JP2958510B2 - How to detect thrombosis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting thrombosis by using a monoclonal antibody against human hepatocyte growth factor in an immunoassay and a diagnostic agent for thrombosis used in the method.

[0002]

2. Description of the Related Art Thrombosis is a phenomenon in which blood coagulates in a heart or a blood vessel due to a change in blood properties, a stagnant blood flow, a change in a blood vessel wall, etc., stops the blood flow, and is nourished by the blood vessel or the like. Refers to the lesion that occurs in the area where it is located. The cause is often arteriosclerosis, and cerebral thrombosis and myocardial infarction are extremely important diseases.

However, a detailed description of myocardial infarction, which is one of the most representative of the above-mentioned thrombosis, is described. Among them, acute myocardial infarction is a basic lesion of which is located in a relatively proximal part of the coronary artery. Atherosclerosis disintegrates in the part where hardening occurs,
It is thought that the contents of the blood partially flow into the blood vessel, the intimal surface becomes unstable, secondary clots are formed there, and luminal obstruction occurs relatively easily in combination with the original stenosis. Have been. Also, if the thrombus is formed, TXA
₂ (thromboxane A ₂ ) is formed, which is also known to cause platelet aggregation, while causing spasm of the coronary arteries (spasm), and this vicious cycle is associated with the myocardial infarction and angina pectoris. It is also thought to be a basic condition.

[0004] Acute myocardial infarction has been treated by CCU, and introduction of an electrocardiogram monitor and cardiopulmonary resuscitation, application of antiarrhythmic drugs and cardiac pacemakers, and the like have contributed greatly to a reduction in acute phase mortality. In addition, acute heart failure, which accounts for the majority of acute deaths in recent years, or shock syndrome,
Efforts are being made to develop more effective treatments.

[0005] In order to provide more rational and effective treatment for acute heart failure associated with myocardial infarction and to maintain and improve cardiac function while preventing the progression of ischemia, it is necessary to first determine hemodynamics in individual infarct cases. It is necessary to accurately understand the degree of disability, and also to be careful in monitoring the progress of myocardial damage and selecting an appropriate treatment method according to the changing degree of disability.

Until recently, the frequency of coronary thrombosis in autopsy cases of myocardial infarction, ranging from 21% to 97%, was markedly different among researchers, and consequently the causal relationship between the two was continually discussed. Naturally, there are differences in the search target and method, the gap between myocardial infarction in a clinical sense and that morphologically confirmed on autopsy, and various pathological changes. It is presumed that the result of intertwining is the presence of an infarct type.

[0007] However, recently, the development of aggressive clinical diagnostic methods and treatments for acute myocardial infarction is about to end this debate. That is, according to DeWood et al.
In a clinical search using coronary angiography and the like in 17 cases of transmural myocardial infarction, coronary artery thrombus was observed in 73%, 80% in cases within 6 hours, 59% in cases of 6 to 12 hours, and 12 to 24.
The time rate is 54%, and the importance of coronary thrombosis as a myocardial infarction onset factor is recognized again, including the validity of wide application of coronary thrombolysis.

[0008] Attempts to perform a biochemical diagnosis by capturing substances flowing out of the necrotic myocardium into the blood with the onset of acute myocardial infarction, in particular, a myocardial escape enzyme, have been attempted to detect GOT (glutamic oxaloacetic transaminase) in the serum during myocardial infarction. ), LD
Increase in H (lactic deydrogenase) etc.
It began in 1979 with the first report by Karmen et al. Then, HBD (α-hydroxybutyrate dehydro
Increases in enzymes such as genase) and CPK (creatine phosphokinase, international name: creatine kinase, CK) have become known and have been widely applied to clinical diagnosis.

However, in order to improve the diagnostic accuracy and to estimate the severity, further improvements are required, and the measurement of CPK-MB, a CPK isozyme, and myosin light chain, a structural protein, has come to be performed. ing.

GOT is an enzyme most contained in the heart and liver, and is also present in skeletal muscle, kidney, pancreas and the like. Elevated GOT is essential for diagnosis of myocardial infarction,
Differentiation from liver disease can be problematic. Maximum value is 12
Appears after ~ 30 hours and rises for 3-5 days. But G
Because OT is increased by hepatitis, liver congestion, and surgery,
The specificity of myocardial infarction diagnosis using this as an index is low.

