JP2002316999A - Monoclonal antibody - Google Patents

Abstract

(57) Abstract: An anti-human thrombin / human antithrombin III complex monoclonal antibody reacting with free human antithrombin III and also reacting with a human thrombin / human antithrombin III complex, and containing the same Immunoassay reagent. [Effect] The TAT concentration in human samples can be accurately, simply,
It can be detected quickly and with high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel monoclonal antibody, and to use the same to accurately determine the concentration of a human thrombin / human antithrombin III complex in a human sample.
The present invention relates to an immunoassay reagent that can be easily measured and is useful for diagnosing a blood coagulation tendency.

[0002]

2. Description of the Related Art Human antithrombin III (hereinafter referred to as ATI)
II) are important inhibitors of serine proteases in the blood coagulation system, and include human thrombin and other activated human blood coagulation factors XII and X.
Inhibits the activity of factor I, human blood coagulation factor X, and human blood coagulation factor IX. The reaction between ATIII and the serine protease proceeds in a molar ratio of 1: 1. The arginine residue of ATIII is ester-bonded to the serine residue, which is the active center of the serine protease, to form a complex. Suppress activity. As one of such complexes, a human thrombin / human antithrombin III complex (hereinafter, referred to as TAT) is known.
An increase in TAT in human blood is believed to be an indication that the initiation of the blood coagulation mechanism and its activation produced human thrombin or other blood coagulation serine proteases. Therefore, it is presumed that by measuring the amount of TAT in the blood, it is possible to know a part of the dynamics of the blood coagulation system, thereby elucidating the pathological condition of the patient from the blood coagulation surface, for example, thrombus formation or generalized blood vessels. It is possible to predict the progress of the pathological condition to internal blood coagulation (DIC) at an early stage and to carry out appropriate treatment.

As a method for immunologically measuring TAT in a human sample, a method for measuring TAT by performing an inhibition assay using an anti-TAT neoantigen-polyclonal antibody using ¹²⁵ I-labeled TAT (Herber)
t, L. Lau, The Journal of Biological Chemistry, 25
5, 5885-5893 (1980)); a method for measuring TAT in human samples by a sandwich system using an anti-thrombin-polyclonal antibody as a solid-phase antibody and an anti-ATIII-polyclonal antibody as an enzyme-labeled antibody (H. Pelzer, Thrombosis & Ha
emostasis, 59, 101-106 (1988)). A sandwich kit using a polyclonal antibody according to the method of H. Pelzer et al.
No.) has been proposed and commercialized.

[0004] However, all of these measurement methods use polyclonal antibodies, and thus have a problem in antibody uniformity. In addition, since the method is a radioimmunoassay or an enzyme immunoassay, there remains a problem in application to a highly versatile automatic analyzer. That is, since the heterogeneous antibody has a high probability of reacting with the cross-reactant contained in the sample and the measurement value is liable to fluctuate, the conventional methods are sensitive, accurate, simple, and further versatile. However, it was hard to say that it met current medical needs.

[0005] To overcome such drawbacks, the use of monoclonal antibodies can be considered. Anti-TAT monoclonal antibodies include S. Asakura et al., Biochemica Biophysic.
a Acta, 952, 37-47 (1988), French Patent No. 2645647
And Japanese Patent Publication No. 6-44876 and Japanese Patent Application Laid-Open No. 7-238099 are known. However, even with these monoclonal antibodies, the present invention can be applied to an immunoassay based on agglutination such as a latex agglutination immunoassay which can easily and quantitatively measure TAT in a clinical sample using a highly versatile automatic analyzer. There was nothing.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a monoclonal antibody suitable for use in an immunoassay based on agglutination such as a latex agglutination immunoassay in the measurement of TAT in a human sample. Is to do.

[0007]

Under such circumstances, the present inventors have conducted intensive studies. As a result, when selecting a monoclonal antibody specific to an antigen, a latex carrying TAT was used in addition to the conventional enzyme immunoassay. By using an insoluble carrier such as, versatility, a monoclonal antibody that can be suitably used in a highly convenient immunoassay is obtained,
If this is used, the TAT concentration in a human sample can be accurately determined,
The inventors have found that measurement can be easily performed using a highly versatile automatic analyzer, and have completed the present invention.

