WO1991008303A1

WO1991008303A1 - Antibody against human double-stranded tissue plasminogen activator

Info

Publication number: WO1991008303A1
Application number: PCT/JP1990/001543
Authority: WO
Inventors: Toshinobu Murakami; Yataro Ichikawa; Yoichi Sakata
Original assignee: Teijin Limited
Priority date: 1989-11-28
Filing date: 1990-11-28
Publication date: 1991-06-13

Abstract

A monoclonal antibody which selectively combines with a human double-stranded tissue plasminogen activator (2-chtPA) but not with a human single-stranded tissue plasminogen activator (1-chtPA) substantially. The use of this novel antibody makes it possible to separate and recover 1-chtPA or 2-chtPA from a solution containing both of 1-chtPA and 2-chtPA or a solution containing 2-chtPA readily and rapidly at a high purity and a good efficiency, and to conduct immunoassay of 2-chtPA in a specimen in a simple manner.

Description

Specification

Antibodies to human double-chain tissue plasminogen activator

The present invention relates to a monoclonal antibody that selectively binds to human double-chain tissue plasminogen activator (hereinafter referred to as 52-cht PA). A monoclonal antibody that does not substantially bind to genactivator 1 (hereinafter referred to as 1-cht PA) and selectively binds to 2-cht PA; a hybridoma that produces the antibody; The present invention relates to a method for separating or recovering 1-cht PA or 2-cht PA using the method, and a method for immunologically measuring 2-cht PA in Atsushi samples using the antibody.

Background art

Human tissue plasminogen activator (tPA) is a fibrinolytic enzyme that converts plasminogen activator to 5-plasmin, a fibrinolytic enzyme.

The purification method of tPA has been reported by Rijken D. C. et al. [Rijken D. C .; et al. J. Biol. Chem., 256, 7035—704.

1 (1981)], its structure, properties, etc. are also analyzed.

This tPA is a type of serine protease, and is composed of plasminogen 0 activator inhibitor 1 (Plasminogen activator inhibitor-

1, hereinafter abbreviated as “PA I-l”) and is known to lose its activity to convert plasminogen to plasmin quickly [Levin. EG Proc. Natl. Acad. Sci. USA, 80, 6804—68 08 (1986), Philips M. et al. Biochem. Biophis. Acta. 80 2, 99-110 (1984)].

In addition, 1? Is a single-chain type 1-1 (1-cht PA) and a light chain with an active center (Light chain; molecular weight) formed by hydrolysis of a specific peptide bond of 1-cht PA. Approximately 33,000 (hereinafter abbreviated as "L chain") and heavy chain (Heavy chain; molecular weight of about 35,000, hereinafter abbreviated as "H chain"). There are two types, PA (2-cht PA), and both types are known to have enzymatic activity

[See Ranby M. et al. Thromb. Res., 27, 175-183 (1982)].

At present, tPA is being clinically applied as a new thrombolytic agent. Regarding thrombolytic therapy, most opinion is that 11 cht PA is good, but it is still a matter of debate whether 1 cht PA is good or 2 cht PA is good. I have. However, it is impossible to separate and purify the 11-cht PA and 2-cht PA used in this method by the conventional method [Rijken DC; et al. J. Biol. Chem., 256, 7035-7041 (1981)]. The operation is very complicated and economical and time-consuming. In the field of basic research on tPA, the conventional method is completely 1-ch t? 8 and 2-. At present, research is being carried out using a mixture of some components because PA cannot be separated. Thus, if it is possible to separate and purify 11-cht PA and 2-cht PA with high purity and efficiency, it will be very important in the fields of basic medicine and clinical medicine.

On the other hand, a large number of monoclonal antibodies to tPA have been reported to date, such as binding to only one cht PA. , Blood, 69, No. 1, 284-289 (1987)] and an antibody that binds to both cht PA and 2-cht PA [Yumiko Takada; et al. , Thrombos. Res., _, 63-72 (1986)]. However, a monoclonal antibody that binds only to 2-cht PA and does not substantially bind to 1-cht PA has not been reported to date.

As a result of intensive studies, the present inventors have succeeded in creating a new monoclonal antibody that does not substantially bind to 1-cht PA but selectively binds to 2-cht PA. If antibodies are used, 1-cht PA or 2-cht PA can be separated from liquids containing 2-cht PA and liquids containing 1-cht PA and 2-cht PA with high purity, efficiently, easily and quickly. The present inventors have found that they can be recovered and that they can easily and immunologically measure 2-cht PA in an Atssey sample by using the monoclonal antibody, thereby completing the present invention.

Disclosure of the invention

Thus, according to one aspect of the present invention, it does not substantially bind to human single-chain tissue plasminogen activator and selects against human double-chain tissue plasminogen activator. A monoclonal antibody that binds specifically (hereinafter, referred to as the monoclonal antibody of the present invention) is provided. According to another aspect of the present invention, there is provided a method for separating or recovering 2-chtPA from a liquid containing 2-chtPA,

(i) contacting the liquid with an insoluble carrier to which the monoclonal antibody of the present invention is immobilized in order to adsorb 2-cht PA to the carrier,

(ii) separating the carrier from the liquid, Elute the desired 2-cht PA from the carrier,

Gv) Retrieve it

And a method for separating and recovering PA.

According to another embodiment of the present invention, in a method for separating and recovering 1-cht PA from a liquid containing 1-cht PA and 2-cht PA 5, (;) 2-cht PA as a carrier For adsorption, the liquid is brought into contact with an insoluble carrier to which the monoclonal antibody of the present invention is immobilized,

(ii) Separating a liquid containing 11 ch htPA that is not adsorbed on the insoluble carrier,

t o Recover the desired 11-ch t PA from the liquid,

(w) If necessary, elute 2-cht PA from the carrier,

(v) retrieve it

1—c ht A method for separating and recovering PA is provided.

According to yet another embodiment of the present invention, the immunization of 2-cht PA in an Atsey sample comprises contacting the first antibody 15 immobilized on an insoluble carrier and a labeled second antibody with an Atsey sample. One of the first antibody and the second antibody is the monoclonal antibody of the present invention, and the other specifically recognizes an antigenic determinant site different from the monoclonal antibody of the present invention; and The present invention provides an immunological assay method for 2-cht PA, which is characterized in that the antibody has the property of binding to 2-cht PA.

According to still another embodiment of the present invention, there is provided a reagent system for immunological measurement of 2-cht PA in an Atssey sample, the reagent system comprising a first antibody immobilized on an insoluble carrier and a labeled second antibody. One of the first antibody and the second antibody is the monoclonal antibody of the present invention, and the other is the monoclonal antibody of the present invention. 2-cht characterized in that it is an antibody that specifically recognizes different antigenic determinants and binds to 2-cht PA.

An immunoassay reagent system for PA is provided.

Hereinafter, the present invention will be described in more detail.

The monoclonal antibodies of the present invention have the basic binding property of not substantially binding to 1_ctPA and selectively binding to 2-ctPA. Further, as a further binding property, it has a property that it does not bind to any of the complex of 1-chtPA and PAI-1 and the complex of 2-chtPA and PAI-1.

Further, the monoclonal antibodies of the present invention can have at least one of the properties described below.

(a) the monoclonal antibody of the present invention generally have a 8x l ^O-8 M or less usually ^{^{1 X 10- 9 ~8 X 10- 8}} M dissociation constant for 2-cht PA in the range of (Kd) it can.