[0011] Although LDH is contained most in the kidney and heart, it is also widely distributed in skeletal muscle, erythrocytes, pancreas, spleen, liver, brain and lung, so it is necessary to distinguish it from other diseases as in GOT. is there. LDH is increased in 90% or more of the cases similarly to GOT, but is similarly low in specificity because it is also abundantly contained in liver, skeletal muscle, and red blood cells.

HBD is an α-hyd among LDH isozymes.
with high affinity fractions to droxybutyrate, LDH _1, LDH
_It is almost the same enzyme as ₂ and has high specificity for myocardium. It is said that when the HBD / LDH ratio is 0.81 or more, the possibility of myocardial infarction is high.

CK is most abundant in skeletal muscle, followed by the heart, brain,
Although the intestinal tract and stomach are included in that order, but the liver, kidney, and blood hardly contain it, the test using this as an index
It is considered to be the most specific test method for the diagnosis of myocardial infarction, and the infarct size can be estimated. The CK rises and becomes a problem due to intramuscular injection, electric shock, surgery, cardiogenic shock, etc. other than the diagnosis of myocardial infarction.
Can be identified by measuring CK-MB, which is an isozyme.

The CK isozymes include MM and B
B and MB are known, MM is specific to skeletal muscle, BB is specific to brain, and MB is specific to myocardium. Myocardium has 1
It contains 0 to 40% of CK-MB and the rest is CK-MM, but the content of CK-MB in skeletal muscle is 3% or less.
When an increase in CK is observed, it is said that in adults, if CK-MB is at least 5% of the total CK value, it may be considered to be derived from myocardium.

However, the value of CK greatly differs depending on the measuring method and the analyzer for measurement, and it is known that even if the same sample is measured, the difference in the measured value between facilities sometimes becomes about four times. Has the disadvantage that it is not possible to compare values measured at different facilities.

As described above, there is no method for simply and more reliably detecting thrombosis such as myocardial infarction, and establishment of such a detection method is demanded in the art.

[0017]

Accordingly, it is an object of the present invention to establish a method for detecting thrombosis as required in the art.

The present inventors have conducted intensive studies for the above purpose, and as a result, obtained using human hepatocyte growth factor (HGF) as an immunogen, utilizing monoclonal antibodies having specific reactivity to human HGF, For example, when measuring HGF in a patient's serum, it was found that the measured value well reflected thrombosis, and the present invention was completed here.

[0019]

That is, according to the present invention,
A method for detecting thrombosis, characterized in that human HGF is obtained as an immunogen and thrombosis is detected by an immunoassay using a monoclonal antibody having specific reactivity to human HGF, and human HGF is used as an immunogen. Obtained human HG
A diagnostic agent for thrombosis, comprising a monoclonal antibody having specific reactivity to F as an essential component is provided.

The present invention makes it possible for the first time to detect a thrombosis patient represented by myocardial infarction or cerebral thrombosis, in particular, early diagnosis, and its clinical value is extremely high.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the monoclonal antibody used in the present invention and an immunoassay method using the antibody will be described in detail.

The antibody used in the present invention is obtained by using human HGF as an immunogen, and can be appropriately selected from monoclonal antibodies having specific reactivity to human HGF. Here, the human HGF used as an immunizing antigen does not need to be natural HGF derived from humans, but is a recombinant HGF obtained by a genetic engineering technique or an equivalent (fragment) having a partial structure thereof. Is also good. In addition, those obtained by using the above HGF or a fragment thereof as a hapten and binding them to a carrier with a binding reagent according to a usual immunizing antigen production technique can also be used. These cells may be separately isolated and prepared from appropriate human cells or the like, or known cells may be appropriately obtained and used.

The production of the desired monoclonal antibody using these as an immunizing antigen is more specifically carried out, for example, in a mammalian plasma cell (immune cell) immunized with the above-mentioned immunizing antigen and a mammalian plasmacytoma cell (myeloma). The fusion can be carried out by preparing a fusion cell (hybridoma) with the cell, selecting a clone producing an antibody (monoclonal antibody) recognizing the desired HGF, and culturing the clone. The production of such monoclonal antibodies
Basically, it can follow the usual law [for example, Hanfland,
P., Chem.Phys.Lipids, 15 , 105 (1975): Hanfland,
P., Chem.Phys.Lipids, 10 , 201 (1976): Koscielak,
J., Eur. J. Biochem., 37 , 214 (1978) etc.].

