JPH04211363A - Hybrid monoclonal antibody having double specificity - Google Patents

Abstract

PURPOSE:To provide an immunological measuring method capable of simply and specifically measuring a tissue plasminogen activator(TPA). CONSTITUTION:A double specific antibody in which either one of double specificity acts on TPA and the other acts on enzyme, fluorescent substance or radioactive nuclide enabling diagnosis of TPA is prepared and TPA is immunologically measured using the prepared antibody. An immunological measuring method capable of quickly, simply and specifically measuring TPA is provided and separation and determination of free type and complex type TPA are made possible by the present double specific measuring method.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to bispecific hybrid monoclonal antibodies. More specifically, one of the bispecifics is for tissue plasminogen activator (hereinafter sometimes abbreviated as TPA).
On the other hand, the present invention relates to polydomas that produce hybrid monoclonal antibodies against substances that enable the diagnosis of TPA, particularly enzymes, fluorescent substances, or radionuclides, and the monoclonal antibodies produced thereby (hereinafter sometimes abbreviated as MoAb). The present invention also provides the above hybrid Mo
The present invention relates to an immunoassay method for rapidly, easily and specifically measuring TPA using Ab. TPA is a type of serine protease that activates plasminogen and converts it to plasmin, and unlike urokinase (hereinafter sometimes abbreviated as UK) and streptokinase, it has a high affinity for fibrin, so it has less tendency to bleed. It is attracting attention as a thrombolytic agent. In particular, recently, the use of genetic recombination technology has opened the door to mass production, and clinical applications are also popular [European Co.
Operative Study Group for
Recombinant Tissue-Type
Plasminogen Activator: The Lancet, 842 (1985)]
. Further, research and development of more effective thrombolytic agents has been carried out, and hybrid proteins of modified TPA, UK, or pro-UK and TPA have been produced. Regarding modified TPA, we genetically engineered a TPA mutein that lacks part of the sugar chain structure, which is thought to be the cause of its short half-life, to avoid capture by sugar chain receptors such as hepatocytes and improve blood dynamics. I'm measuring. Furthermore, with the UK-TPA hybrid protein, we aim to reduce the dosage by combining the strong thrombolytic properties of UK with the fibrin affinity of TPA. These thrombolytic agents are expected to result in a reduced bleeding tendency compared to conventional agents. As TPA and its derivatives have been widely developed as thrombolytic agents, and in order to elucidate its physiological significance in more detail, a highly sensitive method for measuring trace amounts of TPA has become an important issue. Currently, methods for measuring TPA include (1) a method that directly measures its enzymatic activity using a synthetic substrate, (2) a method that measures fibrinolytic plaques observed through activation of plasminogen, and (3) a method that measures fibrinolytic plaques observed through activation of plasminogen. ) There is a radioimmunoassay method or an enzyme immunoassay method (hereinafter sometimes abbreviated as ELISA) using an anti-TPA antibody. The measurement method (1) is simple and allows measurement in a short time, but has low sensitivity. Although the measurement method (2) is important in determining the correlation with the thrombolytic effect, it is somewhat complicated and does not necessarily have high sensitivity. Although the measurement method (3) does not necessarily reflect the biological activity of TPA, it is frequently used because it is relatively simple and highly sensitive. In particular, ELISA can measure trace amounts of TPA in body fluids such as serum in an assay lasting about 5 to 20 hours without requiring any special equipment. In this case, a sandwich (hereinafter sometimes abbreviated as SW) method is advantageously used in which two types of antibodies are used to sandwich and bind TPA in the test liquid. However, such a method requires labeling one of the antibodies with a marker, such as an enzyme, a fluorescent substance, or a radionuclide. All of these operations are carried out by chemical binding methods, but in many cases they are accompanied by a decrease in the antigen-binding ability of the antibody, an increase in aggregation (production of macromolecules), and nonspecific adsorption to the solid phase used in the assay. However, there was also a limit to the stability of labeled antibodies. In addition, in this SW method, TPA in the test solution is first bound to the anti-TPA antibody on the solid phase and washed, then a labeled antibody bound to a marker is added, washed again, and marker measurement is performed at least twice. This method requires multiple binding reactions and washing operations, so it cannot necessarily be called a simple method. On the other hand, recent hybridoma production techniques have enabled the emergence of new types of polydomas such as triomas and tetraomas [C. Milstein et al.: Nature (Na
ture), 305, 537 (1983), M. R
．． Suresh et al.: Proceedings of the National Academy of Sciences, USA (
Proc. Natl. Acad. Sci. U. S. A)
, 83, 7989 (1986)], which enabled the production of bispecific antibodies with new functions [JP-A-63-12276 (Hybritech), U.S. Patent No. 4,714, No. 681 (Univ. of Tex
as System Cancer Center)]
.

