JPH0694A - Anti-human plasmin-alpha2-plasmin inhibitor conjugate specific antibody and immunological measurement method - Google Patents

Abstract

(57) [Summary] [Structure] A monoclonal antibody that specifically reacts with human plasmin-α ₂ -plasmin inhibitor complex (PPI), but does not react with human plasmin and human α ₂ -plasmin inhibitor. The antibody is immobilized as a first monoclonal antibody on an insoluble carrier, and then contacted with a test sample, and subsequently with another labeled antibody that specifically reacts with PPI at a site different from the monoclonal antibody. An immunological measurement method for detecting a signal from the label. [Effect] The PPI can be specifically and quickly measured without requiring dilution of the sample and without being affected by the interfering substances in the sample.

Description

Detailed Description of the Invention

[0001]

The present invention relates to human plasmin-α ₂
-Plasmin inhibitor complex (Plasmin-α
_2- Plasmin Inhibitor Compl
ex: the following abbreviation PPI may be used), but a monoclonal antibody that does not react with human plasmin and human α ₂ -plasmin inhibitor, and a method for immunologically measuring PPI using the monoclonal antibody Regarding More specifically, the present invention can accurately and reproducibly analyze PPI existing in plasma without diluting a plasma sample without being interfered with by coexisting human plasminogen and human α ₂ -plasmin inhibitor. , Relates to a method of measuring immunologically.

[0002]

2. Description of the Related Art Generalized intravascular coagulation syndrome (DIC: Di)
sseminated intravasular
coagulation) plasma, urokinase or tissue plasminogen activator (t-
PA) in the plasma of patients undergoing thrombolytic therapy,
Plasminogen is activated to produce plasmin. The generated plasmin is instantaneously converted into α ₂ plasmin inhibitor (α ₂ PI: Aoki et al., J. Bio, in bloodstream).
l. Chem. , 251, 5956-5965, 197.
6) forms a 1: 1 complex with 6), ie the PPI described above.
Therefore, by measuring PPI, the event of plasmin production, that is, the activation of the fibrinolytic system can be grasped. Recently, PPI in plasma has been emphasized as a molecular marker for diagnosis of DIC and monitoring of thrombolytic therapy. Therefore, it is necessary to measure the amount of PPI in blood or plasma accurately and easily.

The following four methods can be cited as conventionally known PPI measuring methods. The first method is a method using a two-dimensional cross immunoassay method. The second method is a latex agglutination method using a polyclonal antibody that recognizes PPI neoantigen. The third method is an enzyme immunoassay method in which both a polyclonal antibody against plasminogen and a polyclonal antibody against α ₂ -plasmin inhibitor are used, one of which is an immobilized antibody and the other is an enzyme-labeled antibody. A fourth method uses both a monoclonal antibody and a plasmin polyclonal antibody that specifically recognize the site of the α ₂ -plasmin inhibitor that blocks the fibrinolytic action of pramison, one of which is used as the immobilized antibody and the other of which is used. Is an enzyme-labeled antibody, and a plasma sample is diluted 1200 times and measured.

However, the first method has the drawback of low sensitivity and lack of quantitativeness. The second method has a drawback that PPI cannot be specifically measured. The third method has good sensitivity, but has a drawback in that it is difficult to secure a stable antiserum, and the immune reaction has two steps, so that the operation is complicated and the measurement requires a long time.
The fourth method is improved from the third method in that a monoclonal antibody against α ₂ -plasmin inhibitor is used and the immune reaction is manipulated in one step.
It does not solve the drawback of the enzyme immunoreaction, that is, the problem that the operation is complicated and the measurement takes a long time. Further, the fourth method has a drawback that a step of diluting a sample by 1200 times is necessary in order to introduce the immune reaction in one step.

[0005]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present inventor has conducted diligent research to develop a method for simply, accurately and reproducibly measuring PPI.
Monoclonal antibody (especially PPI No. 5)
0), a second monoclonal antibody reactive with both PPI and plasminogen (especially PPI No.
Two monoclonal antibodies of 3) were found. Therefore, the present invention provides a method for simply, accurately and reproducibly measuring PPI using the newly found monoclonal antibody.

[0006]

That is, the present invention relates to a monoclonal antibody which specifically reacts with human plasmin-α ₂ -plasmin inhibitor complex, but does not react with human plasmin and human α ₂ -plasmin inhibitor.

