JPS6134465A - Immunoassay reagent and kit using monoclonal antibody against human alpha2-plasmin inhibitor - Google Patents

Abstract

PURPOSE:To elevate sensitivity, by arranging one of a monoclonal antibody for specifically recognizing a human alpha2-plasmin inhibitor and an antibody against human plasminogen to be an antibody bonded to an insoluble carrier while the other being a labeled antibody. CONSTITUTION:A monoclonal antibody (first antibody) against a human alpha2- plasmin inhibitor is immobilized to an appropriate insoluble carrier and covered with a proper substance. The insoluble carrier having the first antibody immobilized thereto is made to react with a sample material. After washing thereof, a solution of an antibody (second antibody) labeled with a proper label substance against a human plasminogen is brought into contact with the product to react. Then, the amount of the label substance attached to the second antibody existing on the insoluble carrier is measured after washing thereof to calculate the amount of the plasmin-alpha2-plasmin inhibitor in the sample material. Thus, accurate and quick measurement is possible.

Description

DETAILED DESCRIPTION OF THE INVENTION a. Industrial Application Field The present invention relates to a plasmin-human α,-plasmin inhibitor complex (plasmin-a,
-plasmin 1nhibitor complete
x + plasmin-a, -antiplasm
The present invention relates to reagents and kits for immunologically measuring (in complex).

More specifically, monoclonal antibodies that specifically recognize and bind to human α2-plasmin inhibitor and plasmin! Plasmin-human α by sandwich method using antibodies capable of binding
The present invention relates to an immunological assay reagent for 2-plasmin inhibitor complex and a kit thereof.

b. Prior Art Human α,-plasmin inhibitor was first isolated by Qingui and Ryoben.NjIl! It is a strong plasmin inhibitor that instantly inhibits the esterase activity of plasmin, a cytolytic enzyme, and is a single-chain glycoprotein with a molecular weight of approximately 67 (100) containing 11.7N sugar. is known as C Mor
oi&Aokt ; The Journal of
Biological Chemistry, 25
1, 5956-5965 (1976)].

It is known that the -1 human α-plasmin inhibitor has a class 3a active site. The first is a site that blocks the fibrinolytic action of plasmin (hereinafter referred to as the "cutive side") (B, Woman
&D, Co11en; The Journal
of Biological Chemistry
+254.9291-9297 (1979)]
The second is the plasmin binding site CB on the carboxyl group terminal side, 'Woman & D, Co11en
;European Journal of Bioc
hemistry t 84 + 573-578 (1
978)], and the third is the fibrin binding site at the end of the amide 7 group (Y, 5akata + etal-
+ Thrombosis Research + 16
279-282 (1979)].

Human α2-plasmin inhibitor inhibits plasmin activity almost instantaneously, and α,-plasmin inhibitor binds to plasmin at a ratio of 1:1 to form a complex.

(B, Woman & D, Co11en; T1
1r; Journak of Biologica
l iCbemiatry +264 + 9
291-9297 (1979) +D, Co11en
s'l'hramt+ogis and)
faerr + oatasir, + 4 3 +
7 7-89 (1980) #) ￥1) In conditions where plasminogen is activated, such as during disseminated intravascular coagulation (CDIC) or thrombolytic therapy using p-kinase H, the generated plasmin and α, -plasmin inhibitor react and form a complex.

Recently, quantification of plasmin-a,-plasmin inhibitor complex in plasma has been used for monitoring thrombolytic therapy and for DIC.
The diagnosis of isobaric is considered valid (e.g. N, A
, Booth & +B=Bennett:
Br1tish Journal of Haema
tology + 50 *537-541 (1982
)reference).

Therefore, if the amount of plasmin-α,-plasmin inhibitor complex in the 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 plasmin-α,-plasmin inhibitor complexes, the method of Fuki 1 uses two-dimensional cross-immunoelectrophoresis, and the second method involves immobilizing antibodies obtained from antiserum. This method is based on the so-called sandwich method, which is an application of the enzyme-antibody method.

