JP3012666B2 - How to determine myocardial infarction - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for determining myocardial infarction.

Prior Literature and Solution Problems Fatty Acid Binding Protein (FABP) is a protein present in the cytoplasm, has the ability to bind to fatty acids, and is involved in the intracellular metabolism of fatty acids. FABP is distributed in animal liver, myocardial tissue, small intestine, etc.

Recently, FABP derived from human myocardial tissue (hereinafter referred to as hh-FABP) has been separated and purified, and its structural analysis has been performed.
Biochem. J., 252 , 191 (1988) reports that hh-FABP is a protein composed of 132 amino acids and having a molecular weight of 14768.

Circulation Res., 65 , 981 (1989) discloses a monoclonal antibody prepared using FABP derived from rabbit myocardial tissue.
-Using the fact that it shows cross-reactivity with FABP,
It is reported that h-FABP can be quantified. However, this method was based on the fact that FABP derived from rabbit myocardial tissue was used instead of hh-FABP as a standard substance, and that the cross-reactivity of the used antibody to hh-FABP was not equivalent to FABP derived from rabbit myocardial tissue. The value measured by this method does not accurately reflect the amount of hh-FABP.

Therefore, the present inventors have studied variously, established a more accurate immunological quantification method and hh-FA in the body fluid of many patients.
Quantification of the amount of BP revealed that hh-FABP was useful as a marker for myocardial infarction, and completed the present invention.

The present invention relates to a method for determining myocardial infarction.

The antibody used in the present invention recognizes hh-FABP and does not substantially cross-react with FABP and human myoglobin derived from human liver or canine myocardial tissue. The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are more preferable in that they have stricter specificity and can be supplied stably in a homogeneous manner.

The antibody used in the present invention can be produced by a usual method. Polyclonal antibodies can be produced by immunizing animals such as mice, rabbits, goats, and horses with hh-FABP, and monoclonal antibodies can be used to isolate spleen cells and myeloma cells from such immunized animals. Cell fusion, cloning, selection of hybridomas,
It can be produced by culturing ivo or in vitro.
The antigen hh-FABP can be extracted and purified from human cardiac muscle tissue, or can be produced by cell culture or gene recombination techniques.

For immunological quantification of hh-FABP, an anti-hh-FABP antibody obtained using hh-FABP as an antigen (hereinafter referred to as an anti-hh-FABP antibody), a conjugate of an anti-hh-FABP antibody and a label, and A reagent comprising at least one or two of the conjugate or adsorbate of the insoluble carrier and the anti-hh-FABP antibody is used.

Labels include enzymes such as peroxidase, β-galactosidase, and alkaline phosphatase; radioactive substances such as ¹²⁵ I; fluorescent substances such as fluorescein isothiocyanate; and spin compounds. Insoluble carriers include those used in EIA and RIA, such as bacterial cell wall fragments, glass and polystyrene beads, tubes, and microplates, and latex particles for agglutination. The binding of the anti-hh-FABP antibody to the label or the like is carried out by using a carboxyl group, an amino group, an SH group, an OH group, or the like, in the presence or absence of a binder according to a conventional method.

The immunological quantification of hh-FABP may be any method that utilizes the specific antigen-binding ability of the anti-hh-FABP antibody, and is performed, for example, by a method such as the EIA method, the RIA method, or the latex agglutination method. it can.

Reagents used in these immunoassays include:
For example, in the competitive EIA method, an insolubilized antibody against the antibody of the anti-hh-FABP antibody (second antibody) Enzyme-labeled antigen A substrate for the labeling enzyme Standard hh-FABP solution is used, and in the sandwich EIA method, the enzyme-labeled anti-hh-FABP antibody is used. Antibody Insolubilized anti-hh-FABP antibody Substrate for labeled enzyme Standard hh-FABP solution, etc. is used.In the latex agglutination method, a conjugate or adsorbed standard hh-FABP solution of latex particles and anti-hh-FABP antibody is used. Can be

Detecting hh-FABP levels in human body fluids such as serum and urine is useful for determining myocardial infarction. Usually hh-FABP
Immediately after the onset of myocardial infarction, the serum level first peaks, and the urinary level peaks 1-2 hours later. Urine levels are higher than serum levels and can reach 50-100 times serum levels.