Furthermore, in the present invention, the above-mentioned monoclonal antibody is immobilized on an insoluble carrier as a first monoclonal antibody, the immobilized first monoclonal antibody is contacted with a test sample, and then the above-mentioned first monoclonal antibody is contacted. Characterized in that a signal from the label is detected by contacting with a labeled second antibody that specifically reacts with the human plasmin-α ₂ -plasmin inhibitor complex at a site different from that of the monoclonal antibody of 1. The present invention relates to a method for immunologically measuring an anti-human plasmin-α ₂ -plasmin inhibitor complex.

Human P used as an immunogen in preparing a hybridoma secreting the monoclonal antibody of the present invention
PI is, for example, the method of Plow et al. (J. Lab. Cl
in. Med. 93 , 199-209, 1979). Using the purified human PPI immunogen solution, a mammal (eg, mouse or rat) is immunized by the in vivo immunization method by a conventional method, and the spleen is aseptically removed from the immunized animal to prepare spleen cells, Used in the cell fusion process. Various known cell lines can be used as the myeloma cell which is the other parent cell used for cell fusion.

Cell fusion between the immune spleen cells and the myeloma cells can be performed by a usual method. For example, immune spleen cells and myeloma cells, for example, spleen cells are used in an amount of about 1 to 10 times that of myeloma cells in the presence of a known fusion promoter and optionally an adjuvant. Immune spleen cells and myeloma cells were mixed well in a fusion medium, then cells other than hybridomas were removed using a selection medium, and the presence or absence of antibody production in the hybridoma culture supernatant was determined by, for example, immunoprecipitation. After confirmation, the target hybridoma can be isolated.

The hybridoma thus obtained can be subcultured in an ordinary medium and can be easily stored in liquid nitrogen or the like for a long period of time. The culture medium of the hybridoma is not particularly limited, and a medium containing fetal bovine serum, L-glutamine, L-pyruvic acid and an antibiotic is preferably used in DMEM. In the case of in vitro culture of hybridoma, for example, 5% CO _{2 in a} medium is used.
It is preferably carried out at a concentration of 37 ° C. for about 3 days, and for about 14 days when cultured in vivo, for example, in the abdominal cavity of mice.

From the culture solution obtained by culturing the above hybridoma by a conventional method, or from the ascites of a suitable mammal (for example, mouse or rat) to which the hybridoma is administered,
The desired monoclonal antibody can be separated and purified. When the monoclonal antibody is separated and purified from the thus-produced culture medium or mouse ascites fluid, the method generally used for protein isolation and purification, for example, ammonium sulfate salting out, ion exchange chromatography, Molecular sieve column chromatography using a molecular sieve gel, affinity column chromatography using a protein A-bound polysaccharide, dialysis, freeze-drying and the like can be used.

Human plasmin-α ₂ -obtained in this way
Reacts specifically with the plasmin inhibitor complex,
Human plasmin and human alpha ₂ - The plasmin inhibitor using monoclonal antibodies which do not react, it is possible to detect the PPI in a test sample immunologically. In particular,
An insoluble carrier on which a monoclonal antibody having the above-mentioned properties is immobilized as a first monoclonal antibody, and another labeled antibody (this antibody may be a monoclonal antibody or a polyclonal antibody, preferably the first monoclonal antibody) And (using a monoclonal antibody that specifically reacts with PPI at a site different from that) can be suitably performed.

Specifically, the first monoclonal antibody is immobilized on a suitable insoluble carrier (immobilized antibody). Next, in order to avoid nonspecific binding between the insoluble carrier and the specimen sample, the surface of the insoluble carrier is coated with an appropriate substance [eg, bovine albumin (BSA) or rabbit albumin (RSA)]. Subsequently, an undiluted specimen sample is added to the solution for a fixed time (for example, 5 minutes to 3 hours) and a fixed temperature (for example, 4 ° to 4 hours).
The reaction is carried out by contacting at 0 °, preferably near room temperature (1
Next reaction). This is washed with an appropriate washing solution (for example, physiological saline containing a surfactant), a labeled antibody is added thereto, and a certain time (for example, 5 minutes to 3 hours) and a certain temperature (for example, 4 °).
The reaction is carried out by bringing them into contact at -40 °, preferably near room temperature (secondary reaction). After washing with an appropriate washing solution (for example, physiological saline containing a surfactant), the amount of labeled antibody present on the insoluble carrier is quantified. From the value, PP in the sample
The amount of I can be calculated. It is also possible to carry out the primary reaction and the secondary reaction at the same time.