However, the method used by I¥fT had the drawback of low sensitivity and low quantitative performance. Additionally, the latter method is extremely sensitive (
Although this is an effective method, it has the disadvantage that it is extremely difficult to stably obtain an antiserum with a certain level of activity against human α2-plasmin inhibitor.

・0 Structure of the Invention Therefore, the present inventors conducted repeated research on monoclonal antibodies against human α2-plasmin inhibitor, and found that they specifically recognized the site that inhibits the fibrinolytic action of plasmin in human α2-plasmin inhibitor. a. Discovering a monoclonal antibody that has the activity of suppressing the fibrinolysis-inhibiting effect of a plasmin inhibitor,
We can also create hybridoma cells that produce this monoclonal antibody.

As already proposed, the present inventors have proposed that by utilizing the newly discovered specific action of the monoclonal antibody, it is possible to directly and accurately measure the amount of human α2-plasmin inhibitor in a solution state. This discovery led to the present invention.

That is, in the immunoassay reagent using the sandwich method of the present invention, the antibody bound to the insoluble carrier and the labeled antibody are human α2-plasmin inhibitor 1&ii! A monoclonal antibody that recognizes and binds human plasmin, and an anti-plasminoglu antibody component that binds human plasmin.

In general, the method of making antibodies that bind to two different positions on an antigen and measuring the presence or absence of the antigen is called the sandwich method.
ds) J 199-206 (1970). Thus, according to the present invention, there is no difference in the quality of the reagents, and the plasmin-a, -plasmin inhibitor in solution (e.g. in blood Jt) can be used at constant KM. - It becomes possible to measure complexes.

Furthermore, since the plasmin-α2-plasmin inhibitor complex is directly measured, it is not affected by other impurities and can be measured accurately and in a short time. Therefore, according to the present invention, there are provided a reagent and a kit thereof that can accurately and rapidly measure a plasmin-α,-plasmin inhibitor complex.

Next, the method for measuring the content of the measuring reagent and plasmin-α,-plasmin inhibitor complex according to the present invention will be specifically explained.

A monoclonal antibody (first antibody) against human α2-plasmin inhibitor is identified on a suitable insoluble carrier (for example, a plastic container) (hereinafter referred to as "one-directed antibody").

Next, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine blood ff fulbumin) in order to avoid non-specific binding between the insoluble carrier and the reagent or sample material to be measured.

The first antibody obtained in this manner is brought into contact with a sample material in the form of an insoluble carrier immobilized thereon for a certain period of time and at a certain temperature to cause a reaction. During this time, the identifying antibody (kl antibody) and the plasmin-Qt-plasmin inhibitor complex in the specimen material bind. After washing with an appropriate washing solution, an antibody against human plasminogen (secondary antibody) labeled with an appropriate labeling substance is added.
A solution (e.g., an aqueous solution) of the antibody) is brought into contact with the plasmin α-ioplasmin inhibitor complex bound to the immobilized antibody on the insoluble carrier at a certain time and temperature to react with the second antibody 1. This is washed with an appropriate washing solution, Next, the amount of the labeled substance labeled with the second antibody KtlAI and present on the insoluble carrier is measured. Thus, the amount of plasmin-α,-plasmin inhibitor complex in the specimen material can be calculated from this value.

(Thus, the measurement reagent of the present invention mainly consists of an immobilized antibody in which the first antibody is bound to an insoluble carrier, and a labeled second antibody.This reagent can be used efficiently (and conveniently). To do.

In addition to these antibodies, the kit can include various auxiliary agents. Examples of auxiliary agents for preservation include solubilizing agents for dissolving solid reagents, detergents used for washing insolubilized carriers, and enzymes used as labeling substances for antibodies to measure enzyme activity. Substrates and reaction terminators commonly used as kits for immunoassay reagents are included.

Examples of insoluble carriers used in the measurement reagent of the present invention include polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine-fat, crosslinked textran, polysaccharide, other paper, glass, metal, Examples include agarose and combinations thereof.

In addition, the shape of the insoluble carrier is, for example, a tray shape, 9 sphere shape, IIm, cone shape, 9 rod shape, disk shape, or container shape.