[0008] That is, the present invention provides an anti-human thrombin / human antithrombin III complex monoclonal antibody that reacts with free human antithrombin III and also reacts with a human thrombin / human antithrombin III complex. .

[0009] Further, the present invention provides a method of fusing an antibody-producing cell of a mammal immunized with an antigenic substance with a myeloma cell of a mammal, and then performing enzyme immunoassay (hereinafter referred to as "ELISA"). Cells that produce antibodies that react with the substance are selected, and the selected cells are further reacted with an antigen substance by an immunoassay based on agglutination such as latex agglutination immunoassay (hereinafter referred to as "LTIA"). The present invention provides a method for producing a monoclonal antibody, which comprises selecting a cell producing an antibody to be raised and culturing the cell.

The present invention also provides a monoclonal antibody produced by fusing mammalian antibody-producing cells immunized with a human thrombin / human antithrombin III complex with mammalian myeloma cells. It provides hybridomas.

Furthermore, the present invention relates to a human thrombin / human antithrombin III containing the above-mentioned anti-human thrombin / human antithrombin III complex monoclonal antibody.
It provides a complex immunoassay reagent.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
It is produced by a hybridoma obtained by fusing a mammalian antibody-producing cell immunized with TAT with a myeloma cell derived from a mammal, and can be prepared by a conventional method (G.
Kohler and C. Milstein, Nature, 1975, 256; Yarmu
sh M. et al, Proc. Natl. Acad. Sci. USA 77, 2899-,
1980).

TAT used as an immunogen is prepared by binding human thrombin to ATIII. The human thrombin and ATIII used here can be used both as commercial products and purified products from human plasma. Human thrombin and ATIII are 1: 1 to 1
It is preferable that the TAT mixed at a molar ratio of 1: 5 and bound be purified and used by column chromatography or the like.

The hybridoma is prepared, for example, from the obtained TA
It is prepared by injecting T or the like into a mammal as an immunogen. The mammal to be immunized is not particularly limited, and is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion in the subsequent operation. Specific examples include mouse, rat, and the like. It is common to use / c mice.

The method of immunizing a mammal with the above-described immunogen is not particularly limited. For example, TAT alone or an immunogen obtained by combining one or more TAT components can be used subcutaneously in a mammal.
A method of injecting intradermally, intraperitoneally, intravascularly, intramuscularly, intrasplenice, or the like; a method of oral administration in addition to feed or water, and the like can be used. When immunizing, it can be used in combination with an adjuvant, if necessary.

Next, spleen cells collected from the immunized animal are fused with mouse myeloma cells. The mouse myeloma cells, hypoxanthine - guanine - phosphoribosyl transferase deficiency (HGPRT -) - is preferably one having a suitable marker such as ^thymidine kinase-deficient ^(TK). Fusion can be performed according to a known method,
As the fusion promoter, polyethylene glycol, Sendai virus and the like can be used. The mixing ratio of spleen cells to myeloma cells is preferably from 1: 1 to 10: 1. Also,
Cell fusion by an electric fusion method or the like can also be performed.

In the present invention, a monoclonal antibody suitable for use as an immunoassay based on agglutination, such as a latex agglutination immunoassay, is obtained by ELISA after cell fusion.
A cell producing an antibody reacting with the antigenic substance is selected by the method, and a cell producing the antibody reacting with the antigenic substance is further selected from the selected cells by an immunoassay based on aggregation, such as the LTIA method. By culturing the cells,
Can be manufactured.

Specifically, in order to select a monoclonal antibody-producing hybridoma suitable for use as an immunoassay based on the principle of agglutination such as a latex agglutination immunoassay reagent, after cell fusion, an ordinary cell selection medium is used. Hybridomas are selected by culturing in (HAT medium), and selection of hybridomas producing the desired monoclonal antibody by immunoassay based on the principle of agglutination such as ELISA and LTIA and cloning by limiting dilution are repeated. Thereby, the target hybridoma can be established. In the cloning by the limiting dilution method, it is preferable to use a mouse thymocyte, spleen cell, peritoneal macrophage, or a known additive having the same effect as these as a feeder.