The insoluble carrier that can be used in the measuring method of the present invention is not particularly limited, and examples thereof include polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, and polysaccharide. Examples thereof include polymers such as nitrocellulose, paper, agarose, and combinations thereof.

It is advantageous to use an enzyme, a fluorescent substance or a luminescent substance as the labeling substance. Alkaline phosphatase, peroxidase, β-D-galactosidase and the like can be used as the enzyme, fluorescein isothiocyanate and the like as the fluorescent substance, and acridinium ester, luciferin and the like as the luminescent substance.

The test sample used in the immunological quantification method of the present invention is not particularly limited as long as it is a sample that may contain PPI. For example, a biological sample, particularly blood, serum, Plasma or urine, preferably plasma. In the immunological quantification method of the present invention, even if the test sample is used as it is without being diluted, interference with plasminogen and α ₂ -plasmin inhibitor existing in a free state in the test sample is avoided. be able to.

[0017]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. Example 1: Purification of PPI (a) Purification of human plasmin-α ₂ -plasmin inhibitor complex (PPI) was performed according to the method of Plow et al. (J. La.
b. Clin. Med. 93 , 199-209, 197.
9). Briefly, 100 ml of human (healthy) plasma was passed through a lysine-sepharose column (bed volume, 100 ml) to remove plasminogen. To this plasminogen-free plasma, 100 ml of an aqueous plasmin solution (60 μg / ml) was gradually added, and then the mixture was incubated at 37 ° C. for 10 minutes. After the reaction, the aqueous solution was added to a lysine-sepharose column (bed volume, 200 m
It was passed through l) to adsorb PPI. After washing the PPI adsorption column with 0.1 M phosphate buffered saline (PBS), PP was added with PBS containing 50 mM ε-aminocaproic acid.
I was eluted. The PPI obtained was further purified by molecular sieve chromatography on Ultrogel ACA44. This purified PPI was used to screen for immunogen and anti-PPI monoclonal antibody.

(B) Preparation of immunized spleen cells: PPI immunogen solution (A280 nm = 0.1) was mixed with an equal amount of Freund's complete adjuvant until emulsified, and the mixed solution 2
Immunization was performed by intraperitoneally administering 00 μl of BALB / c mouse (first immunization). After 30 days,
200 μl of the same mixed solution as described above was intraperitoneally administered to the mouse (second immunization). Twenty-one days after the second immunization, 200 μl of PPI diluent prepared by diluting a PPI immunogen solution (A280 nm = 0.1) with an equal amount of physiological saline.
1 was administered intravenously to the mice (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse and used in the next cell fusion step.

(C) Cell fusion The aseptically extracted spleen was placed in a petri dish containing 5 ml of DME medium containing 15% fetal bovine serum. Next, the spleen was refluxed with about 15 ml of DME medium containing 15% fetal bovine serum to allow spleen cells to flow out, and the spleen cell suspension was passed through a nylon mesh. The spleen cells were collected in a 50 ml centrifuge tube and centrifuged at 500 xg for 10 minutes. Thus obtained pellets hemolysin Rising solution _{(155mM-NH 4 Cl, 10mM} -KHCO 3, 1
4 ml of mM-Na ₂ EDTA: pH 7.0) was added and suspended. It was left at 0 ° C for 5 minutes to destroy the red blood cells in the suspension. DME medium containing 10 ml of 15% fetal bovine serum was added and then centrifuged. The pellet thus obtained was washed with DME medium by centrifugation, and the number of living spleen cells was measured.