It can be in extreme shapes such as cells, test tubes, etc.

Furthermore, it is advantageous to use a radioactive substance, W element, or a fluorescent substance as the labeling substance. 1251 as a drug-preventing substance
．． 1311.14C, 3H, etc., and the enzyme is alkaline phosphatase.

Peroxidase, β-D-galactosidase, and fluorescent substances such as fluorescein inthiocyanate and tetramethylloguminthiocyanate may be used, but these are not limited to the examples listed above. Is it possible to use other methods as long as it is possible to use it? ! -ru.

Regarding the monoclonal antibody of the present invention and the method for producing the same, a patent application was previously filed (published on April 17, 1982). Manufacturing method °′).

As described in detail in the patent application specification, the monoclonal antibody of the present invention and the method for producing the same specifically recognize and bind to the site that inhibits the fibrinolytic action of plasmin in human α2-plasmin inhibitor. It is a monoclonal antibody that can be used to The contents will be briefly explained below.

Human α2-plasmin inhibitor used as an antigen was isolated and purified from human plasma by the method of Seishi and Roben.

Male Ba1b/c mice are used, but other strains (stra
You can also use a mouse (ins). The immunization regimen and concentration of human α2-plasmin inhibitor should then be chosen such that sufficient antigen-stimulated lymphocytes are formed. For example, a small amount of human α in a mouse
After several intraperitoneal immunizations with Oplasmin inhibitor'l- at intervals, and several more intravenous administrations, pancreatic cells were removed for fusion a few days after the final immunization.

The C6 cell-fused pancreas is aseptically removed and a single cell suspension is prepared from it. These pancreatic cells are fused with mouse bone marrow deer cells from an appropriate line using an appropriate fusion promoter. A preferred ratio of pancreatic cells to myeloma cells ranges from about 20=1 to about 2=1. It is appropriate to use 0.5 to 1.5117 fusion media for about 106 pancreatic cells.

Many myeloma cells used for cell fusion are known, but in the present invention, P3-X63-Ag8-U
l cell (hereinafter abbreviated as P3-Ul) [Yelt
on+ D, E et al, + CurrentTo
pics in Microbiology and
Immunology 811I (1978)] was used. This is an 8-7zag7nin resistant cell line and is an I#L hypoxanthine-guanine phosphoribosyltransferase (hypomnthine-guanine pho
sporibosyltransferase)
is deleted and therefore does not survive in HAT (hyboxanthin, aminopterin, thymidine) medium.

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

For example, a preferable fusion promoter has an average molecular weight of 1
000-4000 polyethylene glyfur may be advantageously used, although other fusion promoters known in the art may also be used.

D. Selection of fused cells: A mixture of unfused pancreatic cells, unfused myeloma cells, and fused cells in a separate container (e.g., microtiter plate) in 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 used is one that is drug resistant (eg, 8-7 zaguanine resistant) and does not support unfused myeloma cells (eg, the above-mentioned HAT medium). Unfused bone marrow abdominal cells die in this selective medium. The unfused pancreatic cells in the cage are non-s1m cells and die after a certain period of time (about 1 week).

Cells fused to these cells can survive in the selective medium because they have both the lung cancerous properties of myeloma parent cells and the properties of pancreatic cells.

After hybridoma cells are detected, the culture supernatant is collected and analyzed for antibodies against human a-plasmin inhibitor by enzyme immunoassay (
EnzymeLinked Immuno 5orbe
nt As5ay ) K.

When hybridoma cells that produce the antibody of interest are cloned using an appropriate method (e.g. limited dilution method), the antibody is
produced in two different ways. According to the first method, monoclonal antibodies produced by the hybridoma cells can be obtained from the culture supernatant by culturing the hybridoma cells in an appropriate medium for a certain period of time. According to a second method/- iploidoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. From the blood and ascites of the host animal after a certain period of time,
It is possible to obtain monoclonal antibodies produced by hybridoma cells.

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 it can be attached with a labeling substance and used as a brittle antibody.