0002

[Means for Solving the Problems] The present inventors have conducted extensive research based on this technical background, and have made it unnecessary to perform chemical bonding operations between markers and antibodies or antigens, which were essential in conventional immunoassay methods. We developed a hybrid MoAb that is easy to operate and shortens the operating time, and completed an immunological diagnosis method for TPA using it. That is, in the present invention, one of the bispecifics is for TPA and the other is for TP.
Hybrid MoAbs with bispecificity for substances, such as enzymes, fluorescent substances or radionuclides, which allow diagnosis of A, and hybrid MoAbs thereof
It concerns a polydoma that produces .

The present invention also provides an anti-TPA antibody-producing hybridoma and anti-horseradish peroxidase (hereinafter referred to as HRP).
(sometimes abbreviated as)) relates to a bispecific hybrid MoAb produced from a tetraoma obtained by fusion with an antibody-producing hybridoma;
Furthermore, the present invention relates to a rapid, simple, and specific immunological diagnosis method for TPA obtained using this hybrid MoAb.

[0004] For the production of the bispecific hybrid MoAb-producing polydomas of the present invention, any hybridoma producing anti-TPA antibodies with high binding activity to TPA can be used. For this purpose, first
Animals are inoculated with TPA, an immunogen, to stimulate the production of anti-TPA antibodies. In this case, the TPA may be either a natural type or a recombinant type, and various TPA muteins can be used as the recombinant TPA. An example of such an immunogen is natural TPA extracted and purified from human melanoma cells [D. C. Rijken et al.: Journal of Biological Chemistry (J. Biol
．． Chem. ), 256, 7035 (1981)] or recombinant N-terminally deleted TPA expressed in various animal cells [K. Wikstrom et al.: Thrombosis and hemostasis (Thromb. Haem
ostas. ), 62, 304 (1989)]. As the inoculated animal, for example, rabbits, rats, mice, guinea pigs, etc. are used, and mice are particularly preferably used in the case of MoAb production.

[0005] The inoculation method may be carried out according to a commonly practiced method, for example, 1 to 100 μg, preferably 5 to 25 μg, is administered to a mouse in an equal volume (0.1 ml) of physiological saline and Freund's complete adjuvant. Emulsify and administer subcutaneously to the back or abdomen or intraperitoneally every 2 to 3 weeks.
A six-dose vaccination method is used.

[0006] Among these immunized animals, such as mice, individuals with high antibody titers are selected, their spleens and/or lymph nodes are collected 3 to 5 days after the final immunization, and the antibody-producing cells contained therein are fused with myeloma cells. The fusion operation can be carried out according to known methods, and examples of the fusion promoter include polyethylene glycol (hereinafter sometimes abbreviated as PEG) and Sendai virus, but PEG is preferably used. Myeloma cells include NS-1, P3U1, S
P2/0, etc., and especially P3U1 are preferably used. For example, the preferred ratio of spleen cells to myeloma cells is 1:1 to
10:1, with a molecular weight of 1,000 to 9,00
0 PEG was added at a concentration of 10-80%, 2
It is best to incubate at 0-37°C, preferably at 30-37°C for 3-10 minutes.

[0007] Various methods can be used to screen for anti-TPA antibody producing hybridomas.
Examples include the ELISA method, in which a hybridoma culture supernatant is added to a microplate on which TPA has been adsorbed, and then an HRP-labeled anti-mouse immunoglobulin antibody is added to detect the anti-TPA antibody bound to the solid phase of the plate. Hybridomas positive for antibody activity that are selected and bred in a HAT (hypoxanthine, aminopterin, and thymidine) supplemented medium are immediately subjected to cloning, which is usually easily carried out by the limiting dilution method or the like. It is possible to measure the antibody titer of the cloned hybridoma culture supernatant using the method described above, select hybridomas that stably produce antibodies with high titers, and obtain the desired monoclonal anti-TPA antibody-producing hybridomas. can.

Anti-T produced according to the above production method
An example of a PA antibody-producing hybridoma is the mouse hybridoma TPA1-39 shown in Example 1 below.