On the other hand, 1 × 10 ⁸ spleen cells were added to about 2 × 10 ⁷ cells of mouse myeloma cells (myeloma cells) SP2 / 0-Ag14 (RIKEN Gene Bank Cell Bank) that had been cultured in advance. , Mix well in DME medium,
Centrifugation was performed (500 xg, 10 minutes). The supernatant is aspirated, the pellet is thoroughly disentangled, and 40% polyethylene glycol 4000 solution (incubated at 38 ° C) 0.5 m
l was added dropwise, and the centrifuge tube was gently rotated by hand for 1 minute to mix the polyethylene glycol solution with the cell pellet. Next, D kept warm at 38 ° C
1 ml of ME medium was added every 30 seconds, and the tube was gently rotated. After repeating this operation 10 times, 1
Add DME medium containing 20 ml of 5% fetal bovine serum,
Centrifugation (500 xg, 10 minutes) was performed. After removing the supernatant, HAT medium containing 15% fetal bovine serum (DM
Aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 in E medium
The cell pellet was washed twice by centrifugation with 10 × 10 ⁻⁵ M and hypoxanthine (1 × 10 ⁻⁴ M), and then suspended in 40 ml of the HAT medium. Add 2 to each well of this 96-well cell culture plate.
It was dispensed in an amount of 00 μl each, and the culture was started at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. During the culture, about 100 μl of the medium in each well was removed at intervals of 2 to 3 days, and the above H
Hybridomas growing in HAT medium were selected by adding 100 μl of AT medium. 15% from the 8th day
Change to HAT medium containing fetal calf serum (DME medium added to thymidine at 1.6 × 10 ^-5 M and hypoxanthine at 1 × 10 ^-4 M), observe the hybridomas, and at day 10 By the immunoprecipitation method described below,
The PPI antibody-producing hybridomas were screened.

(D) Establishment of hybridoma The presence and specificity of the produced antibody in the supernatant of the hybridoma culture broth was examined by immunoprecipitation method. That is, 50 μl (200 μg / ml) of the culture supernatant of each well was mixed and reacted at room temperature for 2 hours. Next, 100 µl of anti-mouse immunoglobulin antibody-immobilized Sepharose 4B (having 1-2 mg of antibody immobilized per 1 ml of gel) and 0.5M-Na.
800 μl of 20 mM Tris-hydrochloric acid buffer solution (pH 7.6) containing Cl (hereinafter referred to as buffer solution A) was added and mixed at 4 ° C.
Then, the mixture was mixed by inversion overnight. After the gel was washed 5 times with the buffer solution A by the centrifugation method, the gel was suspended in 2% SDS-20 mM Tris-HCl (pH 7.6) and mixed by inversion at room temperature for 2 hours.
After centrifugation (700 × g, 5 minutes, 20 ° C.), the supernatant was subjected to SDS-PAGE (3.5-9.0% gel),
Silver staining was performed to examine the reaction specificity with PPI. Similarly, human plasminogen and the human alpha ₂ - were also tested for reactivity with plasmin inhibitors. As a marker, purified PPI, purified human plasminogen, purified human alpha ₂ - a plasmin inhibitor and molecular weight markers were used.

By screening the hybridoma clones producing the anti-PPI-specific antibody by the immunoprecipitation method as described above, the reactivity with the protein antigen contained in the solution in a natural (natural) state can be observed. . That is, E commonly used as a screening method
In the LISA method, a protein antigen is directly bound to a solid phase to check the specificity of the antibody. Therefore, an antibody that recognizes an artificial structural change due to immobilization is a monoclonal antibody that specifically reacts with a natural antibody. The above-mentioned immunoprecipitation method can prevent such misconception, although a false judgment that the

By the above screening, 20 to 40 antibody-producing clones were obtained for each hybridoma. From these clones, a clone having a strong proliferative ability, a high antibody secretory ability, and a stable one was selected and recloned in the same manner as described above, and two kinds of anti-PPI antibody-producing hybridoma PPI No. 50-1-1-1 and PPI No. 3-1-2-2 was established. Two monoclonal antibodies PPI No. 2 secreted from these two hybridomas. 50 and PPI No. 3 and human plasminogen (Atens Research, USA) or human α ₂ -plasmin inhibitor (Atens
(Research, USA) was examined by the same method as the immunoprecipitation method described above. Monoclonal antibody PPI
No. 50 is also human plasminogen, human α ₂ −
It also did not respond to plasmin inhibitors. Monoclonal antibody PPI No. 3 reacted with human plasminogen but not with human α ₂ -plasmin inhibitor.

Each of the above hybridomas has been deposited at the Institute of Microbial Engineering, Institute of Industrial Technology since June 12, 1992. The deposit number is Hybridoma PPI No. 5
0-1-1-1 is Micromachine Research Institute, No. 13001 (FERM
P-13001) and the hybridoma PPI N
o. 3-1-2-2 is Microtechnology Research Institute Bacteria No. 13002 (FE
RM P-13002).

