JPS6197230A - Selective adsorbent for human alpha2-plasmin inhibitor and separation of human alpha2-plasmin inhibitor in human plasma - Google Patents

Abstract

PURPOSE:To separate alpha2-plasmin inhibitor in blood plasma, by using a selective adsorbent for human alpha2-plasmin inhibitor prepared by bonding a specific monoclonal antibody to an insoluble carrier. CONSTITUTION:A selective adsorbent for human alpha2-plasmin inhibitor is produced by bonding an insoluble carrier (e.g. sepharose,polyacrylamide, cellulose, etc.) with a monoclonal antibody (Japanese Patent Application, April 17, 1984) capable of specifically recognizing and bonding the site inhibiting the fibrinolytic activity of plasmin in human alpha2-plasmin inhibitor. The alpha2-plasmin inhibitor in plasma can be separated by the affinity chromatography using said adsorbent. A patient of alpha2-plasmin inhibitor deficient disease is liable to cause grave hemorrhage by the premature disintegration of the thrombus.

Description

DETAILED DESCRIPTION OF THE INVENTION a. Industrial Application Field The present invention provides a selective adsorbent for human α2-plasmin inhibitor and a selective adsorbent for human α2-plasmin inhibitor using the same.
- A method for isolating plasmin inhibitors.

b. The α,-plasmin inhibitor of the prior art was first isolated and purified by Aomizu and Moroi, and is a strong plasmin inhibitor that instantly inhibits the esterase activity of the plasmin lytic enzyme. , 11
．． It is known to be a single-chain glycoprotein with a molecular weight of approximately 67,000 containing 796 sugars (Moral an
d AokiyThe Journal of Bio
Logical Chemistry+251.595
6-5965 (1976)].

In addition, its concentration in plasma is 6.13f0.88 in healthy people.
II9/10 ou (N, A
oki and T, Yamanaka+C1inC1
1nicaChi Acta 84.99-105 (
(1978)].

On the other hand, human α! - It is known that plasmin inhibitors have three types of active sites. The first is a site that inhibits the xenolytic action of plasmin (hereinafter this may be referred to as 1 reactive site') CB, W'iman
and D, Co11en; The Journa
of Biological Chemistry
+254.9291-9297 (1979)] and the second is the plasmin binding site CB on the carboxyl group terminal side, Wiman and D, Co11en;
European Journal of Bioch
emistry+ 84 +573-578 (1978
), and the third is the fibrin-binding site at the end of the amide 7 group (Y, 5akata, et al.
, , Thrombosis Re5earch+
16, 279-282 (1979) #Sho], recently.
Progress has been made in elucidating the physical and biological properties of human α2-plasmin inhibitor, and human α2-plasmin inhibitor.

-Grasumi/Inhibitor is IN! ! It is known that it has my effect on the fibrinolytic structure [for example, Nobuo Aomizu 1 History of Medicine' 126.147-155 (
(1983)].

Furthermore, when the blood of a patient with C2-plasmin inhibitor congenital deficiency is placed in a test tube and left at 37°C, coagulation occurs completely normally, but the blood completely dissolves spontaneously in 4 to 8 hours (N, Aoki + et al. , + Jour
nal of C1inicC11nicalInve
sti+ 63 +877-884 (1979, )].

In fact, in plasmin inhibitor deficiency there is a very severe bleeding tendency due to early disintegration of the hemostatic plug.

This fact suggests that, conversely, in severe thrombosis, C8-
This seems to suggest that by reducing the amount of plasmin inhibitor, the causative thrombus can be rapidly dissolved.

Structure of the C0 Invention The present inventors have conducted extensive research on monoclonal antibodies against human α2-plasmin inhibitor, and have found that they specifically recognize the site that inhibits the fibrinolytic action of plasmin in human α2-plasmin inhibitor.
We have discovered a monoclonal antibody that has the activity of suppressing the fibril lysis inhibition effect of human α2-plasmin inhibitor, and have also created bilidoma cells that produce this monoclonal antibody, which we have already proposed.

As a result of further research into the utilization of the specific action of the monoclonal antibody obtained in this way, the present inventors have developed an adsorbent that can specifically adsorb human a,-plasmin inhibitor and the use of the adsorbent in humans. Human α,-plasmi in plasma.

