refer to Human α.

- Two or more monoclonal antibodies that specifically recognize and bind to different antigen-determining sites of a plasmin inhibitor are immobilized on an insoluble carrier particle, and at least one of them is a monoclonal antibody that specifically recognizes and binds to different antigen-determining sites of a plasmin inhibitor; Aggregation immunological study of human α2-plasmin inhibitor using a monoclonal antibody that can specifically recognize and bind to the site for inhibiting the decomposition of human α2-plasmin (hereinafter sometimes referred to as reactive site). The present invention relates to a measurement method and a kit therefor.

b. Prior Art The human α,-plusξin inhibitor was first isolated by Sesumi and Moroi. It is a single-chain glycoprotein with a molecular weight of approximately 67,000 that contains xl and rt4 sugars and is produced by fI and is a powerful grasmi/inhibitor that instantly inhibits the esterase activity of the fibrinolytic enzyme plasmin. C Moroi is known to be
&Aoki ; Ths Jouroa! of B
iological Chami-stry, 251.
5956-5965 (1976)].

On the other hand, it is known that human α2-plasmin inhibitor has three types of active sites. wJl is a site that blocks the fibrinolytic action of plasmin (reactive side) CB, Wiman & D, Co11en;
The Journal of Biological
Chemistry, 254, 9291-929
7 (1979)], and the second is the plus zy binding site on the terminal side of the carboxyl group (B, Wtman & D,
Co11en;European Journal o
f Biochemistry, 84 *573-
578 (1978)], and the third is the fibrin binding site at the amino terminal (Y, 5akata
, at al, + Thrombosis Re5e
arch.

16.279-282 (1979)].

On the other hand, from a clinical medical point of view, it is known that the blood level of human α2-plasmin inhibitor decreases during hepatic parenchymal cell damage, especially in cases of decompensated liver cirrhosis and severe hepatitis. It has been reported that blood levels decrease very markedly [e.g. N, Aoki & T.
Yamanaka, C11n Chlmica A
cta 84, 99-105 (1978)].

In addition, recent progress has been made in elucidating the physical and biological properties of human α,-plasmin/inhibitor, and human α2-plasmin inhibitor specifically regulates the fibrinolytic mechanism.
It is known that it performs knots and has an important effect on the mechanism [see, for example, Seki F, M, A Gaku no Ayumi, 126, 147-155 (1983)].

Therefore, if the amount of human α2-plasmin inhibitor in blood could be measured accurately and easily, it would be extremely useful for prevention and diagnosis of various diseases.

The first conventionally known method for measuring human α2-plasmin inhibitor is human α.

The second method is an immunodiffusion method using antiserum against plasmin inhibitor.The second method is to add a certain amount of excess plasmin to the specimen sample and measure the plasmin activity present in txLS'A by binding to human α2-plasmin inhibitor. However, in the former method, it is extremely difficult to stably obtain an antiserum with a certain activity because it is used as an animal antiserum, and the activity must be corrected using a standard substance. There were 5 complications. Another drawback is that it takes time for immune diffusion to take place. Furthermore, the latter can be determined by examining the residual t of human α
This is a method for indirectly measuring the amount of human α2-plasmin inhibitor, which is easily influenced by various plasmin activity inhibitors* present in the specimen sample, and does not directly measure the amount of human α2-plasmin inhibitor. Therefore, it is impossible to avoid errors, and in this method, it was also necessary to pay attention to the purity and stability of the plasmin used.

Structure of the C0 Invention Therefore, the present inventors conducted research on seven p-nal antibodies against human α,-plasmin/inhibitor, and found that a specific site of plasmin that inhibits fibrin m-degradation in human α-plasmin inhibitor was found. Recognize,
We have discovered a monoclonal antibody that has the activity of suppressing the fibrinolysis-inhibiting effect of human alpha-plasmin inhibitor, and have also created and previously proposed idlidoma cells that produce this monoclonal antibody.

The present inventors have discovered that by utilizing the newly discovered specific action of the monoclonal antibody, it is possible to directly and accurately measure the amount of human α,-plasmi/inhibitor in solution. We have arrived at the present invention.

That is, the present invention provides an agglutination immunoassay method using insoluble carrier particles, in which two or more antibodies bound to the insoluble carrier particles specifically recognize different antigen-determining sites of human α-ioplasmin inhibitor. ,
Among these antibodies, at least one is a 7-p antibody that can specifically recognize and bind to the site that inhibits the fibrinolytic action of plasmin in human α-ioplasmin inhibitor.
The present invention is an immunological assay method for human αt-plasmin inhibitor using a monoclonal antibody against human α,-plasmin/inhibitor, which is characterized by being a null antibody, and a kit therefor.