Other antibodies that can be used with the monoclonal antibody may be any antibody that can bind human plasmin.

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

The present invention will be described in detail below with reference to Examples.

Example 1 The 1 and 2 antibodies used in this example were obtained from Ming #+Ku (filed on April 17, 1982), which the present inventors had previously applied for;
The following materials obtained by the method described in "Title of the Invention 1: Monoclonal antibodies, hybridoma cells, and methods for producing monoclonal antibodies" were used.

First antibody: Antibody group obtained in Example 3 of the above-mentioned application specification.
IBIOGII" was used and immobilized on an insoluble carrier (microtiter plate) as shown below. This antibody was used in a plasmin inhibitor other than the site (reactive site) that blocks the fibrinolytic action of plasmin. This is a monoclonal antibody that can specifically target the Kll site.

Second antibody An antibody component was purified from an antiserum against human plasminogen obtained by immunizing a rabbit with human plasminogen, labeled with alkaline phosphatase, and used.

A monoclonal antibody (IBI) that specifically recognizes human α2-plasmin inhibitor at a concentration of 20 tttt/g
OGII) was left on a microtiter plate overnight at 4°C to immobilize it. Add to this a buffer containing 1% bovine serum albumin (15mM Na, COa s 35mM N
After adding aHCO3, 3mM NaNB) and leaving it at room temperature for 4 hours, washing solution containing 1% bovine serum albumin (20
mM phosphate buffer + 0.135 M NaC1+2
Washed 5 times with mMNaN, + 0.05% Tween 20 ). Next, plasmin-α,-plasmin inhibitor complexes diluted to various concentrations with a dilution solution (20mM'), 0.135M Na(J), and 0.135M Na(J) were added, and the mixture was left at room temperature for 4 hours. did.

Thereafter, the plate was washed five times with a washing solution, and an antibody against human plasminogen that had been purified with alkaline phos-77ase at a concentration of 350 J/iu was added thereto overnight at 4°C.

After washing with a diluted washing solution, add alkaline phosphatase substrate solution at a concentration of 19% and perform ELISA ANALYZ.
40.5n with ER (Toyo side number @ #ETY-96)
W per minute at a wavelength of m! The change in L-yt degree was measured. The results are shown in Figure 1 of the Usuzuki drawings. From this figure, it can be seen that the relationship between the concentration of the plasmin-α,-plasmin inhibitor complex and the change in absorbance is a linear relationship. Therefore, by using a monoclonal antibody that specifically recognizes a site other than the reactive site in the -plasmin inhibitor as one antibody in the Sand-Deutsch method, the amount of the plasmin-α, -plasmin inhibitor complex can be adjusted to can be measured.

[Brief explanation of drawings]

The accompanying drawings show plasmin-α2 in an embodiment of the present invention.
- shows the relationship between the concentration of the plasmin inhibitor complex and the change in absorbance.

Claims (1)

[Claims] 1. In an immunoassay reagent using a sandwich method,
One of the antibody bound to the insoluble carrier and the labeled antibody is a monoclonal antibody that specifically recognizes human α_2-plasmin inhibitor, and the other is an antibody against human plasminogen. An immunological assay reagent for plasmin-α_2-plasmin inhibitor complex using a monoclonal antibody against α_2-plasmin inhibitor. 2. The measurement reagent according to item 1, wherein the insoluble carrier is a plastic container, plastic beads, latex beads, glass beads, or metal particles. 3. The measurement reagent according to item 1, wherein the labeled antibody is an antibody labeled with an enzyme, a radioisotope, or a fluorescent substance. 4. Contains an antibody bound to an insoluble carrier and a labeled antibody, one of these antibodies is a monoclonal antibody that recognizes and binds to human α_2-plasmin inhibitor as an antigen, and the other one is a monoclonal antibody that recognizes and binds to human plasminogen as an antigen. When an antibody labeled with (a) a lysing agent, (b) a detergent, and an enzyme is used, (c) a substrate for measuring enzyme activity and its reaction. A kit for immunoassay consisting of a combination of a stopping agent.