Speed of appearance of hh-FABP peak in serum (time)
Is as fast as in human myoglobin. Although human myoglobin is a marker evaluated at the point where its peak appears in the early stage of myocardial infarction, it is not possible to distinguish between those derived from myocardial tissue and those derived from skeletal muscle tissue, so that myocardial infarction determination is not always accurate There is a disadvantage that it is not.
Detection of hh-FABP in human serum or urine can be performed by the immunological quantification method described above. Which method is to be selected may be determined according to the urgency of the treatment. For example, when monitoring the onset of myocardial infarction during surgery, a method that can promptly obtain quantitative or qualitative results, such as latex agglutination, is suitable, and patients with suspected myocardial infarction, such as patients complaining of angina When exercise accuracy is required rather than urgency of treatment, such as detecting the hh-FABP level by applying an exercise load to the EI,
Method A is selected.

Specific Examples Next, the present invention will be described in more detail with reference to Reference Examples and Examples.

The buffer represented by the following abbreviations has the following composition.

Buffer A; 0.1% BSA-0.1% NaN ₃ -0.9% NaCl-0.04M phosphate buffer, pH 7.0 Buffer B; 10% glycerol-1mM EDTA-2mM 2-mercaptoethanol-0.9% NaCl-20mM phosphorus Acid buffer, pH 7.4 buffer C; 10% glycerol-1 mM EDTA-2 mM 2-mercaptoethanol-20 mM phosphate buffer, pH 7.4 buffer D; 10% glycerol-1 mM EDTA-2 mM 2-mercaptoethanol 15 mM Tris-HCl buffer, pH 8.4 buffer E; 0.1 M phosphate buffer, pH 7.0 buffer F; 0.02 M phosphate buffer, pH 7.0 buffer G; 1 M sodium acetate buffer, pH 4. 0 Buffer H; 0.9% NaCl-0.1 M phosphate buffer, pH 7.4 Buffer I; 0.05 M phosphate-borate buffer, pH 6.0 Reference Example 1 Preparation of hh-FABP Performed 5 hours after death Hh-FABP was extracted and purified using the following method using 250 g of human myocardial tissue obtained at the time of pathological dissection.

The myocardial tissue is cut with a knife, 2500 ml of buffer B is added, treated with a polytron homogenizer, and centrifuged (100,000 × g, 90 minutes) to obtain a supernatant. The supernatant is fractionated using a Sephacryl S-100HR (Pharmacia) column (2.5 x 95 cm) equilibrated with buffer B. The fraction having a molecular weight of 10,000 to 20,000 is sufficiently dialyzed against buffer C, then added to a hydroxyapatite (Nacalai Tesque) column (2.5 × 30 cm) equilibrated with buffer C, and eluted with buffer C. The first eluting fraction (crude hh-FABP) is obtained. This was used as an immunizing antigen in Reference Example 2 described later.

The crude hh-FABP solution was sufficiently dialyzed against buffer D, added to a DEAE-Sephacel (Pharmacia) column (1 × 50 cm) equilibrated with buffer D, washed sufficiently, and the adsorbed protein was washed with potassium chloride. Elution is performed with buffer C having a linear concentration gradient (0 to 150 mM). 28 eluted protein fractions
Followed by absorbance at 0 nm, each peak was analyzed by electrophoresis, and the fraction showing a single band with a molecular weight of about 16000 was identified as hh-F
ABP solution was used.

Reference Example 2 Preparation of Polyclonal Antibody To the crude hh-FABP (2.5 mg protein / ml) obtained in Reference Example 1, physiological saline was added so that the protein concentration became 1 mg / ml, and an equal amount of Freund's complete adjuvant was further added. To prepare a W / O emulsion, which was subcutaneously applied to two rabbit footpads.
Inject 0.1 ml in each place. Two weeks later, 0.1 ml is injected subcutaneously in 5 places on the back. Thereafter, booster immunization is performed six times every two weeks in the same manner. For the final immunization, a solution prepared by diluting crude hh-FABP with a physiological saline solution to a protein concentration of 2 mg / ml was added to an equal volume of Freund's complete adjuvant to prepare a W / O emulsion, which was subcutaneously applied to the back of the rabbit. It is performed by injecting 0.1 ml in 10 places. Nine days later, whole blood is collected from the carotid artery and serum is separated. The serum diluted with buffer A was used as an anti-hh-FABP polyclonal antibody solution.