The ELISA method can be performed according to a known method. For example, TAT is adsorbed to an ELISA plate directly or via a TAT conjugate, or an anti-mouse Ig antibody is adsorbed directly to the ELISA plate. After that, the culture supernatant of the hybridoma was reacted, and then the enzyme-labeled anti-mouse Ig antibody was reacted. After the unreacted enzyme-labeled anti-mouse Ig antibody was washed away from the ELISA plate, the enzyme reaction was performed, and the reaction was positive. Select the cells contained in the wells. As the TAT conjugate, an anti-ATIII antibody, an antithrombin antibody, an antiprothrombin antibody, heparin and the like can be used. Also, E
For the adsorption to the LISA plate, a physical adsorption method or a chemical bonding method can be used.

In addition, an immunoassay method based on aggregation, such as the LTIA method, uses an automatic analyzer for biochemistry, and uses an insoluble carrier carrying TAT directly or via a TAT conjugate and an insoluble carrier carrying a monoclonal antibody conjugate. This is a method in which a carrier is reacted with a culture supernatant of a hybridoma to select cells contained in the wells in which aggregation has occurred. Anti-ATIII antibodies, antithrombin antibodies, antiprothrombin antibodies, heparin and the like can be used as TAT conjugates, and protein A, protein G, anti-mouse Ig antibodies and the like can be used as monoclonal antibody conjugates. Also,
For the adsorption to the insoluble carrier, a physical adsorption method or a chemical bonding method can be used.

The monoclonal antibody of the present invention can be obtained by culturing the hybridoma selected above in an appropriate medium or intraperitoneally in a mouse or the like. The medium used here includes fetal bovine serum,
RPMI 1640 medium containing antibiotics (penicillin, streptomycin, etc.) is preferred. The cultivation is preferably carried out, for example, by adding the above hybridoma to the medium at a concentration of 10 ⁴ to 10 ⁵ cells / mL and under a condition of 5% carbon dioxide concentration and 37 ° C. for about 2 to 4 days. The monoclonal antibody of the present invention can be obtained by centrifuging the obtained culture supernatant. On the other hand, in the case of intraperitoneal culture, the above hybridoma may be administered intraperitoneally to BALB / c mice, nude mice, nude rats, etc., and the ascites may be collected.

The monoclonal antibody of the present invention in the culture supernatant or ascites thus obtained can be used as it is. For example, fractionation by ammonium sulfate precipitation, ion exchange chromatography, or protein A It is preferable to use the protein after purification by affinity chromatography using a carrier bound with protein G or anti-mouse Ig antibody, or ultrafiltration.

The immunoassay reagent of the present invention contains the anti-TAT monoclonal antibody obtained as described above, and the monoclonal antibody can be used alone or in combination of two or more. Further, a substance having a binding property to TAT, for example, an anti-ATIII antibody, an antithrombin antibody, an antiprothrombin antibody, heparin and the like can be added.

The reagent of the present invention is prepared by suspending a monoclonal antibody-bound insoluble carrier, and the suspension of the insoluble carrier includes, for example, a phosphate buffer,
Buffers such as glycine buffer, Tris buffer and Good buffer are used. The pH of the reaction is 5 to 10, especially pH 6 to
9 is preferred.

The insoluble carrier to which the monoclonal antibody is bound is not particularly limited, and any substance which is usually used for measuring an antigen or an antibody can be used. And inorganic polymer substances such as metal colloids. As the organic high-molecular substance, latex particles in which one or more fine powders selected from resins such as acrylic acid polymer, styrene polymer, methacrylic acid polymer and the like are uniformly suspended are particularly preferable. A latex having a carboxyl group or an amino group is suitably used when a monoclonal antibody is chemically bound using a condensing agent, a dicrosslinking reagent, or the like.

The particle size of the insoluble carrier is not particularly limited, and when the aggregate is directly measured optically or visually observed on the carrier by immunochromatography, the average particle size is generally 1.6 μm. The following are preferred,
In particular, the average particle size is 0.01 to 1 μm, and more preferably 0.01 to 0.
It is preferably 5 μm. When the aggregate is visually observed on the aggregate plate, the average particle diameter is preferably 1 μm or more, particularly preferably 1 to 20 μm.