In general, a method for measuring the presence or absence of an antigen using antibodies bound to insoluble carrier particles is called the latex agglutination method, and the method itself is known (for example, W, P, J
, 5everin; Journal of C1jn
ieal Pathology+ 25.

1079-1082 (1972)).

In the immunoassay method of the present invention, as the two or more types of seven p-nal antibodies used, those that recognize different antigen-determining sites of human alpha-plasmin inhibitor are used. One type uses a monoclonal antibody that can differentially recognize the reactive site ft% in human α-ioplasmin inhibitor. Thus, according to the present invention, it is possible to consistently and accurately measure human α-ioplasmin inhibitor in a solution state (for example, in plasma) without any quality differences in reagents.

In addition, since it directly measures human α-oplasmin inhibitor, it is useful for other purposes! It is completely unaffected by objects and can be measured accurately and in a short time.

According to the present invention, there is provided a kit capable of accurately and rapidly measuring human α,-plasminoid/inhibitor, which has not existed in the past.

Next, the method for measuring the content of A in human αt-plasmin inhibitor according to the present invention will be specifically explained.

A monoclonal antibody against human α-ioplasmin inhibitor is immobilized on appropriate insoluble carrier particles (for example, polyethylene particles) (hereinafter referred to as 1-immobilized antibody'). The insoluble carrier particles are then washed with a washing solution (e.g. 0.0
2 M glycine, 0.03 M NhC6 pH9,
0) and a reaction solution (e.g. 0.1M glycine.

0.15 MNaCI, 1% BSA,
0.05% NaN.

pH 9,0) to obtain an insoluble carrier sample.

This sample and α diluted to various concentrations, −
A plus ξ twin inhibitor solution or a human plasma sample diluted with the above-mentioned anti-filtrate is mixed and reacted for a certain period of time and at a certain temperature. During this time, the immobilized antibody and the human α-ioplasmin inhibitor in the sample bind, and the insoluble carrier particles aggregate. This reaction solution is diluted and the absorbance is measured. Thus, the amount of human α-ioplasmin inhibitor in the sample can be calculated from this value.

The measurement reagent in the present invention is mainly composed of immobilized antibodies bound to insoluble carrier particles. In order to utilize this reagent efficiently and easily, a kit can be formed containing various auxiliary agents in addition to these immobilized antibodies.

Such auxiliary agents include, for example, detergents used to wash insoluble carrier particles, reaction solutions for agglutination reactions, reaction stopper solutions, etc. Commonly used in kits for immunoassay reagents. Examples include things that are done.

Insoluble carrier particles used in the measurement reagent of the present invention include, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, other paper, glass, metal, Examples include agaps and combinations thereof.

Further, the size of the insoluble carrier particles can be selected from various sizes, but is preferably in the range of 0.2 μm to 2.0 μm.

As mentioned above, one of the antibodies used in the assay and kit of the present invention, %at-plasmin inhibitor
A monoclonal antibody that can specifically bind to the site of blocking the fibrinolytic action of plasmin in G fibers was first discovered by the present inventors, and a patent application was previously filed (
Filed on April 17, 1980: Title of the invention 1 Monoclonal antibody, hybridoma cells, and method for producing monoclonal antibody').

The seven Mayuki clonal antibodies of the present invention and the method for producing the same are described in detail in the patent application specification.
The contents will be briefly explained below.

Human α,-plasmin p-HA used as antigen was isolated from human plasma using the method of Yoiki and Moroi.
It was done.

Male Bulb/c mice are used, but other strains (5tra
ins) f) You can also use a mouse. In this case, the immunization regimen and the concentration of human α-plasmin inhibitor should be selected to ensure sufficient antigenic stimulation (i.e., formation of lymphocytes) in the child. For example, a small amount of human α-plasmin/ After several intraperitoneal immunizations with the inhibitor at intervals, several additional doses were administered intravenously. Pancreatic cells were removed for fusion several days after the final immunization.

The C1 cell fused pancreas is aseptically removed and a single cell suspension is prepared from it. These pancreatic cells are fused with mouse myeloma cells from an appropriate line using an appropriate fusion promoter. A preferred ratio of splenic cells to bone marrow gland cells ranges from about 20:1 to about 2:1. For approximately 108 tile fiAMA vesicles, the use of a fusion medium of 0.5 to 51 is appropriate.