Reference Example 3 Preparation of Monoclonal Antibody BALB / C mice were immunized according to Reference Example 2, and their spleen cells were collected. Mouse myeloma cells in logarithmic growth phase
5 of P3-X63-Ag8-U1 (ATCC Catalog No. CRL-1597)
× 10 ⁷ cells and 1 × 10 ⁸ antibody-producing spleen cells were mixed, washed with buffer H by centrifugation (400 × g, 10 minutes), and then kept at 37 ° C. in 0.5 ml of polyethylene glycol 1500- RPMI-1
Slowly add 640 medium (1: 1) and stir slowly. After 90 seconds, 10 ml of serum-free medium for cell fusion (50 U /
ml penicillin G, RPMI containing 50 μg / ml streptomycin
-1640 medium) in the same manner, 10 minutes later, 10 ml of the same medium, and centrifugation (400 xg, 10 minutes) to remove the supernatant. An HAT medium is added to the obtained pellet, and the cells are cultured by a conventional method. A hybridoma having a high antibody titer is selected, cloned by the limiting dilution method, and a cloned hybridoma is established. The assay of the antibody titer is described in Reference Example 4 below.
Was performed using the β-galactosidase-labeled hh-FABP prepared in the above. The cloned hybridoma is inoculated into BALB / C mice previously treated with pristane to obtain ascites containing the desired monoclonal antibody.

 The properties of this monoclonal antibody are as follows.

(A) recognizes hh-FABP (b) does not substantially cross with human liver-derived FABP (c) does not substantially cross with dog heart muscle tissue-derived FABP (d) does not substantially cross with human myoglobin ( e) Competitively binds to both hh-FABP and enzyme-labeled hh-FABP Reference Example 4 Preparation of Enzyme-Labeled hh-FABP Purified hh-FABP (3.5 mg protein / ml) prepared in Reference Example 1.
200 μg of m-MBS (m-MBS) was added to a mixture of 2 ml and 1 ml of buffer E.
0.2 ml of dioxane containing maleimidobenzoyl-N-hydroxysuccinimide (binder) was added dropwise at room temperature.
Stir for 0 minutes. 10 ml of the dried solution F was added to the mixture, and YM-5 was added.
Concentrate with an ultrafiltration membrane (Amicon), and further add Buffer F
Wash and concentrate twice with 7 ml. A mixed solution of 0.2 ml of a buffer F containing 500 μg of β-galactosidase derived from Escherichia coli (Boehringer Mannheim) and 0.2 ml of a saturated ammonium sulfate solution is added dropwise to 1.5 ml of the concentrated solution, followed by stirring at room temperature for 80 minutes. Sepharose 6B (Pharmacia) column (1.5
× 70 cm), eluted with buffer A,
Separate each l. The 25th to 33rd fractions were pooled, and this was diluted 500-fold with buffer A to obtain an enzyme-labeled hh-FABP solution.

Reference Example 5 Preparation of Insolubilized Secondary Antibody 40 mg of bacterial cell wall fragments of Lactobacillus plantarum ATCC8019 was suspended in 4 ml of water, and after sufficiently homogenized, 1 mg of anti-rabbit IgG goat antibody (second antibody) was added, and the mixture was stirred. 60μ
buffer G, 5% aqueous carbodiimide aqueous solution 120μ
l and 10 μl of 25% glutaraldehyde are sequentially added and stirred at room temperature for 1 hour. Centrifuge the reaction mixture (1500 xg, 10
), Add 5 ml of buffer A to the precipitate, and wash by centrifugation.
Repeat this three times to obtain 20 ml of 0.5% cell wall fragments.
In buffer A.