In this specification, the dissociation constant (Kd) of the monoclonal antibody was determined as follows according to the antigen immobilization method [Mark EF, et al. Mol. Immunol., 丄, 101-106 (1979)]. . The antigen was diluted to 5 μg / m with 0.1 M sodium carbonate (pH 9.5), placed in a 96-well polystyrene plastic plate at 100 ^ Z-well, placed at 4 ° C overnight, and coated. Wash the antigen solution with 1% BSAZ 0.1 M sodium carbonate (pH 9.5) at 170 Z-well and block at 37 ° (: 2 hours. Wash solution (0.02% Tween20 / 0.15 MNaCl 2 OmM Tris · HC 1 (pH 7.4)) After washing three times at 170〃 / ゥ, the antibody against ^125- labeled 2-cht PA was 0 to 4 g / e. Was diluted with a 0.1% B SAZ washing solution, added in a 100-z Nowell, and reacted at 37 ° C. for 2 hours. After washing four times with the washing solution, measure the remaining force in each well. From the value, calculate the amount bound to the antigen and the amount not bound to the ¹²⁵ I-labeled antibody added to the well. This value was applied to the equation described in the above document to determine Kd.

In the present specification, the term "antibody does not bind to antigen" refers to the dissociation constant of the antibody for antigen (Kd) is 10 ⁶ or more.

(b) The monoclonal antibody of the present invention inhibits the amide hydrolytic activity of 2-chtPA using the synthetic substrate S2288 by at least 30%, usually within the range of 35 to 50%.

In this specification, the inhibition rate of amide hydrolytic activity was determined as follows.

Antibody diluted with buffer (0.02% B SAZ0.01% Tween80 / TB S (Ph 7.4)) and antigen 1 g / I ^ are mixed in equal volumes to 300 gZm, and left at 4 ° C overnight did. To this reaction mixture 20 was added 100 / ^ of a synthetic substrate S2288 (H-D-isoloucyl-L-prolyl-L-arginme-P-nitroanilide dihydrochloride) diluted to 150 µgZm with the above buffer. After coloring this at 37 ° C for 3-4 hours,

The absorbance at a wavelength of 405 nm was measured. It was determined from the following equation.

405 nm absorbance in the presence of antibody: E i

2—cht PA Absorbance at 405 nm only for Ft: Ft

― P

Inhibition rate (%) = X 100

(c) The monoclonal antibody of the present invention inhibits the complex formation reaction between 2-cht PA and PA I-1 by at least 20%, usually within the range of 25 to 50%. I do. Here, the inhibition rate of the complex formation reaction was determined as follows.

2— c h t PA l / z gZi ^ and 0, 4.68, 18.75, 75, 300

抗体 Mix an equal amount of antibody against 2-cht PA diluted with buffer (0.02% BSA / 0.0 1% Tween80 / TBS (pH 7.4)) to a g / m concentration. Left overnight at 4 ° C. To this mixture 5 // was added 59 cgZm of active PAI-140, and the mixture was reacted at 37 ° C for 30 minutes. After mixing the above buffer solution with the above-mentioned buffer solution, the amount of 2-chtPA.PAI-1 complex in the reaction solution was measured by ELISA and determined by the following equation. Complex amount in the presence of antibody [moles]: E ₂

2 ch—t PA Amount of complex only [mol]: F 2

F ₂ -E ₂

Inhibition rate (%) = X 100

F ₂ (d) The monoclonal antibody of the present invention promotes the plasminogen activating activity of 2-cht PA at least twice, usually about 2 to 5 times, in the absence of BrCN-degraded fibrinogen. Have the ability. Here, the plasminogen activating activity of 2-cht PA was determined as follows. Antibodies to 2-cht PA diluted with a buffer (0.02% B SAZ0.0 1% Tween80 / TBS (pH 7.4)) to 300 〃gZm and 1β gZm 2-cht PA in equal volumes They were mixed and left at 4 ° C for one hour. Synthetic substrate solution 100 [1 87.5 上記 gZn ^ S 2251 (H-D -valyl- L -1 eucyl-isocyanate ysine- P-nitroanilide · dihydrochloride) / 22 fi g / m £ ~ f rasminogen] and color development at 37 ° C for 60 minutes. The absorbance at 405 nm was measured, and it was calculated from the following formula. . Absorbance of 405 in the presence of antibody: E 3

405 Absorbance of 5 2 ch—t PA only: F ₃

E ₃

Promotion effect (times) = one

F ₃

(e) the monoclonal antibody of the present invention further has a plasminogen activating activity of 2-cht PA in the presence of BrCN-degraded fibrinogen of at least 10%, usually in the range of 30-60%. Inhibits. In general, the monoclonal antibody of the present invention having the characteristics described above has a heavy chain class of y! And the class of the light chain can be κ. The monoclonal antibody of the present invention is preferably an intact antibody, but if it substantially possesses the antibody characteristics as described above, a fragment thereof is decomposed, for example, the antibody F of Univalent. ab, Fab ', (Fab') _{2 and the} like. Thus, in the following description, it should be understood that the expression antibody, unless otherwise stated, includes not only the intact antibody but also fragments containing at least the Fab region thereof. . The monoclonal antibody of the present invention can be prepared, for example, by adjusting 2-cht PA by the method of Rijken et al. [Rijken 20 DC et al .; J. Biol. Chem., 256, 7036 to 7041 (1984)], and Kohler and Melstein. Of the hybridoma cells obtained by the method [Kohler and Milstein; Nature, 256, 495-497 (1975)] Do Can be.

Hereinafter, the method for producing a hybridoma producing the monoclonal antibody of the present invention will be described in more detail.

(A) Isolation and purification of antigen

For 2-cht PA and 1-cht PA used for antigen and screening, use the method of Rijken et al. [See Rijken DC et al., J. Biol. Chem., 256, 7035-7041 (1981)]. Can be separated and purified from the culture supernatant of human melanoma cells as follows. The culture supernatant of human melanoma cells is applied to a Zinc chelate agarose column equilibrated with 0.01% Tween80 / 1 M aC 1/2 OmM TrisHC1 (pH 7.5), and then adsorbed to the column. Elute PA with OmM imidazole. After applying this tPA to a concanavalin A agarose column equilibrated with 0.01% Tween 80/1 M NaC 1/1 OmM sodium phosphate (pH 7.5), the tPA adsorbed to the column was reduced to 0.4 M α-D Elution with -methylmannoside / 2 M KSCN. The tPA is subjected to gel filtration using a Sephadex G-150 column to increase the purity. This tPA is applied to a Sepharose 4B column on which an anti-tPA monoclonal antibody is immobilized, and tPA is eluted from the column with 3M NaSCN. The thus obtained tPA contains one cht PA in about 90% or more purity and is used as a one cht PA antigen. The 1-cht PA was immobilized with plasmin and passed through an S-marked harose4 B column to convert the 1-cht PA to 2-cht PA. It contains 2-cht PA at a purity of about 80% or more and is used as a 2-cht PA antigen. (B) Immunization of mammals with 2-cht PA:

The animal used for immunization is not particularly limited, and various mammals, for example, mice, rats, guinea pigs, egrets, sheep, sheep, goats, dogs, cats, and the like can be used, but are easy to handle. Generally for reasons such as

Female BalbZc mice are used, but mice of other strains can be used. The concentration of 2-chtPA used in the immunization scheme and immunization should be selected so that a sufficient amount of antigen-stimulated lymphocytes is formed. For example, mice are immunized intraperitoneally several times at a certain interval with a small amount of 2-chtPA, and then administered several times intravenously to raise antibody titers. More specifically, for example, mice are immunized three times into the peritoneal cavity at 2-week intervals with 50 μg of 2-—chtPA, and then, another 30 // g is intravenously administered.