Example 2: Production of monoclonal antibody (a) In vitro method Mouse hybridoma PPI No. 50-1-1-1
And PPI No. 3-1-2-2, 15% each
The cells were cultured in DME medium containing fetal bovine serum at 37 ° C. in a 5% carbon dioxide atmosphere for 72 to 96 hours. After centrifuging the culture (10,000 xg, 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. The mixture was stirred under ice cooling for 30 minutes and then 6
Centrifuge (10,000 xg, 10 minutes) after standing for 0 minutes, dissolve the resulting precipitate in a small amount of 10 mM phosphate buffer (pH 8.0), and use 1000 times the volume of 10 mM phosphate buffer. The column was packed with DEAE-cellulose equilibrated in. Elution of monoclonal antibody is 10m
It was performed by a concentration gradient method between M phosphate buffer (pH 8.0) and 10 mM phosphate buffer (pH 8.0) containing 0.2 M NaCl. The eluted monoclonal antibody was concentrated by an ultrafiltration method, and 0.1 M phosphate buffer (pH 8.
It was dialyzed against 0). To remove bovine serum IgG,
The dialysate was passed through a goat anti-bovine serum IgG-Sepharose 4B column. Next, the flow-through solution is a 0.1 M phosphate buffer solution (p
H-8.0) Equilibrated with Protein A-Sepharose 4
B column was packed. Purified anti-PPI specific monoclonal antibody PP by eluting the column with a buffer solution of pH 2.5
I No. 50, and also the monoclonal antibody PPI
No. A solution of 3 was obtained.

(B) In Vivo Method 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to 10-12 week old BALB / c mice, and then 14-20. Hybridoma PPI No. 50-1-1-1 or PPI No. Three
-1--2-2 was inoculated so that 2 × 10 ⁶ mice were inoculated. About 10 to 15 ml of ascites was obtained from one mouse for each hybridoma. The antibody concentration was 2-10 mg / ml. Purification of the monoclonal antibody in ascites was performed by the same method as the above-mentioned in vitro purification (except the operation of passing a column of goat anti-bovine serum IgG-Sepharose 4B).

Example 3: Immunoglobulin of monoclonal antibody
Identification of Bryn class and specificity (a) Anti-PPI specific monoclonal antibody PPI N
o. 50 and PPI No. Identification of the 3 immunoglobulin classes and specificities was performed by the Octoney immunodiffusion method and the enzyme immunoassay method, respectively. The results are shown in Table 1.

[0028]

[Table 1]

(B) Monoclonal antibody PPI No. 50 and PPI No. The characteristics of 3 were investigated. That is, the purified monoclonal antibody PPI No. 1 obtained in Example 2 was used. 50 and PPI No. 3 was immobilized on bromocyaninated sepharose 4B (Pharmacia) according to a conventional method. 1-2 mg of antibody was immobilized per ml of gel. Monoclonal antibody-immobilized Sepharose 4B
Gel (MoAb gel) and various antigen samples, ie, purified human plasminogen (Atens Research Co.), α ₂
-Plasmin inhibitor (Atens Research),
PPI [Example 1 (a) above], plasma, and urokinase-treated plasma were mixed, and immunoprecipitation was performed by the following procedure. 100 μl of the antigen sample and the buffer A790
μl and Aprotinin (100
TIU / ml) 10 μl and monoclonal antibody PPI
No. 50 or PPI No. 3 (100 μl of F (ab ′) ₂ -Sepharose 4B) was mixed and mixed by inversion at 5 ° C. overnight. The gel was washed 5 times with buffer A by centrifugation and washed with 2% SDS-20 mM Tris-HCl (pH 7.6; 25).
The gel was suspended in (° C) and mixed by inversion at room temperature for 2 hours. Furthermore, the supernatant obtained by centrifugation (700 × g, 5 minutes, 20 ° C.) was separated by SDS-PAGE (3.5 to 9.0% gel), and silver staining was performed. Monoclonal antibody PPI N
o. 50 and PPI No. Reactivity of 3 (SDS-PAG
E, silver staining) is shown in FIG. As a marker, purified P
PI purified human plasminogen, purified human alpha ₂ - a plasmin inhibitor and molecular weight markers were used.