The inventors of the present invention have discovered that the inhibitor can be selectively separated. That is, the present invention provides a human sham in which a monoclonal antibody capable of specifically recognizing and binding to the site of inhibiting the fibrinolytic action of plasmin in human α2-plasmin inhibitor is bound to an insoluble carrier as a ligand. - A selective adsorbent for plasmin inhibitor, and a method for selectively separating human α2-plasmin inhibitor from human plasma using the adsorbent.

In general, p-chromatography, which utilizes the biological affinity of adsorbents for the separation and purification of biological substances, is called 7-infinity chromatography [for example, Chibata,
Tetsuya Tosa.

"Experimental and Applied Affinity Chromatography" by Yushi Matsuo, published by Koeisha Icontific.

Affinity, ligand in the present invention.

The terms "insoluble carrier" and "adsorbent" shall each have the following meanings.

7 affinity; specific affinity ligand present in the 2m substance question; substance intended for adsorption or a-listing and substance with 7 affinity; insoluble support in water; support insoluble in water (this does not contain the ligand) Adsorption body: one in which the ligand is immobilized on an insoluble carrier Next, the human α-oplasmin inhibitor according to the present invention
The selective adsorbent for and the method for separating human α-oplasmin inhibitor from human plasma will be explained in detail.

A monoclonal antibody against the human σ,-plasmin inhibitor, which will be described later, is chemically bound to an appropriate insoluble carrier (e.g., Sepharose) as a ligand, and this is packed in a column with an appropriate buffer solution (e.g., 5 (ltM)!JS buffer PH7). ,4+ 0.15 M NaC/ ) K.

A horizontal sample (human plasma) is added to this adsorbent to adsorb the human α-2 plasmin inhibitor j' in the specimen sample. Then a suitable wash solution (e.g. 50mM)
Lys buffer, pH 7.4.

Impurities are removed from the adsorbent by 0.15 MNaC/ ). Next, the amount of human α-oplasmin inhibitor in the 1-pass fraction' and the wash fraction' of the home plasma is measured. Thus, the degree of separation of human α-oplasmin inhibitor from human plasma can be calculated from this value.

Various types of insoluble carriers can be used for the selective adsorbent in the present invention, including materials such as Sepharose, polyacryl 7mide 9 cellulose! Text run! Alternatively, a maleic acid polymer or a mixture thereof is preferably used. The shapes of these inert carriers include powder, 9 grains, and pellets! Beaded! As described above, the site for inhibiting the fibrinolytic action of plasmin in human α,-plasmin inhibitor, which is an antibody used in the adsorbent of the present invention, can be in a film-like, fibrous, etc. Monoclonal antibodies capable of specifically recognizing and binding to
Filed on the 7th; Title of the invention: 1. Monoclonal antibodies, noibridoma cells, and methods for producing monoclonal antibodies.

The monoclonal antibody of the present invention and the method for producing the same are explained in detail in the patent application specification,
The contents will be briefly explained below.

Human α used as antigen! -Plasmin inhibitor was purified from human plasma by the method of Aomizu and Moroi.

Male Ba1b/c mice are used, but other strains (5trai
ns) can also be used. The immunization regimen and production of the human σ-plasmin inhibitor should then be chosen such that sufficient antigen-stimulated lymphocytes are formed. For example, after immunizing mice several times intraperitoneally at intervals consisting of a small amount of α,-plasmin inhibitor,
Several more doses were administered intravenously. A few days after the final immunization, lung cells are removed for fusion.

The lungs are aseptically removed and a single cell suspension prepared from it. The lung cells are fused with mouse myeloma cells from an appropriate line by use of an appropriate fusion promoter. The preferred ratio of splenic cells to bone marrow Mm cells is approximately 20=1
~approximately 2=1. It is appropriate to use 0.5 to 1.5117 tons of fusion medium for each splenic visceral cell.

Many bone marrow islet cells used for cell fusion are known, but in the present invention, P3-X63-Ag8-Ul cells (hereinafter referred to as P3
-U 1) C-falton+ D, E, e
tal, lCurrent ToplcainM
icrobiology and immunolog
y+ 81 + 1 (1978) #Sho] was used. This is a cell line resistant to 8-azaguanine, which contains the enzyme hypoxanthine-guanine phosphoribosyltransferase ('hypoxanthinegu-anine
phosphoribosyl transfers
e) is deleted and therefore does not survive in HAT (hypoxanthine, aminopterin, thymidine) medium. In addition, this cell line itself does not shunt antibodies and is a so-called non-secretor.