[0009] The substance that enables diagnosis of TPA in the present invention may be any enzyme, fluorescent substance, radionuclide, or its chelate compound. Examples of these include HRP, β-galactosidase, alkaline phosphatase, fluorescein, tetramethylrhodamine, diethylenetriaminepentaacetic acid (DTPA).
Enzymes are particularly preferred from the viewpoint of sensitivity, safety, ease of handling, etc., and HRP is more preferred.

[0010] Such antibody-producing hybridomas can be produced by the same procedure as in the case of the anti-TPA antibody-producing hybridomas described above. Regarding the screening of these hybridomas, for example, when detecting anti-HRP antibodies, the culture supernatant of the test hybridoma is added to a microplate on which anti-mouse immunoglobulin antibody has been adsorbed, and mouse immunoglobulin is bound to the plate. Next, ELISA or the like is used in which free HRP is added and specific anti-HRP antibodies are detected via HRP enzyme activity. Anti-HRP MoAb thus produced
An example of a producing hybridoma is the mouse hybridoma HR1-32 shown in Example 3 below.

There are several methods for producing polydomas that produce the bispecific hybrid MoAb of the present invention [see, for example, Hiroshi Niimoto et al.: Proteins, Nucleic Acids, and Enzymes, 3
3, 217 (1988)], but for example, the HAT-resistant anti-TPA antibody-producing hybridoma described above is treated with 5-bromodeoxyuridine (
(Hereinafter, it may be abbreviated as 5-BrdU) is gradually adapted to the culture solution, and a thymidine kinase-deficient strain is cloned and made HAT sensitive. Similarly, a HAT-resistant anti-HRP antibody-producing hybridoma is made 8-azaguanine (hereinafter sometimes abbreviated as 8-AZG) resistant, and a hypoxanthine-guanine-phosphoribosyltransferase-deficient strain is cloned and made HAT-sensitive. Next, the tetraoma obtained by fusing the two according to a conventional method is HA
After selection in T-supplemented medium, tetraomas that secrete hybrid MoAbs that have the ability to bind both TPA and HRP are cloned.
After labeling the other anti-HRP antibody-producing hybridoma with tetramethyl rhodamine isothiocyanate (hereinafter sometimes abbreviated as TRITC), the two are fused according to a conventional method. . The obtained cell suspension is subjected to a fluorescein activated cell sorter (hereinafter sometimes abbreviated as FACS) to select and clone tetraomas that simultaneously exhibit the green fluorescence of FITC and the red fluorescence of TRITC. There are several methods. It is also possible to select and clone tetraomas by using completely opposite markers of the parent strains.

[0012] For cell fusion in these operations, methods such as Sendai virus, fusion promoters such as PEG, or electrical stimulation are used. Preferably, PEG is used, and an example thereof is given below, but of course the method is not limited to this. That is, the molecular weight is approximately 1
,000 to 9,000, a concentration of about 10 to 80%, etc. is used, and the processing time is about 0.5 to 30 minutes. As an example of preferable conditions, PEG of about 35 to 55% is used. can be brought into contact with cells at 37° C. for about 4 to 10 minutes to achieve efficient fusion.

[0013] Selection of polydomas can be carried out using the above-mentioned HAT-supplemented medium, but for this purpose, each drug-resistant strain is be obtained. Additionally, various selective media can be used depending on the introduction of new markers into the fused cells. Examples of this include neomycin and hygromycin B-supplemented media [B
．． Sugden. et al.: Molecular and Cellular Biology (Mol. Cell. Biol.)
, 5, 410 (1985)].

Furthermore, as mentioned above, there is also a method of fusing hybridomas labeled with different fluorescent dyes and sorting the double-labeled hybrid hybridomas by FACS [L. Karawajew et al.
Journal of Immunological Methods (J.I.
mmunol. Methods), 96, 265 (19
87)].

Various methods can be used to screen for hybrid antibody-producing polydomas. For example, ■ ELISA for screening the aforementioned anti-TPA antibody-producing hybridomas and anti-HRP antibody-producing hybridomas.
∎ ELISA for detecting a bispecific hybrid antibody by adding the test culture supernatant to a TPA-bound microplate and then adding free HRP, or using an anti-TPA antibody that belongs to a different subclass. When using HRP antibodies, ■ ELISA in which the test culture supernatant is added to a TPA-bound microplate, and then the HRP-labeled anti-mouse IgG subclass-specific antibody is added to detect bispecific antibodies; Modified methods can be used in combination as appropriate.

[0016] Polydomas positive for hybrid antibody activity are immediately subjected to cloning, which is usually easily carried out by a limiting dilution method or the like. The antibody titer of the culture supernatant of the cloned polydoma is measured using the method described above, and polydomas that stably produce antibodies with high titers are selected to obtain the desired monoclonal hybrid antibody-producing polydoma. can be obtained.