Several days after the final immunization, antibody-producing cells, eg, lymphocytes, preferably spleen cells, are removed from the immunized animal for fusion. Hereinafter, the case where spleen cells are used as antibody-producing cells will be described. However, it should be understood that antibody-producing cells other than spleen cells can be used for cell fusion similarly.

(C) Cell fusion:

The spleen is aseptically removed and a single cell suspension is prepared. The spleen cells are fused with myeloma cells from a suitable cell line in a fusion medium using a suitable fusion promoter. The myeloma cells used for the fusion may be from any mammal, but generally those derived from the same species of animal used for the immunization are preferred. The preferred mixing ratio of spleen cells to myeloma cells when fusing is generally in the range of about 20: 1 to about 2: 1, preferably 10: 1 to 2: 1. About 1 0 ⁸ spleen cells with usually, from 0.5 to 1.5 Use of ιη fusion medium is appropriate. As a fusion medium For this purpose, it is appropriate to use physiological saline, a physiological saline buffer solution, a serum-free medium or the like containing a fusion promoter at a concentration of 30 to 70%.

Many bone marrow cells used for cell fusion are known, but in the examples described below, P3-X63-Ag8-U1 cells (hereinafter abbreviated as P3-U1) [Yelton, 5 DE et al., Current Topics in Microbiology and Immunology, 81, 1 (1978)]. This is an 8-azaguanine resistant cell line that lacks the enzymes hypoxanthine-guanine phosphoribosyltransferase and therefore cannot survive in HAT (hypoxanthine, aminobuterin, thymidine) medium . This cell line is a non-secretory type that does not itself secrete antibodies, and is therefore suitable for the production of hybridomas, which is the object of the present invention. However, other myeloma cells, such as P3-NS1-1-Ag4-1, NS1-Ag4Zl, P3-X63-Ag8, (MPC11-45, 6. TGI.7), SP2 / 0-Agl 4 FO, X-63— Ag8— 6.5.3, 2 is 10. RC Y3. Ag 1.2.3, S194 Z5XX O. B U.1, SKO— 007, GM15006 TG— A12, etc. It is possible.

As a preferred fusion accelerator, for example, polyethylene glycol having an average molecular weight of 1,000 to 4,000 can be advantageously used, but other fusion accelerators known in the art, such as Sendai virus, can also be used. 0 Yes. In Examples described later, polyethylene glycol having an average molecular weight of 1,540 was used.

(D) Selection of fused cells:

A mixture of unfused spleen cells, unfused myeloma cells and fused cells in a separate container (eg, microtiter plate) Dilute with a selective medium in which the cells cannot survive, and incubate for a time sufficient to kill unfused cells (about 1 week). The medium used is such that unfused myeloma cells cannot survive (for example, the HAT medium described above). Unfused myeloma cells die in this selective medium. Unfused spleen cells are non-neoplastic cells and die after a certain period of time (about one week). Cells fused to these cells can survive in the selective medium due to the combination of the neoplastic properties of the parental cells of myeloma and the properties of parental spleen cells.

(E) Confirmation of antibody against 2—c ht PA in each container:

After the detection of the hybridoma cells, the culture supernatant was collected and subjected to enzyme immunoassay for antibodies to 2-cht PA (Enzyme Linked Immuno Sorbent Assay) [eg, Schuurs, AHWM and van Weenie n, BK : See Clin. Chim. Acta, 81, 1—40 (1977)]

(F) Cloning of hybridomas producing monoclonal antibodies against the desired 2—cht PA:

The hybridoma producing the desired antibody is cloned by an appropriate method (eg, limiting dilution method).

An example of a hybridoma that can produce the monoclonal antibody of the present invention produced by the method described above includes FERM BP-3109, which was deposited under the Budapest Treaty with the Institute of Microbial Industry and Technology of the Ministry of Industry and Technology. Hypri-Dorma J TA-T, which is given the number However, the hybridoma producing the monoclonal antibody of the present invention is not limited to the above-mentioned JTA-T as long as it produces a monoclonal antibody having the above-mentioned properties, and other substances can be used.

(G) Preparation of the monoclonal antibody of the present invention For the monoclonal antibody produced by the hybridoma, the clone is grown in the abdominal cavity of a BalbZ C mouse, and the monoclonal antibody is purified from the ascites using ProteinA-Sepharose4B. The purification method is as follows. A buffer (3M NaC 1 / 1.5M glycine) in 5m of ascites

5 (pH 8.9)) and centrifuge at 3000 rpm for 5 minutes. Did the supernatant equilibrate with eight buffers? 1 "016 ^ 8-56 1131" 05648 (5 mi) slowly and slowly. Then, wash well with buffer A and elute the monoclonal antibody with buffer B (0.1 M citric acid (pH 3.0)). The eluted monoclonal antibody was immediately adjusted to pH 7 with 3M TrisHC1.

After 10 minutes, add 1 OmM sodium phosphate 0.15 M sodium chloride (pH 7.

4) Thoroughly dialyze to purify the monoclonal antibody.

The monoclonal antibody of the present invention prepared as described above does not substantially bind to 1-cht PA, but has the property of selectively binding to 2-cht PA. 2-cht PA can be separated or recovered from a liquid is-containing body containing 2-cht PA.

The separation and recovery of the 2-chtPA can be carried out, for example, as described below using an insoluble carrier having the monoclonal antibody of the present invention immobilized thereon as a selective adsorbent.

The monoclonal antibody of the present invention is chemically bound to a suitable insoluble carrier (for example, Sepharose), packed in a column, and equilibrated with a suitable buffer solution (for example, 50 mM Tris buffer pH 7.4, 0.15 M NaCl). Become A liquid containing 2-cht PA to be treated (for example, human plasma or human serum) is added to the adsorbent to adsorb 2-cht PA in the liquid containing 2-cht PA. Then use a suitable washing solution (eg, 5 OmM Tris buffer). Impurities are removed from the adsorbent by a buffer, pH 7.4, 0.15 MNaCl). Next, the amount of 2-cht PA in the "flow-through fraction" and the "wash fraction" of the liquid is measured. Thus, the degree of separation of human 2-cht PA from the human 2-cht PA-containing liquid can be calculated from the value.

5 As the insoluble carrier used for the selective adsorbent in the present invention, various ones can be used.For example, agarose, polyacrylamide, cellulose, dextran, or maleic acid polymer or a mixture thereof is preferably used as a material. Can be Examples of the shape of these insoluble carriers include powders, granules, pellets, beads, films, and fibers.

It can be in 10 different forms.

Immobilization of a monoclonal antibody or a fragment thereof to these insoluble carriers is generally performed by chemically binding the monoclonal antibody or a fragment thereof to the insoluble carrier. For example, it is effectively performed by sepharose activated by CNBr treatment and an antibody immobilized thereon [R. Axea et al; Nature, 214, 1302-1304 (1967)].

As described above, if the adsorbent that has adsorbed 2-ChtPA by contact with a 2-ChtPA-containing liquid is separated from the liquid, 2-ChtPA in the liquid is removed. As a result, when human plasma or human serum is used as the liquid, human plasma or human blood substantially free of 2-chtPA is used.