Preferred fusion promoters include those with an average molecular weight of i, o.
oo to 4,000 polyethylene glycol can be advantageously used, although other fusion promoters known in the art can also be used.

In a separate container (e.g., a microtiter plate), dilute the mixture of unfused pancreatic cells, unfused myeloma cells, and fused cells with selective medium that does not support unfused myeloma cells, and remove the unfused cells. sufficient time to kill (
Culture for about 1 week). The medium should be drug resistant (e.g. 8-7
A medium (such as the above-mentioned HAT medium) that is resistant to Zag7nin and does not support unfused myeloma cells is used.

Unfused bone marrow Il cells die in this selective medium.

These unfused pancreatic cells are non-neoplastic cells and die after a certain period of time (approximately one week). Cells fused to these cells can survive in a selective medium because they have both the PIk neoplastic properties of myeloma cells and the properties of parent pancreatic cells.

After hybridoma cells have been detected in this manner, their culture supernatants are collected and analyzed for antibodies against human alpha-yoplasmin inhibitor by enzyme immunoassay (Enzyme Li).
nked Immun.

5orbent A+! say) Screening from K.

Noiplidoma cells producing antibodies against human α-ioplasmin inhibitor were cultured in a serum-free medium, and the culture supernatant containing antibodies was concentrated.
! - incubated with plasmin inhibitor for a certain period of time. Furthermore, plasmin was added to this human α-ioplasmin inhibitor mixture, and the plate was placed on a fibrin plate, and the area of fibrin scales was measured. In this way, hygridoma cells producing antibodies with activity against human α-plasmin inhibitor are selected.

When hybridoma cells producing antibodies of interest are cloned by an appropriate method (eg, limited dilution), antibodies can be produced in two different ways. According to the first method, the seven clonal 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. Second
According to this method, hybridoma 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,
Monoclonal antibodies produced by the hybridoma cells can be obtained.

The monoclonal antibody obtained as described above is human α
It specifically recognizes the site in the ioplasmin inhibitor that inhibits the fibrinolytic action of plasmin and selectively binds to that site.

In the adsorbent of the present invention used for separating α,-plasmin inhibitor in human plasma.

Such a monoclonal antibody is used as a ligand by chemically bonding it to an insoluble carrier.

In general, the binding method may be any method in which a monoclonal antibody is chemically bound to an inert carrier.

As described above, according to the present invention, α from human plasma.

- It is possible to easily and selectively separate plasmin inhibitors.

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

Example α: An adsorbent (0,51111) chemically bound to a monoclonal antibody against plasmin inhibitor IDl0CI as a ligand was packed in a column, and a washing solution (
50m M41@Liquid pH 7.4, 0.15M NaCl
), then 1.0117 ml of human plasma was added.

Furthermore, the inner wall of the column was washed with 1.0 lj of the above washing solution, and both eluted fractions were designated as "pass-through fractions". Next, the unadsorbed substances were eluted with the above-mentioned washing solution 2.0IIj and designated as a "washing fraction." All the above operations were performed at 4°C. The fractionated “
The "pass-through fraction" and "washing fraction" are immediately desalted and concentrated at 4°C.Creating a human α2-plasmin inhibitor calibration curve α! −
The amount of plasmin inhibitor is described in the specification previously filed by the present inventors, filed on May 1, 1980; Title of Invention 1.
Immunoassay reagent and Kira) using monoclonal antibody against human α,7 blasmin inhibitor
It was measured using the Sanderuch method described in C.

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: 1 Monoclonal antibodies, hybridoma cells, and monoclonal antibodies). The following product obtained by the method described in "Manufacturing method") was used.

First antibody Antibody name obtained in Example 3 of the above application specification
IDl0CI' was used and immobilized on an insoluble carrier (microtiter plate) as described below. This antibody is a monoclonal antibody that can specifically target the site (reactive site) of the fibrinolytic action of plasmin in α,-plasmin inhibitor.

1a2 antibody Antibody name obtained in Example 3 of the above application specification
IBIOGII' was used. This antibody is a monoclonal antibody that specifically recognizes Kg at a site other than the ri7 cuttape site in α,-plasmin inhibitor, and was used after being converted into 1iAR with alkaline phosphatase.