[0017] The polydoma of the present invention described above can be generally cultured in a liquid medium or intraperitoneally in an animal (eg, intraperitoneally in a mammal such as a mouse) by a known method. Antibodies in culture fluid and ascites can be purified using a combination of known biochemical techniques. For example, the cell culture fluid or ascites fluid is centrifuged, the supernatant is removed, and salting out (usually using ammonium sulfate or sodium sulfate) is performed. The obtained protein precipitate was dissolved in an appropriate solution, and after dialysis, column chromatography (ion exchange column, gel filtration column, protein A
column, hydroxyapatite column, etc.) to separate and purify the antibody of interest. By the above separation and purification operation, for example, hybrid Mo with a purity of 80% or more in terms of protein weight ratio can be obtained from 1 liter of culture supernatant.
Approximately 1-5 mg of Ab can be obtained. Also, 20ml
5 to 20 mg of the same antibody can be obtained from ascites fluid.

Hybrid M obtained as above
By treating oAb with a protease (such as pepsin), it is possible to obtain F(ab')2 fragments that retain the ability to bind to TPA and substances that enable diagnosis of TPA, and these fragments can be used as hybrid MoAbs of the present invention. It can be used for the same purpose.

An example of a hybrid antibody-producing polydoma produced according to the above production method is trioma HT2-263 shown in Example 4 below. In addition, as polydomas producing the hybrid MoAb of the present invention, anti-TPA MoAb producing hybridomas and TP
We have given an example of a tetraoma with a MoAb-producing hybridoma for a substance that allows diagnosis of A.
A trioma of an Ab-producing hybridoma and another MoAb-producing cell, or each MoAb
Even hybridomas obtained by immortalizing cells that produce MoAb with Epstein-Barr virus etc. and cell fusion can be used for the same purpose as the above-mentioned tetraomas, as long as they produce the hybrid MoAb of the present invention. Can be used.

[0020] In the immunoassay method for TPA of the present invention, various assay systems are used. For example, (1) add a certain amount of the hybrid antibody of the present invention, an enzyme, and TPA bound to a solid phase to a test solution containing an unknown amount of TPA;
A method for measuring TPA in a test solution by measuring the activity of an enzyme bound to a solid phase (competitive binding method); TP in the test solution is determined by adding a TPA-containing test solution and a certain amount of the hybrid antibody and enzyme of the present invention to the phase, and measuring the activity of the enzyme bound to the solid phase.
There are methods for measuring A (sandwich method). In either method, measurement can be performed more easily and in a shorter time than conventional measurement methods. For example, the TPA concentration can be measured within 1 to 2 hours using the methods (1) and (2). Furthermore, there is no need to chemically bond enzymes to antibodies or antigens in advance, and free enzymes can be used at the time of use. Moreover, in this case, it is not necessary to use a purified enzyme preparation. The test sample used in the assay of the present invention can be any serum, plasma, urine, tissue extract, etc.

Solid-phase anti-TPA used in the assay system (■)
Any antibody may be used as long as the antigen-determining site does not overlap with the hybrid antibody of the present invention, but rabbit PoAb or mouse MoAb, which has TPA-neutralizing activity and is prepared using TPA as an immunogen, is preferably used. It will be done. An example of such an anti-TPA antibody is mouse MoAb TPA1-70 shown in Example 1 below.
Examples include. It also reacts with free TPA, but
When a mouse MoAb that does not react with the complex of TPA and plasminogen activator inhibitor 1 (hereinafter sometimes abbreviated as PAI-1) is used as a solid-phase antibody, free TPA in the test sample is detected. It enables selective detection and separate quantification of free and complex TPA. As an example of such an anti-TPA antibody, mouse MoAb TPA2-14 (Japanese Patent Application No. 1993-1991) shown in Example 1 below is used.
72935).

[0022]

Effects of the Invention As described above, the bispecific hybrid antibody of the present invention is extremely useful for clinical diagnosis of TPA in the sense that TPA can be measured simply, quickly, and specifically, and is superior to conventional MoAb. It has superior characteristics compared to other materials.