20 serum is obtained and such 2-ch PA free human plasma or human serum can be advantageously used, for example, for plasma exchange or serum exchange therapy. On the other hand, the adsorbent adsorbing 2-cht PA separated from the liquid can be subjected to an elution treatment to elute 2-cht PA from the adsorbent and recover 2-cht PA. The recovered 2— cht PA is an example For example, it can be used as a thrombolytic agent or the like.

The above-mentioned elution treatment can be performed by treating the adsorbent having adsorbed 2-cht PA with an eluent. As an eluent that can be used, an aqueous solution of ethylene glycol having a pH of 2.5 to 12.5, preferably 4.0 to 9.0 is advantageously used. In addition, an aqueous solution of glycerin, an aqueous solution of glycine, an aqueous solution of propionic acid, an aqueous solution of thiocyanate, and an aqueous solution of guanidine are used. Etc. can also be used.

The concentration of sodium thiocyanate in the aqueous solution of sodium thiocyanate is 0.5 M to 6 M, preferably 2 M to 4 M. In addition, this aqueous solution may be appropriately adjusted to adjust the pH, such as sodium hydroxide, hydroxide such as hydroxide, salts such as Tris salt, phosphate, veronal salt, hydrochloric acid, sulfuric acid, nitric acid, acetic acid, and the like. Acids such as citric acid and oxalic acid, amines such as ethanolamine, ammonia, and urea can be contained. The elution treatment is carried out at a temperature between the freezing point and 37 ° C, preferably 2-10 ° C. The elution is performed by a column method, batch method, etc., and the time required for elution should be short.

Separation and purification of 2-chtPA from the eluate containing the eluted 2-chtPA can be performed by a method known per se, for example, dialysis, concentrated liquid chromatography, and the like.

Further, by using the selective adsorbent comprising the insoluble carrier to which the monoclonal antibody of the present invention is immobilized, it is possible to separate or recover 11-cht PA from a liquid containing 1-cht PA and 2-cht PA. Becomes possible. For example, passing a liquid containing 11 cht PA and 2-cht PA through a column containing the selective adsorbent and equilibrated as described above. By selectively adsorbing 2-cht PA to the column, an eluate from which 2-cht PA has been substantially removed can be obtained.

As a result, 2-chtPA can be completely separated and removed from a liquid containing both 1-chtPA and 2-chtPA, for example, cell culture supernatant, blood, and plasma. The liquid from which 2-cht PA has been removed in this manner may be further processed by a method known per se, such as dialysis, concentration, or liquid chromatography, if desired, to recover purified 1-cht PA. be able to.

On the other hand, if necessary, the column adsorbed with 2-cht PA can be treated as described above to elute 2-ch PA from the column and recover it.

By the method described above, 2-cht PA can be virtually completely separated from 1-cht PA, which has been considered difficult to separate, so that 2-cht PA-free 1-cht PA can be obtained. Can be provided. This is2-chtPA free 1_chtPA can be used, for example, as a thrombolytic agent.

Furthermore, the use of the monoclonal antibody of the present invention makes it possible to directly and accurately quantify 2-chtPA in Atsushi samples by the so-called "sandwich method".

20 In general, a method for measuring the presence or absence or the amount of an antigen using an antibody that binds to two different sites of the antigen is referred to as a “sandwich method”. For example, the “radioimmunoassay (fide)” Radioimunoassay Methods) J 199-206 (1970).

The immunological assay method of the present invention uses two kinds of antibodies (a primary antibody and a secondary antibody). Antibodies) that specifically recognize and bind to different antigen-recognition sites of 2-cht PA. In particular, one of them does not bind to 1-cht PA and 2-cht PA The present invention uses the monoclonal antibody of the present invention which can selectively bind to the antibody. Thus, according to the present invention, it is possible to constantly and accurately measure 2-cht PA in a solution state (for example, in plasma) without any difference in the _five qualities of the reagents. In addition, since 2-cht PA is measured directly, 2-cht PA can be measured accurately and in a short time without being affected by other crowds. Therefore, according to the present invention, there is provided a method capable of accurately and rapidly measuring 2-cht PA, which did not previously exist.

In the method of the present invention, one of the above two antibodies (primary antibody) is immobilized on an insoluble carrier, and the other (secondary antibody) is labeled and used. Monoclonal antibodies of the present invention may be used as the primary antibody fixed to an insoluble carrier, some have may be used as labels to secondary antibody, either way, the measurement of ₅ 2-cht PA obtained The result is not substantially affected.

Other anti-tPA antibodies used in combination with one of the monoclonal antibodies of the present invention are those of the present invention that specifically recognize and bind to an antigenic determinant site different from the one described above. Should be fine. Antibodies that bind to tPA, which are conventionally known, namely, 1-chtPA and 2-chtP. Those that bind to both A can be used in combination with the monoclonal antibody of the present invention. They can be monoclonal or polyclonal. Monoclonal antibodies that bind to both 1-cht PA and 2-cht PA include, for example, the monoclonal antibody J TA-1 (FERM BP-2316) described in EP-A-0339302. And JTA-4 (FERMP-10402).

The immobilization of the primary antibody on the insoluble carrier can be carried out by a method known per se, for example, a method in which the primary antibody solution is brought into contact with the insoluble carrier and allowed to stand for physical adsorption, or the antibody is constituted. It can be carried out by a method in which a functional group such as a carboxyl group, an amino group or a hydroxyl group of an amino acid to be chemically bonded to an insoluble carrier. The carrier to which the primary antibody has been immobilized in this manner is preferably coated on the surface of the insoluble carrier with a suitable substance (for example, bovine serum albumin) to avoid nonspecific binding to the secondary antibody or the sample to be measured. Cover. Examples of the insoluble carrier used for immobilizing the primary antibody include polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, nitrocellulose, cross-linked dextran, polysaccharide, and agarose. Examples include substances, inorganic substances such as glass and metals, and combinations thereof. Examples of the shape of the insoluble carrier include trays, spheres, fibrous is, granules, beads, rods, plates, and containers. And various shapes such as a cell shape and a test tube, and specific examples include a plastic container, plastic beads, glass beads, and metal particles.

On the other hand, secondary antibodies are labeled with radioactive substances, enzymes or fluorescent substances. Etc. Examples of the radioactive substance for example ^{^{1 25 I, 1 31 I,}} 1 4 C, 3 H and the like, as the enzyme 20, for example, alkaline phosphatase, Paokishidaze, such as β one D- galactosidase is included, also Examples of the fluorescent substance include fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, and the like, but these are merely examples, and those that are used in the immunoassay method may be used. If other labeling substances are used it can. Binding of these labeling substances to the secondary antibody can be performed by a method known per se, for example, GS David; Biochem. Biophys. Res. Co. π., 48, 46 4-471 (1972), Imagawa et al., Anal. Lett. , J_6, 1509-1523 (1983) and Nishioka et al., Cancer Res., 3_2, 162-1665 (1972).

The immobilized primary antibody and the labeled secondary antibody described above are contacted with an analyte sample using 2-ch h PA measurement. The contact method can be a two-step method in which the immobilized primary antibody is first brought into contact with the sample and then with the labeled secondary antibody, or the sample is contacted with the primary antibody and the sample simultaneously with the secondary antibody. Any one-step method can be used, but the latter one-step method is advantageous because 2-cht PA can be measured in a short time and easily.