In the method, the mammal to be immunized with the immunizing antigen is not particularly limited, but in consideration of compatibility with the plasmacytoma cell used for cell fusion, generally,
Mice, rats and the like are advantageously used. Immunization can be performed by a general method, for example, by administering the above immunizing antigen to a mammal by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like. More specifically, for example, in the case of a mouse, the immunizing antigen is diluted to an appropriate concentration with a phosphate buffer solution (PBS) containing physiological saline, physiological saline, or the like, and, if desired, used in combination with a normal adjuvant. Administered several times every 2-14 days,
It is preferred that the total dosage be on the order of about 100-500 μg / mouse. As the adjuvant, pertussis vaccine, complete Freund's adjuvant, alum and the like can be preferably used. As the immunizing antigen, it is preferable to use splenocytes extracted about 3 days after the final administration.

Various mammalian plasmacytoma cells as the other parent cells to be fused with the above-mentioned immune cells are known in the art, for example, p3 / × 63-Ag8 (X63)
[Nature, 256 , 495-497 (1975)], p3 / X63-A
g8. U1 (P3U1) [Current Topics in Microbio
logy and Imunology, 81 , 1-7 (1978)], P3 / NSI
-1-Ag4-1 (NS-1) [Eur. Immunol., 6 ,,
511-519 (1976)], Sp2 / 0-Ag14 (Sp2 /
0) [Nature, 276 , 269-270 (1978)], FO [J.
unol. Meth., 35 , 1-21 (1980)] et al. RCY3. Ag 1.2.3. (Y3) [Natu
re, 277 , 131-133 (1979)].

The fusion reaction between the above immune cells and plasmacytoma cells can be performed by a known method, for example, by using the method of Milstein.
The method can be carried out according to the method [Method in Enzymology, 73 , 3 (1981)] and the like. More specifically, the above-mentioned fusion reaction is carried out in a normal medium in the presence of a normal fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like. An auxiliary agent such as dimethyl sulfoxide can be added as needed to increase the amount. In addition, a method by electric processing (electric fusion) or the like can be appropriately adopted. The use ratio of immune cells to plasmacytoma cells is not different from the usual method.
It is common to use about 10 to 10 times. Examples of the culture medium for the fusion reaction include various culture mediums usually used for the growth of the above-described plasmacytoma cells, such as RPMI-1640 medium, MEM medium, and other culture mediums commonly used for such cell culture. It is preferable that serum replacement fluid such as fetal calf serum (FCS) be removed from the medium. Fusion, a predetermined amount of the immune cells and plasmacytoma cells, well mixed in the medium,
A PEG solution pre-heated to about 37 ° C., for example, one having an average molecular weight of about 1,000 to 6,000,
It is performed by adding and mixing at a concentration of 60 W / V%. Thereafter, a desired hybridoma is formed by repeating the operation of successively adding an appropriate medium, centrifuging, and removing the supernatant.

The desired hybridoma separation obtained is
It is performed by culturing in a usual selection medium, for example, a HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). The culture in the HAT medium may be performed for a period of time sufficient to kill cells (unfused cells and the like) other than the target hybridoma, usually several days to several weeks. The hybridoma thus obtained is subjected to a search for a target antibody and a single cloning by a usual limiting dilution method.

The search for the target antibody-producing strain is performed, for example, by ELISA
Method A [Engvall, E., Meth. Enzymol., 70 , 419-439 (198
0)], plaque method, spot method, agglutination method, uchterlony method, radioimmunoassay (RI
A) Various methods generally used for the detection of antibodies such as the method ["Hybridoma method and monoclonal antibody", published by R & D Planning, pp. 30-53, March 1982
5), and the immunizing antigen can be used for this search.

The hybridoma producing the desired monoclonal antibody recognizing HGF thus obtained can be subcultured in a usual medium, and can be stored for a long time in liquid nitrogen. Collection of the monoclonal antibody from the hybridoma is performed by culturing the hybridoma according to a conventional method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal compatible with the hybridoma and proliferating the hybridoma to obtain the ascites. Is adopted. The former method is suitable for obtaining high-purity antibodies, and the latter method is suitable for mass production of antibodies.