[0023]

[Examples] The present invention will be specifically explained below with reference examples and examples, but it goes without saying that these are not intended to limit the scope of the present invention. The animal cells used in the Examples have been deposited as shown in Table 1 below. Table 1
(IFO) (F
RI) animal cells
IFO No. FERM
No. mouse hybridoma
50233 BP-2819
TPA 1-39 mouse hybridoma
50179 BP-2086
TPA 1-70 mouse hybridoma
50194 BP-2519
TPA 2-14
mouse hybridoma
50234 BP-2818
HR 1-32 mouse hybrid hybridoma 50235
BP-2817HT 2
-263
I
FO: Fermentation Research Institute (Osaka) FR
I: Ministry of International Trade and Industry, Microbial Technology Research Institute.

Reference example 1. ELISA for anti-TPA antibody measurement
Dispense 100 μl of TPA 5 μg/ml solution into a 96-well microplate, leave it overnight at 4°C, and then incubate for 2 more days.
A sensitized plate was prepared by adding 150 μl of a phosphate saline solution (hereinafter sometimes abbreviated as PBS) containing % casein and 0.01% thimerosal. Remove the above liquid and
．． After washing with phosphate saline buffer containing 0.05% Tween 20 (hereinafter sometimes abbreviated as PBS-Tw), 100 μl of test hybridoma culture supernatant was added and reacted at room temperature for 2 hours. After washing the plate again with PBS-Tw,
A horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG antibody was added and allowed to react at room temperature for 2 hours. After washing, 0.1M citrate buffer containing orthophenylenediamine and H2O2 as enzyme substrates was added to each well, and the enzyme reaction was performed at room temperature. After stopping the reaction with 1N sulfuric acid, the amount of colored dye was measured at a wavelength of 492 nm using Multiscan (manufactured by Flow).

Reference example 2. ELISA for anti-HRP antibody measurement
Dispense 100 μl of rabbit anti-mouse IgG antibody 5 μg/ml solution into a 96-well microplate, leave it overnight at 4°C, and add P containing 2% bovine serum albumin (BSA).
A sensitized plate was prepared by adding BS. At the time of ELISA measurement, the above solution was removed, and after washing with PBS, the test hybridoma culture supernatant was added and reacted at room temperature for 2 hours. After washing with PBS, HRP was added, and the mixture was further reacted at room temperature for 2 hours. Hereinafter, an enzyme reaction was performed by the method described in Reference Example 1, and the antibody titer was measured.

Reference example 3. ELISA reference example 1 for hybrid antibody measurement
The test hybridoma culture supernatant was added to the TPA-sensitized plate prepared above, and the reaction was allowed to proceed at room temperature for 2 hours. Then P.B.
After washing with S, HRP was added and the mixture was further reacted at room temperature for 2 hours. Hereinafter, an enzyme reaction was performed by the method described in Reference Example 1, and the antibody titer was measured.

Reference example 4. Fibrinolytic reaction neutralization test T
The test MoAb solution was added to the PA solution (final concentration 20 μg/ml), and after reaction at 37° C. for 1 hour, 5 μl of the reaction mixture was injected per well of a fibrin agarose plate. After 2 to 6 hours at 37°C, the fibrin lysis plaque (diameter) was measured, and the ability of MoAb to neutralize the enzymatic activity of TPA was determined.

Reference example 5. Preparation of TPA-PAI-1 complex PAI-1
(Sold by American Diagnostics) was activated by treatment with 8M guanidine/hydrochloric acid solution at 37°C for 30 minutes, then half the amount of TPA was added and further incubated at 37°C for 30 minutes to prepare a complex. . When the obtained complex solution was subjected to the fibrinolytic reaction described in Reference Example 4, almost 100% of TPA activity was lost.

Example 1. Preparation of mouse anti-TPA monoclonal antibody-producing hybridoma ■ Commercially available single-chain TPA (sold by Chuo Kagaku Kogyo K.K.) 20
After adding an equal volume of Freund's complete adjuvant to a 0 μg/ml physiological saline solution and thoroughly emulsifying it, BALB/c mice (
20 μg/0.2 ml/mouse) were administered intraperitoneally and subcutaneously to the back, and booster immunizations were performed at 2-3 week intervals. After three booster immunizations, a TPA antigen solution (50 μg/0.1 ml physiological saline/mouse) was intravenously administered to the individual that showed the maximum serum antibody titer 10 days later.

■Cell Fusion Three days after the final immunization, the spleen was removed, and a spleen cell suspension was prepared by a conventional method (approximately 108 cells). Next, 2 x 107 mouse myeloma cells (P3U1) were added, and PEG 600
0 was used for cell fusion according to the method of Kohler and Milstein [Nature, 256, 495 (1975)]. After completion of the fusion, the cell mixture was suspended in so-called HAT medium containing hypoxanthine, aminopterin, and thymidine, and cultured for 10 days. Thereafter, as soon as the selection of parent cells was completed, the HAT medium was replaced with an HT medium in which aminopterin was removed, and culture was continued.