The method for binding the monoclonal antibody to the insoluble carrier is not particularly limited, and the monoclonal antibody can be directly physically adsorbed or chemically bound to the insoluble carrier.
Further, it can be bound to an insoluble carrier via a monoclonal antibody conjugate. Here, as the monoclonal antibody conjugate, protein A, protein G, anti-mouse Ig antibody and the like can be used, and the binding of these monoclonal antibody conjugates to the insoluble carrier may be either physical adsorption or chemical bonding. .

The immunoassay reagent of the present invention is used by mixing it with a sample to be measured (a human sample such as blood), and the TAT present in the sample reacts with the monoclonal antibody of the present invention to aggregate. Can measure the TAT. The measurement of the degree of agglomerates generated by the reaction with TAT in the sample can be measured optically, and the optical measurement method of such agglomerates is not particularly limited as long as it is a commonly used method. A general-purpose spectrophotometer, an automatic analyzer for biochemistry using spectrophotometry as a measurement principle, a device using near-infrared as a measurement wavelength, a device using integration sphere turbidity as a measurement principle, and measuring scattered light intensity An optical measuring device such as a device can be used. The immunoassay reagent of the present invention is particularly suitable as a latex agglutination immunoassay reagent.

[0029]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 (1) Preparation of TAT: α-Thrombin of thrombin preparation (manufactured by Midori Cross) and ATIII preparation (manufactured by Hoechst)
Of ATIII in 0.02 M Tris buffer (pH 7.4)
And mixed at a molar ratio of α-thrombin: ATIII = 1: 1.5, and reacted at 37 ° C. for 10 minutes.
The reaction product was washed with Heparin-Sepharose (Pharmacia) 2
Apply to a × 6 cm column and filter the adsorbed fraction to 0.1-2M
Elution was performed with a linear gradient of NaCl / 0.02M Tris buffer (pH 7.4). Each fraction is 4-
Analysis was performed by 20% SDS-PAGE (manufactured by Daiichi Kagaku) and the TAT fraction was collected. TAT concentration is ELISA
As a result of measurement using Enzygnost TAT Beringberg Co., it was 750 μg / mL.

(2) Immunization: 100 μg of the above TAT was
Used for one immunization. Freund's complete adjuvant was used for both the primary and booster immunizations. TAT100
μL and 100 μL of Freund's adjuvant were mixed, and 200 μL of the obtained emulsion was injected into the peritoneal cavity of one BALB / c male mouse per immunization, and the immunization was repeated four times at two-week intervals. Blood was collected from the fundus vein of the mouse, the antibody titer was measured by ELISA, and a mouse with a high antibody titer was selected and used for cell fusion.

(3) Cell fusion: Two weeks after the fourth immunization, 100 μg of TAT diluted in 200 μL of physiological saline
Was intraperitoneally injected into the mouse, and three days later, the spleen was removed from the mouse. The spleen cells were collected from the excised spleen, and 10 ⁸ live spleen cells were mixed with 10 ⁷ logarithmically growing mouse myeloma cells (SP2 / O or P3U1) which had been cultured beforehand. GKN solution containing (w / v) polyethylene glycol 1540 (NaCl: 8 g, KC
l: 0.4 g, glucose: 2 g, Na ₂ HPO ₄ : 1.
41 g, NaH ₂ PO ₄ .2H ₂ O: 0.78 g was dissolved in 1 L of purified water) to fuse the cells. The cells after the fusion were suspended in a 15% FCS RPMI medium containing HAT (10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.5 × 10 ⁻⁵ M thymidine) and feeder cells.
37 ° C, 5% using a 6-well microculture plate
The cells were cultured in a carbon dioxide incubator.