The antibody-producing hybridoma culture supernatant and mouse ascites obtained according to the above method can be used as such as a crude antibody solution without any modification. Aminonium sulfate fractionation, salting out, gel filtration can be performed according to a conventional method. The antibody can be purified by affinity chromatography such as a method, ion exchange chromatography, and protein A column chromatography to obtain a purified antibody. A particularly preferred specific example of the above monoclonal antibody is, for example, that described in Japanese Patent Publication No. 5-60359.

Next, the immunoassay for detecting thrombosis of the present invention will be described in detail. For example, the immunoassay can be performed according to a sandwich method or the like as an ordinary enzyme immunoassay.
In addition, the RIA method using a normal competitive method, a sandwich method, etc.,
An ELISA method, an agglutination method, or the like can also be used. The operation, procedure, etc. of each of these methods can be in accordance with ordinary methods.

In particular, the method of the present invention is preferably a three-step sandwich method using an HGF monoclonal antibody because of its high sensitivity. This method is typically performed, for example, as follows. That is, an HGF monoclonal antibody immobilized on a suitable carrier such as a 96-well plate is used as a first antibody, and the HGF standard solution and a measurement substance (sample such as an experimental sample) are allowed to stand at room temperature overnight. Reaction [first step], and then anti-HGF rabbit serum as second antibody (rabbit anti-HGF polyclonal antibody)
Is added to the above plate, and reacted at room temperature for about 2 hours, whereby the second antibody reacts with the reactant in the first step (reactant between the monoclonal antibody and the substance to be measured) (second step), Further, a fixed amount of a labeled antibody such as an enzyme-labeled anti-rabbit IgG antibody is reacted with the reaction product (reaction complex of the monoclonal antibody, the test substance and the second antibody) in the second step for about 2 hours at room temperature. [3rd step],
Next, after the unbound labeled antibody is separated and removed from the conjugate of the reaction complex and the labeled antibody obtained in the above third step, a color developing solution is added, and a color developing reaction is performed, and the color developing reaction is stopped with 2N sulfuric acid. It is carried out by measuring the absorbance of the resulting color reaction solution. Thus, HGF in the sample can be quantified.

In the above, the immobilization (insolubilization) of each antibody to be used can be carried out by physically or chemically binding these antibodies to an insoluble carrier according to a conventional method. As the carrier for the insolubilization, for example, polystyrene, Sephadex, ion exchange resin, plastic tube,
Amino copolymers and the like can be used, and insolubilization is carried out by chemical reaction such as diazo method, peptide method, alkylation method, carrier binding method by cross-linking reagent, carrier binding method by Ugi reaction, etc. An ion bonding method using such a carrier, a physical adsorption method using porous glass such as glass beads as a carrier, or the like can be used.

The polyclonal antibodies include HGF
There is no particular limitation, as long as the antibody recognizes the antibody of the present invention, and the antiserum produced in vivo by administering the antibody of the present invention or the immunizing antigen disclosed in the production of the antibody to a mammal can be used. Can be collected according to the law.

The labeling antibody used for labeling in the above may be a known antibody, and is obtained, for example, by immunizing animals such as mice, rats, guinea pigs, rabbits, sheep, goats, horses, and cows which are already commercially available. The antiserum was prepared from peroxidase (POD), alkaline phosphatase, β-
Anti-immunoglobulin antibodies labeled with enzymes such as D-galactosidase and acid phosphatase, that is, POD-labeled anti-rabbit IgG antibodies, POD-labeled anti-mouse IgG antibodies, and the like can be used. This enzyme labeling can be performed according to a conventional method.

In the detection method of the present invention, the sample is preferably, for example, blood in the form of serum or plasma, but may also be cell tissue fluid, lymph fluid, thymic fluid, ascites fluid, amniotic fluid, gastric fluid, urine,
Various body fluids such as pancreatic fluid, bone marrow fluid, saliva and the like can also be used. Further, the plasma is citrate plasma, EDTA plasma,
It may be naphamostat mesylate plasma or the like.