Example 4: By enzyme immunoassay (EIA)
Measurement of PPI (a) Preparation of solid phase antibody Monoclonal antibody PPI No. It is prepared in 50 mM phosphate buffer saline (pH 7.5) so that the IgG fractionated protein concentration of 50 is 5 μg / ml. 50 μl each of this antibody solution was added to a 96-well ELISA plate (I
Mmulon II, Nippon Dynatech) was dispensed into each well and left at 4 ° C. for 24 hours. (B) Preparation of labeled antibody Monoclonal antibody PPI No. IgG fraction of 3
Horseradish peroxidase (HR
Labeling with P) gave a labeled antibody. (C) Preparation of calibration curve Monoclonal antibody PPI N obtained in (a) above
o. 50-fixed 96-well ELISA plate was added to 0.
After washing three times with 05% Tween-20 physiological saline,
Purified PPI of Example 1 (a) 40 μg / ml, 20 μm
g / ml, 10 μg / ml, 5 μg / ml, 2 μg / ml, 1 μ
50 μl of a solution having a concentration of g / ml and 0.5 μg / ml was added to each well and reacted at 25 ° C. for 10 minutes. 0.05% T
After washing three times with a ween-20-saline solution, the horseradish peroxidase (HRP) of the above (b) was used.
Labeled monoclonal antibody PPI No. 0.05% of 3
50 μl of a Tween-20-50 mM phosphate buffer-physiological saline (pH 7.5) solution was added, and the mixture was reacted at 25 ° C. for 10 minutes. Then, after washing three times with 0.05% Tween-20-physiological saline, a substrate solution for HRP [0.5 mM aminoantipyrine, 10 mM phenol and 0.005%
50 mM phosphate buffer (pH 7.5) containing hydrogen peroxide solution]
200 μl was added to each well and reacted at 25 ° C. for 10 minutes, and the absorbance at 490 nm of each well was measured. The obtained calibration curve is shown in FIG.

Example 5: Addition and recovery test of purified PPI Five kinds of samples (healthy humans A and B, and DIC patients C and D)
And P) in plasma collected from E), and then purified PPI of 2.0 μg / ml for each sample.
0 μg / ml and 8.0 μg / ml were added and a recovery test was conducted. As shown in Table 2, the results show a recovery rate of 94-10.
It was as good as 4%.

[0032]

[Table 2]

Example 6: PPI of healthy subjects and DIC patients
According to the measuring method of the value Example 5, healthy human plasma 12 specimens, DIC
The PPI of 15 patient plasma samples was measured. The results are shown in Fig. 3. The PPI value in the healthy group was less than 1 μg / ml in all cases. In contrast, the DIC patient group was 2 μg / ml or more in all cases.

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, the PPI in a patient's plasma can be identified without interfering with the free plasminogen and the free α ₂ -plasmin inhibitor in the plasma even if the plasma sample is not diluted. Therefore, it is possible to measure accurately and quickly. This was made possible for the first time by the present invention. Therefore, the present invention provides a useful means for diagnosis and pathological research such as DIC.

[Brief description of drawings]

FIG. 1: Monoclonal antibody PPI No. 50 and P
PI No. FIG. 3 is an explanatory view showing the reactivity with plasminogen, α ₂ -plasmin inhibitor and PPI by the immunoprecipitation method of 3.

FIG. 2 is a graph showing a calibration curve for quantification of PPI.

FIG. 3 is a graph showing measured values of a complex of a healthy person and a DIC patient.

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Claims (4)

[Claims] 1. A monoclonal antibody which specifically reacts with human plasmin-α ₂ -plasmin inhibitor complex, but does not react with human plasmin and human α ₂ -plasmin inhibitor. 2. A human plasmin-α ₂ -plasmin inhibitor complex formed by fusion between a mammalian spleen cell and a mammalian myeloma cell immunized with the human plasmin-α ₂ -plasmin inhibitor complex, and specifically with the human plasmin-α ₂ -plasmin inhibitor complex. reactions, but human plasmin and human alpha ₂ - hybridomas secreting monoclonal antibodies which do not react with plasmin inhibitors or cell lines derived therefrom. 3. The monoclonal antibody according to claim 1 is immobilized on an insoluble carrier as a first monoclonal antibody, the immobilized first monoclonal antibody is contacted with a test sample, and then the first monoclonal antibody is contacted. Characterized in that it is contacted with a labeled second antibody that specifically reacts with human plasmin-α ₂ -plasmin inhibitor complex at a site different from that of the monoclonal antibody, and the signal from said label is detected. An immunological assay method for an anti-human plasmin-α ₂ -plasmin inhibitor complex. 4. The immunological assay method according to claim 3, wherein a monoclonal antibody is used as the labeled second antibody.