A specific Kl! ! Orisuki monoclonal antibody (IDIOCI) was immobilized on a microtiter plate by leaving it at 4°C overnight. Add to this a buffer containing IX bovine serum albumin (15mM N
12CO3+ 35 m M NaHCO2+3 m
After adding M NaN, ) and leaving it at room temperature for 4 hours, 19
Wash solution containing 6 bovine serum albumin (20mM phosphate buffer + 0.135M NaCl + 2mM Na
N, +0.05 j%' Tween 20) and 5
Washed twice. Next, the dilution solution (20mM phosphorus buffer p
α-plasmin inhibitor diluted to various concentrations with H7,4sO, 135M NaCl) was added and left at room temperature for 4 hours.

After that, it was washed 5 times with the washing solution, and then the alkaline phosphatase-labeled α,-plasmin inhibitor was added.
A monoclonal antibody (IBIOGII) that recognizes a site other than the reactive site was added and left overnight at 4°C. After washing with the washing solution, the alkaline phosphatase group J [ffz Wi t I W / 1LtcD
Density Y: Add, MICROPLATE PHOTO part T
K after 20 minutes with ER (manufactured by Corona Denki, MTP-20)
Absorbance was measured at a wavelength of 405 nm. The results are shown in the attached Figure 1. From this figure, it can be concluded that the relationship between the concentration of σ,-plasmin inhibitor and the absorbance is a linear relationship. Therefore, by using a monoclonal antibody that specifically targets the reactive site of α,-plasmin inhibitor as one antibody in the sandwich method, the amount of α,-plasmin inhibitor can be easily measured.

Using the attached drawing as a standard curve, human plasma [pass-through fraction]
and the amount of α,-plasmin inhibitor in the “washed fraction” was measured.

Measurement of the amount of α,-plasmin inhibitor in a sample Human α
A monoclonal antibody that specifically recognizes the reactive site in aoplasmin inhibitor (IDIOC)
I) was immobilized at a concentration of 20 μm and 17 M on a microtiter plate by standing overnight at 4°C. Add to this a buffer containing 1% bovine serum fulpmin (15mM Na1CO3135
mMNaHco@+3 m MNaNs) was added and left at room temperature for 4 h.

Washing solution containing IN bovine serum fulpmin (20mM phosphate a
! Solution 10.135 M NaC/+2 mM NaN
Washed 5 times with 10.05% Tvreen 2 G). Next, dilute the solution (20mM phosphate buffer + 0.5mM phosphate buffer).
Samples (the above-mentioned "pass-through fraction", "washed fraction" and human plasma) diluted with 135 MNaC/ ) to varying concentrations were added and left at room temperature for 4 hours.

Thereafter, it was washed 5 times with the washing solution, and the alkaline phosphatase-labeled human α-oplasmin inhibitor was removed.
A seven-clonal antibody (IBIOGII) that recognizes a site other than the reactive site was added and left at 4°C overnight.

After washing with the washing solution, alkaline phosphatase substrate solution 119/red was added, and MICROPLATEPHO
After 20 minutes, absorbance at a wavelength of K 405 nm was measured using TOMETER (Corona Electric #M, MTP-12).

The results are shown in the table below. The "Weighing Fraction" and "Washing Fraction" buttons indicate α, -plasmin inhibitor is not detected, and by adding human plasma to the adsorbent, α
It was confirmed that the -plasmin inhibitor was completely separated.

Table (α,-plasmin inhibitor profile in each sample)

[Brief explanation of the drawing]

Rimura V564 is human α-oplasmin inhibitor 2
This shows the relationship between [4] and absorbance.

Claims (1)

[Scope of Claims] 1. A selective method for human α_2-plasmin inhibitor in which a monoclonal antibody capable of specifically recognizing and binding to the site for inhibiting the fibrinolytic action of plasmin in human α_2-plasmin inhibitor is bound to an insoluble carrier. adsorbent. 2. The selective adsorbent according to item 1, wherein the insoluble carrier is at least one selected from the group consisting of Sepharose, polyacrylamide, cellulose, dextran, and maleic acid polymer. 3. Using a selective adsorbent for human α_2-plasmin inhibitor, in which a monoclonal antibody capable of specifically recognizing and binding to the site for inhibiting the fibrinolytic action of plasmin in human α_2-plasmin inhibitor is bound to an insoluble carrier, A method for separating human α_2-plasmin inhibitor from human α_2-plasmin inhibitor-containing plasma.