As the solvent used in the above-mentioned measuring system, any of various ordinary solvents which do not adversely affect the reaction can be used. For example, citrate buffer, phosphate buffer, Tris-HCl buffer, acetate buffer It is preferable to use a buffer having a pH of about 5.0 to about 9.0. In the present invention, about 0.1 to 30% w / v of serum (containing no HGF to be measured) and / or about 0.1%
It is preferable to include about 2 M of NaCl because it is more suitable for the purpose of the above-mentioned assay.

The immunoreaction conditions for the immunoassay are not particularly limited, and can be the same as those used in a usual measurement method of this kind. Generally less than about 45 ° C, preferably about 4-40.
The immunoreaction may be performed at a temperature of about ℃ for about 1 to 80 hours.

In the detection method of the present invention, the separation of the solid-liquid phase (combination of the reaction complex and the labeled antibody-unbound labeled antibody in the above-described three-step sandwich) after the completion of the immune reaction is performed, for example, by centrifugation, It can be performed by a usual method such as filtration, decantation, and washing.

The measurement of the enzyme labeling activity of each substance thus separated can be carried out according to various known methods depending on the type of the enzyme used. As a coloring solution used at that time, when a normal solution such as peroxidase is used, o-phenylenediamine (OP
D) and the like can be used, and the coloring reaction can be stopped according to a conventional method, for example, by adding a suitable enzyme activity inhibitor such as 1 to 4N sulfuric acid to the reaction solution.

Thus, according to the method of the present invention, HGF in a sample can be quantified with high accuracy and high sensitivity by a simple operation, and thereby thrombosis can be detected.

That is, the HGF value of a healthy person averaged 0.19 ±
It was 0.05 ng / ml, no significant difference was observed between males and females, and the average value for fulminant hepatitis was 10.17 ± 24.77 n.
g / ml, and the average value for subacute hepatitis exhibiting a pathological condition similar to fulminant hepatitis “subacute type” is 2.71 ± 2.52 ng.
/ Ml, and acute hepatitis (0.40 ± 0.16 ng
/ Ml), chronic hepatitis (0.35 ± 0.16 ng / m
l), cirrhosis (0.60 ± 0.35 ng / ml), liver cancer (0.56 ± 0.36 ng / ml), etc. show low values, whereas myocardial infarction patients measured according to the method of the present invention According to the results of 16.39 ± 16.05 ng in the patient
/ Ml, which remained high for about 15 hours and was still 1.91 ± 1.72 ng / ml after 2 days. Further, according to the method of the present invention, not only the above-mentioned patients with myocardial infarction but also various thrombosis patients showed almost the same high values.

From the above, according to the method of the present invention,
Not only can thrombosis patients be detected, but also in cases of myocardial infarction patients whose time has elapsed since the onset of seizure, the diagnosis can be easily made, and diagnosis of various thrombosis and observation of the prognosis can be performed. It has been found that there are also advantages that can be performed.

The present invention also provides a thrombosis diagnostic agent suitable for the above immunoassay.

The diagnostic agent contains an HGF monoclonal antibody as an essential reagent component, and may further contain a labeled antibody or a second antibody, for example, in the case of the above-described three-step method. A stabilizing agent such as glycerol or bovine serum protein and / or a preservative may be added to the monoclonal antibody reagent in the diagnostic agent. The antibody reagent is preferably lyophilized,
The diagnostic agent may contain a water-soluble or water-miscible solvent. Further, the antibody reagent may contain a buffer solution for keeping the reconstituted reagent system at a constant pH and a preservative and / or stabilizer for preventing deterioration of the sample. It is preferable to use a buffer that has a pH of about 5.0 to 9.0 when performing the present measurement method. The reconstituting agent preferably contains water, but part or all of the water can be replaced with a solvent miscible with water. Examples of the water-miscible solvent include:
Well-known examples include glycerin, alcohols and glycol ethers.

[0046]

According to the present invention, there are provided a diagnostic agent and a detection method capable of diagnosing and detecting thrombosis such as myocardial infarction and cerebral thrombosis, particularly, early diagnosis.