In the two-step method, first, an immobilized primary antibody is brought into contact with a sample for a certain period of time and at a predetermined temperature for reaction. During this time, the immobilized primary antibody and 2—chtPA in the sample sample are bound. Then, after washing with an appropriate washing solution, a solution containing the labeled secondary antibody (for example, contact with an aqueous solution for a certain period of time and at a temperature to react with the secondary antibody. This is washed with an appropriate washing solution and then insoluble. Measure the amount of labeled substance on the secondary antibody present on the carrier, and compare that value to the calibration curve generated using a sample containing 20-cht PA at a known concentration. By doing so, the amount of 2-cht PA in the sample can be determined.

In the one-step method, the immobilized primary antibody is brought into contact with the sample sample and the labeled secondary antibody at the same time, preferably with a mixture of the sample sample and the labeled secondary antibody for a certain period of time and at a certain temperature. And react. Then appropriate After washing with the washing solution, the amount of the 2-cht PA in the sample can be determined by measuring the amount of the labeling substance labeled on the secondary antibody present on the insoluble carrier in the same manner as described above.

According to the method described above, 2-cht PA in a sample can be measured accurately and easily with reproducibility. Samples that can be measured by this method include human plasma, human serum, and cell culture. Supernatants and the like can be mentioned. In carrying out the above method, the present invention provides a reagent system comprising the insoluble carrier on which the above-mentioned primary antibody is immobilized and the above-mentioned labeled secondary antibody. This reagent system can form a kit containing various auxiliary agents in addition to these antibodies, in order to make the system efficient and easy to use. Such adjuvants include, for example, a solubilizer for dissolving a solid secondary antibody, a detergent used for washing an insolubilized carrier, and an enzyme activity when an enzyme is used as a labeling substance for an antibody. As a kit for an immunological measurement reagent such as a substrate for measurement and a reaction terminator thereof, there can be mentioned those usually used as kits.

Currently, tPA is being clinically applied as a new thrombolytic agent. However, these studies require that tPA be administered in sustained large doses, similar to perokinase, which is currently used as a thrombolytic agent. This may be because the plasminogen activating activity of tPA is strong in the presence of fibrin, but very weak in the absence of fibrin. If the efficiency of the plasminogen activating activity of tPA can be increased, the dose of tPA can be reduced, and the adverse effect of a large dose can be eliminated. Further, in the present invention, the monoclonal antibody of the present invention comprises 2-cht The finding that PA has the property of promoting the plasminogen activating activity enables the binding product of the monoclonal antibody of the present invention to 2-cht PA to be used as a thrombolytic agent. There is. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of examining the ability of JTA-T to bind to 1-chtPA or 2-chtPA in Example 2 using an antigen immobilization technique.

FIG. 2 is a calibration curve of 2-chtPA used in Example 4.

FIG. 3 shows that, in Example 5, a column using TA-T as an immunoadsorbent was coated with tPA containing a mixture of 11-cht PA and 2-cht PA, and the non-adsorbed fraction and the adsorbed fraction were analyzed. After reduction with 1-mercaptoethanol, 9% SDS-polyacrylamide gel electrophoresis was performed, and the gel was stained with silver.

FIG. 4 shows the results of a study in Example 6 on the effect of JTA-T on the amide hydrolytic activity of 2-chtPA using the synthetic substrate S2288.

Fig. 5 shows the effect of JTA-T on the formation of a complex of 2-cht PA with PA I-1 in Example 7, which was examined using a tPA · PAI-1 complex measurement enzyme. The result.

6 and 7 show the results of examining the effects of JTA-T on the plasminogen activation activity of 2-chtPA in Examples 8 and 9 using the synthetic substrate S2251.

FIG. 8 shows the activation of plasminogen to plasmin by 2-cht PA in the presence of JTA-T in Example 11, using plasminogen labeled with ¹²⁵ I, reducing with I-mercaptoethanol, 9% SD s—The band when subjected to autoradiography after polyacrylamide gel electrophoresis.

FIG. 9 is a graph showing the results of measurement of the effect of 6-amino-n-bronic acid pentanoic acid on the effect of JTA-T on the activation of plasminogen activation of 2-cht PA by the synthetic substrate S 2251 in Example 12. is there. Embodiments The present invention will be described more specifically below.

Example 1 (Acquisition of monoclonal antibody)

For 2-cht PA and 1-cht PA10 used for antigen and screening, use the method of Rijken et al. [See Rijken DC et al .; Biol. Chem., 256, 70 35-7041 (1981)]. Then, it was separated and purified from the culture supernatant of human melanoma cells as follows. The culture supernatant of human melanoma cells was applied to a Zinc chelate-agarose force column equilibrated with 0.0 l% Tween80Z1M NaC1 / 2OmM Tris · HC1 (ph 7.5) and then adsorbed to the column. tPA was eluted with 5 OmM imidazole. The tPA was applied to a concanavalin A- agarose column equilibrated with 0.0 l% Tween80Z1M NaC1 / 1 OmM sodium phosphate (ph 7.5), and the tPA adsorbed to the column was reduced to 0.4 M Eluted with methy lmannoside / 2 M KSCN. The tPA was subjected to gel filtration on a S-marked hadex G20-150 column to increase purity. This tPA was applied to a Sepharose 4B column on which an anti-one cht PA monoclonal antibody was immobilized, and tPA was eluted from the column with 3M NaSCN. The thus obtained tPA contained 1-cht PA at a purity of about 90% or more and was used as a 1-cht PA antigen. Also, this 1 cht PA The 1-cht PA was converted to 2-cht PA by immobilizing Sumin and passing through a Sepharose 4B column. Two 2-cht PAs thus obtained were immunized 5 times at intervals of 14 to 2 female BalbZC mice (4 weeks old). The first immunization was performed by adding 2〃1121 118 of 15〃8 dissolved in 0.1 M Lysin / 0.9% NaC1 / 0.01% Tween80 (pH 3.0) to Freund's Complete adjuvant (Freund's Complete) in an equal volume of Freund's. adjuvant) and the emulzine was administered intraperitoneally (0. Sn ^ Zhead ^ 2nd to 4th times, 10〃g of 2-cht PA was added to Freund's incomplete adjuvant). The final immunization was performed by dissolving 10 g of 2-cht PA inactivated with diisopropyl fluorophosphate in 50 mL of PBS solution and administering the solution through the tail vein of mice. Spleen cells of mice that were subsequently immunized were prepared and used for cell fusion.

Spleen cells of immunized mice and myeloma cells (P3U1) of syngeneic mice were mixed at a ratio of about 2: 1 to about 15: 1, and 50% polyethylene glycol 1540 was used as a fusion promoter with KOhler] (ilstein's The cells were fused according to the following procedure: The cells after the fusion were suspended in 10% FCS 'RPMI-1640 medium to a cell concentration of 1 × 10 ⁶ cells / m, and placed in a 96-well microplate (Coster). 100 Dispensed 100 per aell.

The fused cells were cultured in C0 ₂ incubator _{(5% C0 2, 37 °} C), hypoxanthine; aminopterin: thymidine the medium was replaced with medium (HAT culture locations) including, grown in HAT medium, Screening was performed on hybridomas consisting of spleen cells and myeloma cells.