Many myeloma cells used for cell fusion are known, but in the present invention, P3-X6'3-Ag8-
Ul cells (hereinafter abbreviated as P3-Ul) (Yelto
n+ D, E et al, + CurrentTop
ics in Mjcrobiology and I
mmunology 81, I (1978)] was used. This is an 8-7 Zaguani/resistant cell line that is resistant to the enzyme hypoxanthine-guanjne phosphoribosyltransferase (hypoxanthine-guanjne pho
sporibosyltransferase) is deleted, and therefore HAT (hyboxanthin,
Aminopterin does not survive in thymidine medium.

Furthermore, this cell line itself does not secrete antibodies, and is a so-called non-secretor.

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.

Dilute the mixture of unfused splenic cells, unfused myeloma cells, and fused cells in a separate container (e.g., a microtiter plate) with selective medium that does not support unfused myeloma cells, and Culture for a sufficient period of time (approximately 1 week) to kill the cells. The medium used is fAT medium which is drug resistant (e.g. 8-azabuanine resistant and does not support unfused myeloma cells (e.g. mentioned above). In this selective medium, unfused bone marrow Il!I cells are In addition, unfused pancreatic cells are non-tumor cells and die after a certain period of time (about one week).

Cells fused to these cells can survive in a selective medium because they have both the intestinal properties of myeloma parent cells and the properties of parent tibia cells.

Hybridoma cells are thus detected. After that, the culture supernatant was collected and cultured with human α.

- Enzyme Linked Immunoassay (Enzyma Linked Immunoassay) for antibodies against plasmin inhibitors
5orbent As5ay).

The antibody-containing culture supernatant obtained by culturing No. 1 hybridoma cells producing antibodies against human α-plasmi/inhibitor in a serum-free medium was tea-condensed and
The cells were incubated with a plasmin inhibitor for a certain period of time. Furthermore, this α was added to a mixture of antibodies against plasmin inhibitors, and the plate was placed on a fibrin plate to measure the fibril/dissolution area. In this way, hybridoma cells producing antibodies with activity against the α2-plasmin inhibitor are selected.

Once a hybridoma cell producing an antibody of interest is cloned by an appropriate method (e.g. limited dilution method), the antibody can be produced in two different ways. By culturing in a suitable medium, the /'
It is possible to obtain 7/Rp-nal antibodies produced by T iglidoma cells. According to a second method, I. hybridoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. After a certain period of time, the seven p-nal antibodies produced by the ibridoma cells can be obtained from the host's proboscis blood and ascites.

The monoclonal antibody obtained as described above is human α
Two plasmin inhibitors specifically recognize the site of inhibition of plasmin's fibrinolytic action and selectively bind to that site.

Other antibodies used in conjunction with the monoclonal antibody may be any antibody that can recognize and bind to a site other than the fibrinolytic action blocking site in α,-plasmi/inhibitor.

As described above, according to the present invention, it is possible to accurately and easily measure the amount of human α2-plasmin inhibitor in a sample containing the inhibitor (for example, human plasma).

The two types of antibodies used in the following examples are described in the specification previously filed by the present inventors (filed on April 17, 1982; title of the invention: 17 p-nal antibodies, 8 p-nal antibodies, 8 p-plidoma cells, and monoclonal antibodies). The following product obtained by the method described in 1) was used.

Antibody name: ID10CI; This is a monoclonal antibody that can specifically recognize the site (reactive site) of the fibrinolytic action of plasmid in α-plasmin inhibitor.

Antibody name: 1B10Gtl; This is a monoclonal antibody that selectively recognizes a site other than the reactive site in α,-plasmid/inhibitor.

The present invention 1 will be described in detail below with examples.

Example 1 Seven drugs against human α-oplasmin inhibitor
null antibodies IDl0CI and IBIOGII each separately at a concentration of 50 μi/Ml, 0.02 M glycine,
Dissolved in a solution of 0.03 M NaCl pH 9,0, and added a diameter of 0.60 to 1.0317 of these two types of antibody solutions.
A polystyrene latex of μm was suspended to a concentration of 2% and left overnight at room temperature to adsorb the antibody to the polystyrene latex. The above polystyrene latex was mixed with 0.0% polystyrene latex. (12M glycine.