[0047]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

[0048]

[Example 1] Detection of myocardial infarction patient Chest pain lasting more than 30 minutes, S in two lead abnormalities on electrocardiogram
Blood was collected from nine patients determined to have myocardial infarction based on an increase in T of 0.1 mV or more and an increase in serum CPK at 3, 9, 15, 24, and 48 hours after onset of chest pain. HGF in the obtained serum was measured as follows.

That is, 350 μl of a washing solution was added to each well of a 96-well antibody plate, washed three times, and placed on a paper towel to sufficiently remove water. Next, 50 μl of a phosphate buffer solution was added to each well, and 50 μl of an HGF standard solution or the above-mentioned sample was further added thereto. After sealing with a seal, the mixture was reacted for 1 hour at room temperature (25 ° C.) while shaking with a shaker. After the reaction, the reaction solution was removed by suction and washed five times with a washing solution. After washing, 100 μl of the first antibody (anti-HGF monoclonal antibody) was added to each well, sealed with a seal, and then shaken with a shaker at room temperature (25 ° C.).
For 1 hour. After the reaction, the reaction solution was removed by suction and washed five times with a washing solution. Next, 100 μl of a second antibody solution (anti-HGF polyclonal antibody, rabbit) was added to each well, sealed with a seal, and allowed to react at room temperature (25 ° C.) for 1 hour while shaking with a shaker. The reaction solution was removed by suction and washed five times with a washing solution. After washing, add OPD to each well
Add 100 μl of the substrate solution at room temperature (25 ° C.) for 10 minutes.
Allowed to react for minutes. The reaction was stopped by adding 100 μl of a reaction stop solution to the reaction solution, and the absorbance of each well was measured at a wavelength of 492 nm using a microplate absorbance meter.

The above results are shown in Table 1 below.

[0051]

[Table 1]

From the table, it is clear that the myocardial infarction patient can be detected by the ELISA system using the HGF monoclonal antibody of the present invention.

[0053]

EXAMPLE 2 Male rats (250-300 g) were anesthetized with pentobarbital sodium, and after opening the chest, the left coronary artery was ligated. After 1 hour of ischemia, reperfusion was performed to induce myocardial infarction and ligature. 1 hour after, 1,3,6,12 after reperfusion
After 24 hours, plasma was collected, and HGF was measured using a rat HGF EIA kit manufactured by Tokushu Immune Laboratory.

The results (mean ± SE of n = 3) are shown in Table 2 below.
Shown in

[0055]

[Table 2]

Table 2 also shows the same measured values of the control without ligation and reperfusion.

From Table 2, it can be seen that the plasma HGF level in the rat myocardial infarction model shows its maximum value very early, ie, 3 hours after reperfusion, and this measurement enables the detection and early diagnosis of myocardial infarction. It became clear.

[0058]

Example 3 In this test, a thrombus model (SIAT model) in which a thrombus was formed by exfoliating rat carotid artery endothelial cells and adding stenosis was used [Circulation Researc].
h, Vol.77, No.2, August 1995, pp.310-316]. Before the treatment (Pre), 15 minutes, 60 minutes and 120 minutes after the treatment (endothelial cell detachment + stenosis treatment), the plasma HGF level of each rat (4 to 5 rats per group) was determined by the special immunological laboratory. Was measured using a rat HGF EIA measurement kit.

For each rat, the treated blood vessel weight (L) and the untreated blood vessel weight (R) per 13 mm of the carotid artery were determined, and the thrombus weight (mean of n = 5 ± S) was calculated according to the following equation.
E) was calculated.

Thrombus weight (mg / 13 mm, mean ± SE) = [LR] The results thus obtained are shown in Table 3 below.

[0061]

[Table 3]

As shown in Table 3, the plasma HG of thrombosis model rat
The amount of F and the weight of the thrombus are well correlated, and thus it can be seen that thrombosis can be detected and diagnosed by measuring the amount of plasma HGF.

Claims (2)

(57) [Claims] 1. A obtained hepatocyte growth factor of human as an immunogen, myocardium using a monoclonal antibody with specificity reactive with human hepatocyte growth factor, and detecting a myocardial infarction by immunoassay How to detect infarction . 2. A diagnostic agent for myocardial infarction characterized by comprising a monoclonal antibody having human hepatocyte growth factor as an immunogen and having specific reactivity with human hepatocyte growth factor as an essential component.