Antibodies in the culture supernatant of the hybridoma are detected using the antigen (2-cht PA Alternatively, ELISA was performed by a double antibody method using a microtiter plate coated with 11-cht PA). As a secondary antibody, horseradish peroxidase (hereinafter abbreviated as HRP) -labeled goat anti-mouse IgG antibody was used.

Among them, the cloning by limiting dilution was repeated twice for a hypolipoma of Pell, which was positive for 2-chtPA and not positive for 1-chtPA, to obtain one clone. The monoclonal antibody produced from this clone was named JTA-T. The hybridoma that produces this monoclonal antibody has been deposited with the Institute of Microbial Industry and Technology of the Agency of Industrial Science and Technology under the number FERM BP-3109 under the Budapest Treaty.

The resulting clone was stored in liquid nitrogen suspended in 90% FCS-10% DMS0. The monoclonal antibody produced by the clone was obtained by growing the clone in the abdominal cavity of a BalbZ C mouse and purifying the resulting ascites using a protein A-Sepharose 4B column.

Example 2 (Ability to bind to 1-cht PA or 2-cht PA in enzymimino atsee)

The binding ability of the obtained monoclonal antibody to 1-cht PA or 2-cht PA was assayed using the antigen immobilization method. Each antigen

The protein concentration was adjusted, added to polystyrene plastic gel, and coated overnight at 4 ° C. Wash the wells, block with 1% bovine serum albumin (hereinafter abbreviated as BSA) at 37 ° C for 2 hours, and prepare a monoclonal antibody (JTA-T) prepared at a protein concentration of 1.6 ng gZm to 5000 ng / n ^. ) At 37 ° C for 2 hours Warmed. After washing, the plate was incubated at 37 ° C for 2 hours with an HRP-labeled goat anti-mouse antibody. After washing, a peroxidase substrate solution was added, and after reacting at room temperature for 30 minutes, the absorbance was measured at a wavelength of 415 nm.

The results are shown in FIG. Even when the amount of the antibody used in the reaction was increased to 5 gZn ^, this antibody did not show binding to 1-ctPA. This indicates that JTA-T is a monoclonal antibody selective for 2-ChtPA that binds to 2-ChtPA and does not bind to 1-ChtPA.

Example 3 (Immunoglobulin class of monoclonal antibodies and dissociation constants for various antigens)

The immunoglobulin class of JTA-T was performed using a mouse monoclonal single-typing kit (The BINDING SITE LTD.). In addition, the dissociation constant (Kd) for 11-cht PA, 2-cht PA and their respective PA I-11 complexes was determined by the antigen immobilization method [Mark EF; et al. Mol. Immunol., 16, 101]. -106 (1979)]. Each antigen was diluted with 0.1 M sodium carbonate (ρΗ9.5) to 5j «gZm £, put into a 96-well polystyrene plastic plate at 100 ゥ -well, allowed to stand at 4 ° C overnight, and coated. 37. Add the antigen solution and add 1% BS AZ 0.1M sodium carbonate (pH 9.5) at 170 ° Z-well. C, blocking for 2 hours. Wash solution (0.02% Tween 20 0.15 M NaCl 2 OmM Tris · HC 1 (pH7.4)) 1Ί ϋ After washing 3 times with β-well, 0Τ4〃 g of ¹²⁵ I-labeled JΤΑ—Τ / m 2, diluted with 0.1% BSA / wash solution, added at 100 Z-well, and reacted at 37 ° C for 2 hours. After washing 4 times with washing solution, count remaining in each well Kd was determined. Table 1 shows the results.

Example 4 (2—cht PA immunological assay method)

JTA-T was coated on a polystyrene plastic well with a protein concentration of 10 g / me at 4 ° C overnight, and coated. The wells were washed and blocked with 1% 83-8 at 37 ° C for 2 hours. Wash the wells and mix 0, 4, 18, 90n / m ^ 2-cht PA solution and 100ng / ^ t PA concentration adjusted l-cht PA and 2-cht PA for the standard curve. Solution NO.;! 33 was placed in a well and kept at 37 ° C. for 2 hours. After washing, the amount of 2-cht PA remaining in the wells is determined using an HRP-labeled anti-tPA antibody (JTA-1) that reacts similarly to both single-chain tPA and double-chain tPA. Tested. A calibration curve (Fig. 2) was created from the measurement results of 2-cht PA for the calibration curve, and the amount of 2-cht PA contained in the mixed solutions No. 1 to 3 was measured using the calibration curve. The results are shown in Table 2.

Table 2

In order to confirm that the measurement of 2-cht PA using the antibody of the present invention was performed accurately, the above mixed solutions Nos. 1 to 3 were electrophoresed in a reduced state, and stained with Coomassie brilliant blue. The ratio of the amount of stained 1-cht PA to that of 2-cht PA was measured with a densitometer and compared with the ratio of 2-cht PA determined by ELISA.

, Table 3

From the above results, the ratio of 2-cht PA determined by electrophoresis and the ratio of 2-cht PA determined by ELISA were almost the same. Therefore, the ELISA system using the antibody of the present invention showed that cht PA and 2— cht P It was confirmed that only the amount of 2-cht PA in the mixed solution in which A was mixed could be accurately measured.

Example 5 (Separation and purification of 1-chtPA and 2-chtPA)

For separation and purification, 500 TA of JTA-T was immobilized on CNBr-activated Sepharose 4B 1.6m and used as an immunoadsorbent. JTA-T immobilized Sepharose 4B 1.6m is packed in a column (10 x 20 marauder), and A buffer (50 UZni aprotinin / ^ O.5 NaC1 / 0.01% Tween80 / 0.01M Lysin / 0.01M Tris (pH 7.4)).

To this column, a tPA sample (150 g of protein) mixed with 11-cht PA and 2-cht PA separated and purified from the culture supernatant of human melanose cells by the method described in Example 1 was used. It was added and left at 4 ° C for 8 hours. After washing with 6 m of A buffer and eluting non-adsorbed 1-cht PA, 2-cht PA adsorbed to the column with 1 A buffer containing 3N NaSCN was eluted. The non-adsorbed 1-cht PA and the adsorbed 2-cht PA on this column were reduced with / S-mercaptoethanol, and then subjected to SDS-polyacrylamide gel electrophoresis. Indicated. Here, 1 to 4 indicate the following. 1: t-PA mixed with 1-cht PA and 2-cht PA before being applied to the column, 2: 1-cht PA not adsorbed to the column, 3: 2-cht PA adsorbed to the column, 4: After reducing the molecular weight marker tPA and subjecting it to SDS-polyacrylamide gel electrophoresis, 1-cht PA is converted into one band with a molecular weight of 68000, and 2-cht PA is converted into two bands with a molecular weight of 33000 and 35000. Become a band. As shown in Figure 3, the tPA before applying to the column was calculated using the force JTA-T, which is a mixture of 1-cht PA and 2-cht PA, as the immunoadsorbent. It was confirmed that 1-cht PA containing no PA could be purified. In addition, it was confirmed that 2-cht PA applied to the column was eluted in the adsorption fraction of column 5.

From this, it is clear that 1-cht PA can be separated and purified as non-adsorbed tPA fraction and 2-cht PA as adsorbed tPA fraction by using JTA-T as an immunoadsorbent. It became.