0.03 M NaCl pH 9,0 solution 1.01 to 7
After washing twice, add 0.1M Gly'/7. 0.15
M NaCl, 1 tBS A, 0.05%
NaN, pH 9,0 solution 1. OMl was suspended. next,
Human α! - 50 μl of polystyrene latex adsorbed with monoclonal antibody ID10C1'l' against Blasmi/inhibitor and 350 μl of polystyrene latex adsorbed with IBIOGIIV were mixed to prepare a latex standard. This latex standard is 100μ! And various concentration Nohito α! - Plasmin inhibitor standard or 0.1M
Glycine, 0.15 MNmCl, 1% BS
A, 0.05% NaN, pH9, OR11'El
100 μl of human plasma sample diluted with 100 μl was mixed and reacted at 37° C. for 30 minutes.

Reaction solution 11'0. IM glycine, 0.15M NaC
l, 1% BSA, 0.05'I NaN,
Diluted 125 times with pH 9,0 solution g, wavelength 600nm
The absorbance was measured at A calibration curve was created using human α,-plasmin/inhibitor samples at various concentrations, and the human CTL-plasmin inhibitor concentration in human plasma samples was
t was calculated.

Since the absorbance value at a wavelength of 600 nm for a 620-fold diluted healthy human plasma sample was 1,068, the amount of human α,-blasmi/inhibitor was calculated using the calibration side shown in Figure 1 to be 60.8 μi. /Ml.

Example 2 Monoclonal antibodies against α2-plasmin inhibitor IDl0CI and IBIOGII were mixed at a concentration ratio of 1=1, and 0.02M glycine and 0.03M glycine were added.
myrtrtcm solution of M NaCl pH 9,0;
A solution 114 with an antibody concentration of 50 μm/ml was prepared. This antibody solution 1. Polystine latex with a diameter of 0.60 μm? : The two types of monoclonal antibodies were suspended to a concentration of 2% and allowed to stand at room temperature until the two types of monoclonal antibodies were adsorbed onto polystyrene latex. This Ur Tsuru: x, 0.
02 Mglycy, 0.03MNmC1p)i 9,0
#1 liquid 1. After washing twice with O+aj, 0.1 M glycine, Oot 5 M NaCl, 1% BSA.

0.05% NaN, pH 9.0 soluble fi 1. Suspended in Omu, dry) 5 100μj and various concentrations of
Plasmin inhibitor preparation, or 0.1 M glyciso, 0.15 M Na (J, 1% BSA, 0
．． 100aJtt of human plasma sample diluted with U5% NaN, pH 9.0 solution was mixed and incubated at 37°C for 30 minutes.
I reacted. Reaction solution fjtO0I M glycine, 0.
15M NaCl, 1% H8A, 0.05T Na
Diluted 125 times with N, pH 9, Of# solution, wavelength 60
Absorbance at 0 nm was measured. A calibration curve was created using αt-plasmin/inhibitor samples at various concentrations, and the amount of α,-plasmin inhibitor in the human plasma sample was calculated.

Since the absorbance value of the 620-fold diluted healthy human plasma sample at a wavelength of 600 nm was 1,002, the amount of α,-plasmi/inhibitor was calculated using the test 1td shown in Figure 2 to be 60.1 μI/ It was Go.

[Brief explanation of drawings]

FIGS. 1 and 2 show a calibration M showing the relationship between human α,-plasmin inhibitor concentration and absorbance (600 nm) used in Example 1 and Example 2 of the present invention, respectively. Patent applicant: Teijin Limited. -5, "・Shino' Figure 1 t14z-79 people (,4 int bitashi 4 each (η9
/vn9) Figure 2

Claims (1)

[Scope of Claims] 1. In the agglutination immunoassay method, two or more antibodies bound to insoluble carrier particles specifically recognize different antigen-determining sites of human α_2-plasmin inhibitor, and A human α_2-plasmin inhibitor characterized in that at least one of these antibodies is a monoclonal antibody capable of specifically recognizing and binding to a site for inhibiting the fibrinolytic action of plasmin in the human α_2-plasmin inhibitor. An immunological assay method for human α_2-plasmin inhibitor using a monoclonal antibody against it. 2. Contains antibodies bound to insoluble carrier particles, these antibodies specifically recognize different antigen-determining sites of human α_2-plasmin inhibitor, and at least one antibody recognizes plasmin in human α_2-plasmin inhibitor. A monoclonal antibody that can specifically recognize and bind to a site that blocks the fibrinolytic action of
A kit for immunoassay consisting of this in combination with a reaction initiator, a detergent, and a reaction stopper. 3. Item 2, wherein the insoluble carrier particles are made of polymers such as polystyrene, polyester, polyethylene, polypropylene, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, cellulose, glass, metal, agarose, and combinations thereof. Kit for the immunological assay described.