(4) Selection of anti-TAT monoclonal antibody by ELISA method: To select anti-TAT monoclonal antibody in the culture supernatant, TAT (1
μg / mL), ATIII (1 μg / mL), or goat anti-mouse Ig antibody (1 μg / mL: manufactured by Kappel Products)
The culture supernatant was added to a 96-well microplate on which was solid-phased, incubated at 37 ° C. for 1 hour, washed with PBS, and washed with 0.1%
A peroxidase-labeled anti-mouse Ig antibody (derived from goat: manufactured by Kappel Products) diluted 1000-fold with BSA-PBS was added, and the mixture was kept at 37 ° C for 1 hour. This is PB
After washing with S, a substrate solution (citrate-phosphate buffer containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide; pH 5.0) was added and reacted at room temperature for 30 minutes.
After stopping the reaction by adding sulfuric acid, the absorbance was measured.
The wells of the supernatant giving a high absorbance were selected.

(5) Preparation of heparin-carrying latex liquid:
Aminated polystyrene latex (average particle size: 0. 0%) adjusted to a 2% concentration with a 0.05 M borate buffer (pH 7.5).
To a 6 mL suspension of 2 μm, manufactured by Interhetic Dynamics, was added a 0.05 M borate buffer (pH 7.0).
3) Heparin sodium (manufactured by Daiichi Kagaku Chemical Co., Ltd.) solution (3 mL) prepared in 5) to a concentration of 8 mg / mL and 2-mg / mL prepared in a 0.05 M borate buffer (pH 7.5)
3 mL of ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojindo Laboratories) solution was added, and
The reaction was performed at 25 ° C. for 4 hours. Further, 4 mL of a 0.05 M borate buffer (pH 7.5) containing 2% of glycine was added, and the mixture was stirred at 25 ° C overnight. After performing centrifugal washing twice using 0.05 M glycine buffer (pH 8.4),
The suspension was redispersed in 84 mL of a 0.05 M glycine buffer (pH 8.4), and a latex solution containing heparin (Heparin-L) was added.
x) was obtained.

(6) Preparation of latex solution carrying protein A: 60 mg of protein A (manufactured by Sigma) was purified water.
After dissolving in 4 mL, 0.25 M glycine buffer (pH
Dialysis was performed overnight at 8.4). 7.7m of this protein A solution
To the L, 0.25 M glycine buffer (pH 8.4) was added to make 13 mL, and 0.25 M glycine buffer (p
13 mL of a suspension of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.2 μm and adjusted to a concentration of 2% with H8.4) was added, followed by stirring at 4 ° C. overnight. Next, 0.05M glycine buffer containing 2% bovine serum albumin (pH
8.4) was added and the mixture was stirred at 4 ° C. overnight. After removing the supernatant by centrifugal washing, 0.05M glycine buffer (pH
8.4) 182 mL was added and mixed well to obtain a protein A-supported latex liquid (protein A-Lx).

(7) Preparation of TAT-carrying latex solution: 10 mg of TAT prepared in the above (1) was dialyzed overnight against a 0.25 M glycine buffer solution (pH 8.4). This TAT
To the solution, 0.25 M glycine buffer (pH 8.4) was added to make 13 mL, and an average particle diameter of 0.2 μm adjusted to 2% concentration with 0.25 M glycine buffer (pH 8.4) was added thereto.
13 mL of a suspension of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) was added, followed by stirring at 4 ° C. overnight. Next, 2%
0.05M glycine buffer containing bovine serum albumin (p
H8.4) was added and the mixture was stirred at 4 ° C. overnight. After removing the supernatant by centrifugal washing, 0.05M glycine buffer (pH
8.4) 182 mL was added and mixed well to obtain a TAT-supported latex liquid (TAT-Lx).

(8) Selection of anti-TAT monoclonal antibody by LTIA method: In the above (4), the hybridoma for which the presence of the anti-TAT monoclonal antibody was confirmed in the culture supernatant by ELISA was further subjected to latex agglutination immunoassay. A monoclonal antibody suitable for the reagent was selected. That is, as the first reagent, 10% glycerin,
Using a solution obtained by dissolving 1 μg / mL of TAT or ATIII in 0.02 M Tris buffer (pH 9.0) containing 0.1% albumin, add 20 μL of the culture supernatant to 200 μL of the first reagent, and add 5 μl at 37 ° C. After heating for 2 minutes, as a second reagent, 100 μL of a solution obtained by mixing 2 mL of the heparin-supported latex solution prepared above and 2 mL of the protein A-supported latex solution was added and stirred. In addition, the first excluding TAT and ATIII
After adding 20 μL of the culture supernatant to 200 μL of the reagent and heating at 37 ° C. for 5 minutes, 2 mL of the TA prepared above was used as a second reagent.
100 μL of a solution obtained by mixing a T-supported latex solution and 2 mL of a protein A-supported latex solution was added and stirred. Thereafter, the amount of change in absorbance at a wavelength of 600 nm for 1 to 5 minutes was measured, and in each case, those having a high absorbance were selected.