Example 6 (Effect of JTA-T on Amide Hydrolysis Activity of Double-Stranded tPA) A buffer (0.02% BSA / 0.0 l%) was adjusted to 10 2 -cht PA Lytz g / m and 300〃 gZm. Mix JTA-T [or JTA-4 (anti-tPA monoclonal antibody), anti-tPA polyclonal antibody or normal mouse IgG] diluted with Tween80 ZT BS (pH 7.4)) in equal amounts. And left at 4 ° C. 100 Z of the synthetic substrate S2288 diluted to 50 gZ of 1 is with the above buffer was added to the reaction mixture 20. After color development at 37 ° C for 3 to 4 hours, the results of measuring the absorbance at a wavelength of 405 nm are shown in FIG. In the presence of JTA-T, the absorbance was reduced by about 40% compared to the case of 2-cht PA alone without IgG. This indicates that JTA-T inhibited the amide hydrolytic activity of 2-chtPA by about 40%.

Example 7 (Effect of JTA-IT on complex formation of 2-cht PA with PA I-1)

2—Cht PA Dilute with 1 gZn ^ buffer (0.02% BS l / T.0 l% Tween80 / TB S (pH 7.4)) to a suitable concentration. The diluted JTA-T (or anti-tPA polyclonal antibody, mouse IgG) was mixed in equal amounts and left at 4 ° C overnight. 59 ngm of active PAI-1 was added to 5 / of the reaction mixture, and the mixture was reacted at 37 ° C for 30 minutes. After the above buffer (115 1) was mixed with the reaction solution, the amount of tPA · PAI-1 complex at 5 in the reaction solution was measured by ELISA.

The results are shown in FIG. The vertical axis represents the concentration of the tPA · PAI-1 complex, and the horizontal axis represents the molar ratio between the reacted antibody and 2-chtPA. In JTA-T, the concentration of the tPA / PAI-1 complex decreased by about 30% as the amount of the mixed antibody increased. This shows that JTA-T inhibited the complex formation of 2-chP10A with PAI-1 by about 30%.

Example 8 (J T against the plasminogen activating activity of 2-cht PA

A—Effect of T①)

2— dilute J TA— T [or J TA— with 1 buffer (0.02% BSA / 0.0 l% Tween80 / TBS (pH 7.4)) so that cht PA 1 β g / n ^ and 300 z gZra 4 (anti-tPA monoclonal antibody), anti-tPA polyclonal antibody or normal mouse IgG] were mixed in equal amounts, and left to stand at 4 ° C. To this reaction mixture 20 was added a synthetic substrate solution 100 [181.5 g / m £ S 2251/22 / gZffl plasminogen] diluted with the above buffer, and the color was developed at 37 ° C for 60 minutes.

The absorbance at 20 405 nm was measured. The results are shown in FIG. As shown in Fig. 6, the plasminogen activating activity in the absence of BrCN-degraded fibrinogen showed a decrease in the absorbance when JTA-T was present compared to the case of 2-cht PA alone without IgG. Increased about 4 times. Thus, under these conditions, JTA-T increases the plasminogen activating activity of double-stranded tPA by about 4 times. It can be seen that it was promoted.

Example 9 (J T for the plasminogen-activating activity of 2--cht PA

A—Effect of T②)

2—Cht PA 1 gZm and J diluted with a buffer [0.02% BS AZ0.0.1% Tween80 / TBS (pH 7.4)] to make 0 to 300 0g〃πι An equal amount of TA-T [or anti-tPA polyclonal antibody or normal mouse IgG] was mixed and left at 4 ° C overnight. Synthetic substrate solution 100 «(18.7 7.5 β g / m S 2 25 1/2 2 ^ gZm Plasminogen 50 0 β / mio B diluted with the above buffer in this reaction mixture 15 r CN-decomposed fibrinogen) was added thereto, and the color was developed at 37 ° C for 30 minutes, and then the absorbance at 405 nm was measured. The results are shown in FIG. In the presence of BrCN-degraded fibrinogen in the presence of plasminogen, as shown in Fig. 7, the absorbance decreased by about 50% as the amount of JTA-T mixed antibody increased. did. This indicates that under these conditions, J TAis-I T inhibited the plasminogen activating activity of double-stranded tPA by about 50%.

Example 10 0 (2—c h t JTA for binding of PA to fibrin clots

T effect)

¹²⁵ 1 Labeled 2— cht PA 1 β g / a £ 50 / and J TA— T [or 0 or mouse IgG or buffer] 300 / gZm 50

Mix and leave at 4 overnight. The buffer used was 50 O UZm aprotinin Z0.01%, BSA / 0.01% Tween80 / TBS (pH 7.5). Mix the above reaction mixture 10 and 15 mg Zm fibrinogen 26.7 / buffer 163.3 ^, 0.1 MC a C 1 z 5 and 37 ° C The reaction was performed for 5 minutes. Thereafter, 5 〃 of 40 OU / m thrombin was added and reacted at 37 ° C for 5 minutes to form a fibrin clot. The fibrin mass was taken up with a bamboo skewer and washed with a buffer, and the amount of ¹²⁵ I-labeled 2-cht PA bound to the fibrin mass was measured with a gamma counter, and the results in Table 4 were obtained.

Table 4

From Table 4, it can be seen that about 40% of 2-cht PA normally binds to fibrin clots, but decreased to about 30% in the presence of JTA-T. This indicates that JTA-IT inhibited binding of 2-cht PA to fibrin clots by about 30%.

Example 11 (Activation of plasminogen to plasmin by 2-chtPA in the presence of JTA-T)

JTA—T, mouse IgG or buffer diluted with 1〃 gZm of 2-ch t PA and buffer (0.02% BS AZ0.01% Tween80 / TBS (pH7.5)) to 30 gZn ^ or 300 g / ni Each was mixed in an equal amount and left at 4 ° C. To the reaction mixture 50, a plasminogen one Gen labeled with 1 ²⁵ I about 1 Bruno 20 44 gZm including flop Rasminogen 50 was added and reacted at 37 ° C for 60 minutes. After reducing this sample with 3-mercaptoethanol, SDS-polyacrylamide gel electrophoresis was performed. After drying the gel, it was subjected to autoradiography, and the results are shown in FIG. Here, (1) to (6) indicate the following. (L) When J TA-T is added to tPA at 30〃g / ne! (T When JTA-T is added to tPA at 300〃g / m, (3) Mouse IgG is added to tPA 3 0 ng / me when added, (4) when the addition of mouse I gG with 300〃 g / m to t PA, (5) when addition of buffer to t PA, it was labeled with (6) ¹²⁵ 1 Plasminogen.

After reduction of plasminogen, it is subjected to SDS-polyacrylamide gel electrophoresis to form a single band with a molecular weight of 87,000. However, when plasminogen is activated to plasmin by tPA, it forms two bands, a heavy chain with a molecular weight of about 60,000 and a light chain with a molecular weight of about 26,000.

From FIG. 8, it is clear that when JTA-T was added to tPA, more plasmin was produced than when mouse IgG or a buffer was added. This indicates that JTA-T promotes the activation of plasminogen to plasmin by 2-chtPA.

Example 12 (Effect of 6-amino-1n-caproic acid on the promotion of plasminogen activating activity of 2-chtPA by JTA-T)

2-cht PA 1 iL gZm and 300 gZm in JTA-T diluted with buffer (0.02% BSA / 0.0 l% Tween80 / TBS (pH7.5)), mouse IgG or buffer respectively. And left overnight at 4 ° C. The final concentration of this reaction is 15 OmM in 15 // 150 meS 2251/22 μg Zm plasminogen 100 was added in the presence or absence of 6-amino-n-caproic acid and reacted at 37 ° C. for 60 minutes. Thereafter, the absorbance at 405 nm was measured. The results are shown in FIG. In FIG. 9, JTA-T enhanced the plasminogen activating activity of tPA by about 2-fold, regardless of the presence or absence of 6-amino-n-cabronic acid. This indicates that the effect of J TA-T on the promotion of tPA plasminogen activation activity by tPA is not affected by 6-amino-1n-caproic acid.