■ Hybridoma selection and cloning fusion 10~
Since the appearance of hybridomas was observed after 20 days, the ELISA described in Reference Example 1 using a TPA-bound microplate was performed.
The antibody titer of the hybridoma culture supernatant was measured. For hybridomas that showed particularly strong antibody activity,
It was subjected to cloning using the limiting dilution method. The culture supernatant of the cloned hybridoma was subjected to ELI in the same manner as in Reference Example 1.
Mouse hybridomas producing three types of anti-TPA antibodies with strong TPA binding ability for SA screening TPA 1
-39, TPA 1-70, TPA 2-14 were obtained. These immunoglobulin classes and subclasses were all IgG1 as determined by the Ouchterlony method.

■ Preparation of monoclonal antibody 0.5 m
BALB/c mice were administered intraperitoneal mineral oil with 5 x 10
Six anti-TPA MoAb-producing hybridomas were inoculated intraperitoneally. Approximately 10 to 15 days later, accumulation of ascites was observed. Antibodies were purified by a conventional method by fractionation with 45-50% saturated ammonium sulfate and then subjected to DEAE-cellulose and protein A column chromatography. Anti-TPA MoAb respectively
TPA 1-39, TPA 1-70 and TPA2-
14 was obtained.

Example 2. Neutralizing activity of anti-TPA monoclonal antibody against TPA fibrinolytic ability Example 1-■
The anti-TPA MoAb prepared above was subjected to a fibrinolytic reaction neutralization test using a fibrin agarose plate described in Reference Example 4, and the neutralizing activity against TPA was measured. The results were as shown in Table 2. Antibody TPA 1-
Antibody TPA 1-70 showed strong neutralizing activity, while antibody TPA 2-14 showed no neutralizing activity at all. Table 2

Antibody concentration %fibrinolytic activity

Anti-TPA monoclonal antibody (ng/ml)
TPA1-39 TPA1-
70 TPA2-14 0
．． 012 100 86
100
0.12 86
30 86
1.2 86
8 100
1 2 63
0 116
Example 3. Preparation of mouse anti-HRP monoclonal antibody-producing hybridoma ■ Immune commercially available single chain HRP (sold by Seikagaku Corporation) 200
Add an equal volume of Freund's complete adjuvant to μg/ml physiological saline solution and thoroughly emulsify it, then add BALB/c mice (♀
, 20 μg/0.2 ml/mouse) was administered intraperitoneally and subcutaneously to the back, and booster immunizations were performed at 2-3 week intervals. After three booster immunizations, HRP antigen solution (50 μg/0.1 ml physiological saline/mouse) was intravenously administered to the individual that showed the maximum serum antibody titer 10 days later. (2) Cell fusion Three days after the final immunization, the spleen was removed, spleen cells were prepared, and cell fusion was carried out in the same manner as in Example 1-(2).

■ Hybridoma selection and cloning fusion 10~
The culture supernatant of the hybridoma that appeared after 20 days was subjected to ELISA as described in Reference Example 2 using an anti-mouse IgG antibody sensitized plate, and the anti-HRP antibody titer was measured. Hybridomas that showed particularly strong antibody activity were subjected to cloning by limiting dilution method. The culture supernatant of the cloned hybridoma was subjected to ELISA as described in Reference Example 2.
RP antibody-producing mouse hybridoma HR1-32 was obtained. The antibody produced was IgG1.

Example 4. Preparation of hybrid monoclonal antibody with anti-TPA-anti-HRP bispecificity ■ Anti-TPA antibody-producing hybridoma T obtained in Cell Fusion Example 1
PA1-39 and anti-HRP antibody producing hybridoma HR1-32 obtained in Example 3 were each given at 0.5 μg.
/ml FITC and 1.5μg/ml TRITC
The cells were incubated with Iscove-Ham's F.12 mixed medium at 37° C. for 30 minutes, and fluorescently stained. Next, after adding LSM solution (sold by Wako Pure Chemical Industries KK) to remove dead cells, both hybridomas were mixed at a ratio of 1:1, and PE
Cell fusion was carried out using G6000 according to the method described in Example 1-■. After incubation at 37°C for 2 hours, 25,000 cells double-stained with fluorescein and rhodamine were collected by FACS, and then placed in a 96-well microplate in which mouse thymocytes were seeded at 5 x 10 cells/well as feeders. Add 10 of the above double-stained cells to
Cells/well were seeded and cultured.