(9) Establishment of anti-TAT monoclonal antibody-producing cells: Cells that were positive by ELISA and LTIA were cloned by the standard method using a limiting dilution method. That is, 9 cells containing BALB / c mouse thymocytes and RPMI1640 medium containing 15% FCS.
Specific antibody positive cells were cultured in a 5% carbon dioxide incubator at 37 ° C. using a 6-well microculture plate. EL
The selection of specific antibody positive wells by the ISA method and the monocloning operation by the limiting dilution method were repeated three times each to obtain an anti-TA
T monoclonal antibody producing cells were established. Three of the resulting hybridomas were 26221 and 26, respectively.
223, 26403 and deposited with the National Institute of Advanced Industrial Science and Technology. Table 1 shows the strain names and accession numbers.

[0039]

[Table 1]

(10) Isolation and purification of anti-TAT monoclonal antibody: The anti-TAT monoclonal antibody-producing hybridoma obtained above was cultured in the mouse intraperitoneal cavity to prepare a monoclonal antibody. BALB 8 weeks old as pretreatment
0.5 mL of pristane (2, 6,
10,14-tetramethylpentadecane).
Eight days later, 5 × 10 ⁵ cells suspended in 0.5 mL of RPMI1640 medium were intraperitoneally administered to the mouse. From day 9 after administration, ascites was repeatedly collected and pooled. The collected ascites was centrifuged at 3,000 rpm for 10 minutes to remove insolubles such as cells. An equal volume of a saturated ammonium sulfate solution was added to the supernatant while stirring, and the mixture was allowed to stand overnight at 4 ° C., and the resulting precipitate was collected by centrifugation. The precipitate was dissolved in a 20 mM Tris-HCl buffer (pH 8.0) and dialyzed. After the dialysis contents were adsorbed on a DEAE-Sephacel (Pharmacia) column equilibrated with the same buffer, the column was eluted with a NaCl 0-0.3 M gradient in the same buffer to obtain a purified antibody.

(11) Reaction Specificity of Anti-TAT Monoclonal Antibody: The reaction specificity of the monoclonal antibody obtained above was determined by measuring the reactivity with immobilized TAT, ATIII, thrombin and prothrombin by ELISA. Reactivity with TAT and ATIII was confirmed by the LTIA method. That is, the ELISA method uses TAT (1 μg / m
L), ATIII (1 μg / mL), thrombin (1 μg / m
L) and prothrombin (1 μg / mL) were immobilized on a 96-well microplate, and the purification was performed in the same manner as in (4) above, using a purified monoclonal antibody (5 μg / mL) instead of the culture supernatant. In addition, the LTIA method uses 1
0.0% containing 0% glycerin, 0.1% albumin
1 μg / mL TAT in 2 M Tris buffer (pH 9.0)
Alternatively, 5 μg / mL purified anti-monoclonal antibody 20 was used as a sample in 200 μL of the first reagent using a solution in which ATIII was dissolved.
Then, after heating at 37 ° C. for 5 minutes, 100 μl of a solution obtained by mixing 2 mL of the heparin-supported latex solution and 2 mL of the protein A-supported latex solution prepared above as the second reagent was added.
L was added and stirred. Thereafter, the absorbance at a wavelength of 600 nm was measured. Table 2 shows the results.

[0042]

[Table 2]

(12) Reactivity of anti-TAT monoclonal antibody: Purified anti-monoclonal antibody (26221, 26223, 264) was prepared in the same manner as in the above-mentioned (11) LTIA method.
03 or 26210 (as described in JP-A-7-238099)
Was used to confirm the reactivity. 1 to 600 nm wavelength
Table 3 shows the results of measuring the change in absorbance for 5 minutes.