Industrial applicability

According to the present invention, a new monoclonal antibody that does not substantially bind to 1-cht PA but selectively binds to 2-cht PA has been successfully created. If this novel monoclonal is used, 2_c ht PA, 1-cht PA or 2-cht PA can be easily and quickly separated and recovered from liquids containing 1-cht PA and 2-cht PA with high purity and efficiency. By using the monoclonal antibody, 2-cht PA in an Atssey sample can be easily measured immunologically.

Claims

The scope of the claims

(1) A substance that does not substantially bind to human single-chain tissue plasminogen activator but selectively binds to human double-chain tissue plasminogen activator Clonal antibodies.

(2) Human single-chain tissue plasminogen activator complex with plasminogen activator inhibitor-11 and human double-stranded tissue plasminogen activator and plasminogen 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody does not substantially bind to any of the complexes with the activator inhibitor.

(3) human 2-chain tissue plasminogen one plasminogen § Kuti beta - dissociation constant for (K d) is 8 X 1 0 - ⁸ monoclonal饥体of claim 1, wherein M is less.

(4) The monoclonal antibody according to (1), which inhibits at least 30% of the amide hydrolytic activity of human double-chain tissue plasminogen activator beta using the synthetic substrate S2288.

(5) The monoclonal antibody according to claim 1, which inhibits at least 20% of a complex formation reaction of human double-chain tissue plasminogenogen activator-1 and plasminogenactivator-1 inhibitor-11.

(6) The method according to claim 1, wherein in the absence of BrCN-degraded fibrinogen, the plasminogen activating activity of the human double-chain tissue plasmaminactivator is promoted at least twice. Monoclonal antibodies.

(7) The monoclonal antibody according to claim 1, which inhibits at least 30% of the plasminogen activating activity of human double-chain tissue plasminogen activator in the presence of BrCN-degraded fibrinogen. . (8) Heavy chain class! The monoclonal antibody according to claim 1, wherein the class of the light chain is / c.

(9) A hybridoma that produces the monoclonal antibody according to claim 1.

(10) A method for separating or recovering human double-stranded tissue plasminogen activator from a liquid containing two human tissue plasminogen activators,

(Iii) contacting the liquid with an insoluble carrier to which the monoclonal antibody according to claim 1 is immobilized, in order to adsorb human double-chain tissue plasminogen activator to the carrier;

() Separating the carrier from the liquid,

010 Elute the desired human double-stranded tissue plasminogen activator beta from the carrier,

Civ) Retrieve it

A method for separating and recovering human double-stranded tissue plasminogen activator.

(11) From a liquid containing human single-chain tissue plasminogen activator and human double-chain tissue plasminogen activator, human single-chain tissue plasminogen activator In the method of separating and recovering

(i) contacting the liquid with an insoluble carrier to which the monoclonal antibody according to claim 1 is immobilized in order to adsorb human double-chain tissue plasminogen activator to the carrier;

(ii) separating a liquid containing a single-chain tissue plasminogen activator beta that is not adsorbed to the insoluble carrier, dn) collecting the desired human single-chain tissue plasminogen activator from the liquid,

(ίν) If necessary, elute double-stranded tissue plasminogen activator from the carrier,

(v) retrieve it

A method for separating and recovering human single-chain tissue plasminogen activator.

(12) A selective adsorbent for human double-stranded tissue plasminogen activator, comprising an insoluble carrier and the monoclonal antibody according to claim 1 fixed thereto.

(13) The selective adsorbent according to claim 12, wherein the insoluble carrier is agarose, polyacrylamide, cellulose, dextran, maleic acid polymer or a mixture thereof.

(14) contacting a first antibody immobilized on an insoluble carrier with a labeled second antibody with an assay sample, comprising a human double-stranded tissue plasminogen activator in an assay sample; In the immunoassay, one of the first antibody and the second antibody is selected from the monoclonal antibodies according to claim 1, and the other antibody is selected from the monoclonal antibodies according to claim 1. A human double-chain tissue that specifically recognizes an antigenic determinant site different from that of a monoclonal antibody and has the property of binding to a human double-chain tissue plasminogen activator. An immunoassay for plasminogen activator.

(15) The immunological assay method according to claim 14, wherein the other antibody is a human single-chain tissue plasmid and a human double-chain tissue plasmid. An immunological assay method for a human double-chain tissue plasminogen activator, which is an antibody having a property of binding to both a minogen activator.

(16) The method according to claim 14, wherein the label of the second antibody is an enzyme, a fluorescent substance, or a radioisotope.

(17) The method according to (14), wherein the labeled second antibody and the Atsushi sample are simultaneously brought into contact with the first antibody immobilized on an insoluble carrier.

(18) A reagent consisting of a first antibody fixed to an insoluble carrier and a labeled second antibody for immunoassay of human double-chain tissue plasminogen activator in an Atsushi sample In the system, one of the first antibody and the second antibody is selected from the monoclonal antibody according to claim 1, and the other antibody is different from the monoclonal antibody according to claim 1 selected above. An antibody that specifically recognizes an antigenic determinant site and has the property of binding to human double-chain tissue plasminogen activator. Immunological measurement reagent system.

(19) In the immunoassay reagent system according to claim 18, wherein the other antibody comprises a human single-chain tissue plasminogen activator and a human double-chain tissue plasminogen activator. An immunoassay reagent system for human double-chain tissue plasminogen activator, which is an antibody having a property of binding to both. International style INTERNATI ONAL FORM

BUDAPEST TREATY ON THE I NTERNAT I O—

NAL RECOGNITI ON OF THE DEPOS IT OF

MI CROORGANI SMS FOR THE PURPOSES OF

International recognition of deposit of microorganisms in patent proceedings PATENT PROCEDURE

Budapest Treaty on

RECEI PT IN THE CASE OF AN OR I GI NAL DEPOS IT

According to Rule 7.1 by the International Depositary Authority:

Issued is s sued pur s uan t t o u l e 7.1 by t he

INTERNATIONAL DEPOS ITARY AUTHOR ITY

Deposit receipt for the original deposit i den t i f i ed a 1 t he bo t t om o f t h i s a g e.

(Name) Teijin Limited

ft¾ 者 Hiroshi Itagaki

Depositary

Name © 541

1-6-7 Minamihoncho, Chuo-ku, IS City, Oitafu University

I. Display of organisms

(Indication by the depositor for distinction) (Accession number)

JTA-T

(FERM BP-3109)

Π. Scientific and taxonomic rank S

Itsuki's animal stated the following; sc «was attached.

Figure Scientific properties

Cause Taxonomic rank E

I. Receipt and Consignment

The International Depositary Authority accepts the I-fed goods received on November 16, 1989 (the original deposit date) _c

(Transferred from No. P-11118, deposited on November 16, 1980)

IV. Special Depositary Authorities

Ministry of International Trade and Industry, National Institute of Industrial Science and Technology, Microorganisms and Industrial Technology Institute

Name

(Address 3 0 5)

305. JAPAN 1990 (1990) September 21