① Selection and cloning of hybrid hybridomas Culture supernatants of wells in which cell proliferation was observed 1-2 weeks after fusion were each subjected to ELISA as described in Reference Example 3 to measure antibody activity. Cloning by limiting dilution method was performed on the wells showing high hybrid antibody activity, and the desired bispecific antibody-producing mouse hybrid hybridoma (tetraoma) HT2-263 was obtained.

■Purification of hybrid antibody: 0.5 m
5x to 6 BALB/c mice intraperitoneally administered mineral oil.
106 mice/mouse of mouse hybridoma (tetraoma) HT2-263 were inoculated intraperitoneally. Approximately 10 to 20 days later, 23 ml of ascites was collected and salted out with 50% saturated ammonium sulfate.
gG fraction was obtained. Then 20mM PBS (pH 7.5
), then subjected to HRP-coupled Cellulofine column,
Elution was performed with 0.2M glycine/hydrochloric acid buffer at pH 3.0. The acid-eluted fraction was dialyzed against PBS, then applied to a TPA Sepharose 4B column, and eluted with 0.2M glycine/hydrochloric acid buffer at pH 2.3. 12.3 mg of bispecific antibody HT2-263 was obtained by dialysis with PBS. The obtained antibody was subjected to ELISA as described in Reference Example 3, and a dilution curve of the bispecific antibody was prepared. The results were as shown in FIG.

Example 5. Specificity of anti-TPA monoclonal antibody ■ Preparation of solid-phase antibodies Example 1-■ Three types of anti-TPA MoAb prepared in
Purification was performed using the TPA-bonded Sepharose 4B column described in Example 4-■. Each of the three purified antibodies was used in place of the rabbit anti-mouse IgG in Reference Example 2 to prepare an anti-TPA antibody sensitized plate. (2) Preparation of biotinylated antibodies Each of the purified antibodies obtained in (1) above was biotinylated using commercially available N-hydroxysuccinimide biotin (manufactured by Funakoshi) according to a conventional method [K. Hoffmann et al.: Journal of the American Chemical Society (
J. Am. Chem. Soc. ), 100, 35
85 (1978)]. ■ Search for specificity ■ Add TPA25n to the anti-TPA antibody sensitized plate prepared in ■.
After adding 50 μl of PBS-T and reacting for 2 hours at room temperature,
Washed with w. Next, biotinylated antibody 5 prepared in
After adding 0 μl and further reacting at 37°C for 1.5 hours, PB
Washed with S-Tw. Add 50 μl of a 500-fold diluted solution of HRP-labeled avidin D (sold by Funakoshi) and incubate at 37°C for 3
After reacting for 0 minutes, the plate was washed with PBS-Tw, and an enzyme reaction was carried out as described in Reference Example 1 below. The results were as shown in Table 3. Three types of anti-TPA antibodies TPA1-39,1-
Both antibodies 70 and 2-14 were determined to have no overlapping antigen recognition sites with each other.

[0039]
Table 3
Enzyme activity bound to solid phase [absorbance (492 nm)] Biotin Solid phase Antibody
Antibody TPA1-39 TPA1-70
TPA2-14 TPA1-39
0.1 2.7 2
．． 6 TPA1-70 2.6
0.1 2.7
TPA2-14 2.2
2.7 0.0
Example 6. Setting of ELISA for TPA Quantification The anti-TPA antibody TPA2-14 sensitized plate prepared in Example 5-■ was reacted with various concentrations of TPA at room temperature for 2 hours.
Washed with BS-Tw. Next, the bispecific antibody HT2-263 (1 μg/ml) obtained in Example 4 was added to the commercially available H
It was mixed in advance with RP solution (100 μg/ml), added to the above plate, and reacted at room temperature for 2 hours. After washing with PBS-Tw, an enzyme reaction was performed by the method described in Reference Example 1 to prepare a TPA standard curve. The results were as shown in FIG. TPA of 1 ng/ml or less was quantifiable.

Example 7. Example 5 of setting up ELISA for quantifying free TPA
- Anti-TPA antibodies TPA1-70 and TP prepared in
TPA solutions of various concentrations or TPA-PAI-1 complex solution 5 prepared in Reference Example 5 were placed on the A2-14 sensitized plate.
0 μl of the immunoconjugate solution of bispecific antibody HT2-263 and HRP described in Example 6.
was added and reacted at room temperature for 1 hour. After washing with PBS-Tw, an enzymatic reaction was carried out by the method described in Reference Example 1, and TP
Standard curves for A and TPA-PAI-1 complexes were created. The results were as shown in FIG. TPA2-14
Free TPA is detected in ELISA using antibody-sensitized plates.
was quantifiable, and the TPA-PAI-1 complex was hardly detected.