[0044]

[Table 3]

From the results in Table 3, it was confirmed that the monoclonal antibody of the present invention has high reactivity with TAT and ATIII, and is suitable for a latex agglutination immunoassay reagent.

(13) Preparation of latex carrying anti-TAT monoclonal antibody: The anti-TAT monoclonal antibody purified above (26221, 26223, 26403 or 2403)
6210) in 0.25M glycine buffer (pH 8.
After dialysis overnight in 4), 5 mL of a solution prepared to a concentration of 1.4 mg / mL was mixed with 0.25 M glycine buffer (pH 8.4) to a concentration of 2%, and polystyrene having an average particle size of 0.2 μm. 5 mL of a latex suspension was added, and the mixture was stirred at 4 ° C overnight. Next, 50 mL of 0.05 M glycine buffer (pH 8.4) containing 2% bovine serum albumin was added and mixed well,
Each monoclonal antibody-carrying latex solution was obtained.

(14) TAT measurement using latex agglutination immunoassay reagent: 0.02 M Tris buffer (pH 9.0) containing 2% NaCl, 2% polyethylene glycol 6000 and 0.1% albumin as the first reagent ) Was prepared. On the other hand, as the second reagent, a 1: 1 mixture of each of the monoclonal antibody-supported latex solutions prepared above and heparin-supported latex solution was prepared. First reagent 200μL
20 μL of 1, 10 or 100 ng / mL TAT lysate
After heating at 37 ° C. for 5 minutes, 100 μL of the second reagent was added and stirred. Thereafter, 1 to 5 at a wavelength of 600 nm
The change in absorbance per minute was measured. Table 4 shows the results.

[0048]

[Table 4]

[0049]

The monoclonal antibody of the present invention has a TAT
And high reactivity with ATIII, suitable as an immunoassay reagent, and accurately and simply TAT concentration in a human sample,
It can be detected quickly and with high sensitivity.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) G01N 33/543 581 C12N 5/00 33/577 15/00 CF term (reference) 4B024 AA11 BA44 GA03 HA15 4B064 AG27 CA10 CA20 CC24 DA01 DA13 4B065 AA92X AB05 CA25 CA44 CA46 4H045 AA11 AA20 AA30 CA42 DA76 EA24 EA50 FA72 GA10 GA15

Claims (8)

[Claims] 1. An anti-human thrombin / human antithrombin III complex monoclonal antibody that reacts with free human antithrombin III and also reacts with a human thrombin / human antithrombin III complex. 2. After fusing mammalian antibody-producing cells immunized with an antigenic substance with mammalian myeloma cells, a cell producing an antibody that reacts with the antigenic substance is selected by enzyme immunoassay, A method for producing a monoclonal antibody, comprising selecting cells producing an antibody that reacts with an antigenic substance by latex agglutination immunoassay from the selected cells, and culturing the cells. 3. The production method according to claim 2, wherein the monoclonal antibody is used as a latex agglutination immunoassay reagent. 4. A human thrombin / human antithrombin
After fusing mammalian antibody-producing cells immunized with the III complex with mammalian myeloma cells, cells that produce antibodies that react with the human thrombin / human antithrombin III complex by enzyme-linked immunosorbent assay The method according to claim 1, further comprising selecting cells that produce an antibody that reacts with a human thrombin / human antithrombin III complex by latex agglutination immunoassay from the selected cells, and culturing the cells. 2. The method for producing an anti-human thrombin / human antithrombin III complex monoclonal antibody according to 1. 5. A human thrombin / human antithrombin
The hybridoma producing the monoclonal antibody according to claim 1, obtained by fusing a mammalian antibody-producing cell immunized with the III complex with a myeloma cell of the mammal. 6. The accession number is FERM P-1827.
6, FERM P-18277 or FERM P-1
The hybridoma according to claim 5, which is 8278. 7. An immunoassay for a human thrombin / human antithrombin III complex, comprising the anti-human thrombin / human antithrombin III complex monoclonal antibody according to claim 1. 8. A latex agglutination immunoassay for a human thrombin / human antithrombin III complex, comprising the anti-human thrombin / human antithrombin III complex monoclonal antibody according to claim 1.