Example 8. Blood dynamics of TPA TPA (0.
15 mg/kg) was administered intravenously to rabbits, and blood was collected over time. The TPA concentration in the rabbit plasma was then measured by ELISA using the TPA1-70 and TPA2-14 antibody sensitized plates described in Example 7. The results were as shown in FIG. Free T using TPA2-14 antibody
In ELISA for measuring PA, 1/4-1 of the value obtained in ELISA for measuring total TPA using TPA1-70 antibody.
The measured value was /2.

Example 9. TPA amount in TPA-producing cell culture supernatant Human umbilical vein endothelial cells HUVEC, human fibroblasts IMR-
90, human melanoma cells G-361 were cultured in a medium containing fetal bovine serum, and the culture supernatant was treated with TPA described in Example 7.
Free TPA-ELISA using 2-14 antibody and total TPA-ELISA using TPA1-70 antibody were performed. The results were as shown in Table 4. Various human TP
It has become clear that most or a part of TPA exists as a complex during the culture of A-producing cells.
Table 4
cell
Free TPA (ng/ml) Total TPA
(ng/ml) HUVEC
< 1.0
6.4 IMR-40
2.3
9.8 G-361
5.5 28.
2

[Brief explanation of the drawing]

FIG. 1: Bispecific antibody HT2-26 described in Example 4.
3 is a dilution curve obtained when 3 was subjected to the ELISA described in Reference Example 3 (see Example 4).

FIG. 2: Anti-TPA antibody described in Example 1 (TPA2-1
4) as a solid-phase antibody, the bispecific antibody HT described in Example 4
2-263 is a TPA standard curve prepared by ELISA using 2-263 as a labeled antibody (see Example 6).

FIG. 3: Anti-TPA antibody TPA1-70 described in Example 1
Free TPA (○, ●) and TPA2-14 (□, △) were prepared by ELISA using solid-phase antibodies and the bispecific antibody HT2-263 described in Example 4 as a labeled antibody.
○, □) and the standard curve of TPA-PAI-1 complex (●, △) (see Example 7).

[Figure 4] Plasma from TPA-treated rabbits was treated with anti-TPA antibody TP.
The blood dynamics curves when subjected to the ELISA described in Example 7 using A1-70 (●) and TPA2-14 (○) as solid-phase antibodies are shown (see Example 8).

Claims (9)

[Claims] Claims: 1. A dual specificity, one of which is for tissue plasminogen activator and the other for an enzyme, fluorophore or radionuclide that allows the diagnosis of tissue plasminogen activator. A polydoma that produces hybrid monoclonal antibodies with specific characteristics. 2. The polydoma according to claim 1, wherein the enzyme is horseradish peroxidase. 3. A hybridoma obtained by fusing an anti-horseradish peroxidase antibody-producing hybridoma with an anti-tissue plasminogen activator antibody-producing hybridoma, and capable of binding to both horseradish peroxidase and tissue plasminogen activator in one molecule. The polydoma according to claim 1, which is a tetraoma that produces a heavy specific hybrid monoclonal antibody. 4. The tetraoma according to claim 3, wherein the anti-horseradish peroxidase antibody-producing hybridoma is HR1-32. 5. The tetraoma according to claim 3, wherein the anti-tissue plasminogen activator antibody-producing hybridoma is TPA1-39. 6. The anti-horseradish peroxidase antibody-producing hybridoma is HR1-32, and the anti-tissue plasminogen activator antibody-producing hybridoma is T.
The tetraoma according to claim 3, which is tetraoma HT2-263, which is PA1-39. 7. A dual specificity, one of which is for tissue plasminogen activator and the other for an enzyme, fluorophore or radionuclide that allows diagnosis of tissue plasminogen activator. A hybrid monoclonal antibody with specific characteristics. Claim 8: A bispecific hybrid monoclonal antibody H capable of binding to both horseradish peroxidase and tissue plasminogen activator in one molecule.
8. The bispecific hybrid monoclonal antibody of claim 7, which is T2-263. 9. Dual specificity, one of which is for tissue plasminogen activator and the other is for an enzyme, fluorescent substance or radionuclide that allows diagnosis of tissue plasminogen activator. An immunoassay method for tissue plasminogen activator, characterized by using a hybrid monoclonal antibody having the following.