Reference Example 6 Competitive EIA method Take 50 μl of standard hh-FABP solution or sample in a test tube,
To this, 200 μl of the anti-hh-FABP polyclonal antibody solution obtained in Reference Example 2 is added, stirred, and left at room temperature for 15 to 20 hours. To this, 200 μl of the enzyme-labeled hh-FABP solution obtained in Reference Example 4 was added.
and left at 37 ° C. for 30 minutes. Next, 200 µl of the suspension of the insolubilized second antibody obtained in Reference Example 5 was added, and the mixture was allowed to stand at 37 ° C for 30 minutes. Then, 2 ml of 0.9% NaCl solution was added and centrifuged (1500 × g, 10
And remove the supernatant. This washing operation is repeated once more. 0.5 ml of buffer A was added to the precipitate, and the mixture was stirred with a mixer to completely disperse the precipitate. The mixture was preheated at 37 ° C. for 3 minutes, and 100 μl of the enzyme substrate solution [0.3 mM 4-methylumbelliferyl-β-D -Galactopyranoside-1 mM MgCl ₂ -0.0
4M phosphate buffer (pH 7.0)] and leave at 37 ° C. 6
After 0 minutes, the enzyme reaction stop solution (0.1 MK ₂ HPO ₄ -NaOH buffer, pH
Add 11), stir, and measure the fluorescence intensity (excitation wavelength 365 nm, fluorescence wavelength 450 nm).

 FIG. 1 is a quantitative curve in this method.

Example 1 hh-FABP Level in Patients with Myocardial Infarction According to Reference Example 6, the hh-FABP levels in blood and urine of a patient with myocardial infarction were quantified, and the results shown in FIG. 2 were obtained. For the same sample, the 26th Annual Meeting of the Japanese Society of Clinical Chemistry
, P. 89 (1986). In FIG. 2, "s-" indicates a case where serum was used as a sample, "u-" indicates a case where urine was used as a sample, and Mb indicates myoglobin. Thus, for example, "u-hh-FABP" means hh-FABP in urine.

As shown in FIG. 2, in the case of this patient, s-hh-FABP peaked about 5 hours after the onset of myocardial infarction, and u-hh-FABP peaked about 3 hours later. Appears and the peak of s-hh-FABP coincides with the peak of s-Mb. The peak concentration of u-hh-FABP is s-hh-FABP
About 100 times.

Reference Example 7 Latex agglutination method (1) Preparation of antibody-sensitized latex suspension 10% carboxylic acid-modified latex H0 suspended in dry solution I
901 (particle diameter 0.93μ, Nippon Synthetic Rubber Co., Ltd.) 2.5m in 350μl
50 μl of an aqueous solution containing g of a water-soluble carbodiimide is added dropwise and stirred at room temperature. After 30 minutes, 100 μl of the mouse ascites (anti-hh-FABP monoclonal antibody solution) obtained in Reference Example 3 is added dropwise, and the mixture is stirred at room temperature for 30 minutes. After centrifugation, the precipitate is washed three times with 5 ml of buffer A, the latex is dispersed by sonication, suspended in buffer A, and a 1% anti-hh-FABP monoclonal antibody-sensitized latex suspension is prepared. .

(2) Aggregation reaction The reaction was performed by a latex slide method. Anti-hh-FA
20 μl of the BP monoclonal antibody-sensitized latex suspension was dropped on a slide for judgment, and 20 μl of urine from a healthy person or various patients with myocardial infarction was added thereto, mixed well, and agglutination was determined by a slide aggregation image after 3 minutes at room temperature. The degree was determined, and the results in the following table were obtained. The following table also shows the results of the same sample quantified by the competitive EIA method of Reference Example 6.

As shown in the preceding table, the qualitative results (degree of aggregation) of the latex agglutination reaction method almost corresponded to the quantitative results of the competitive EIA method. This method is considered to be useful as an emergency judgment method.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a standard curve for quantification of hh-FABP by competitive EIA, and FIG. 2 is hh-FABP in blood (s−) and urine (u−) in patients with myocardial infarction. And blood myoglobin (s
-Mb) shows a fluctuation pattern.

Continuation of front page (56) References Int. J. Biochem. Vol. 22, No. 4 (1990.4) p. 393-398 Circulation Research vol. 65, no. 4, (1989) p. 981-988 (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/53 G01N 33/50 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. The level of fatty acid binding protein (hereinafter referred to as hh-FABP) derived from human myocardial tissue in human blood isolated from a human suspected of acute myocardial infarction within 6 hours after the onset of seizure. Anti-hh-FABP antibody obtained using FABP as an antigen (hereinafter referred to as anti-hh-FABP anti), a conjugate of an anti-hh-FABP antibody and a label, and a conjugate of an insoluble carrier and an anti-hh-FABP antibody, or A method for judging acute myocardial infarction, which comprises detecting using a reagent containing at least one or two of the adsorbates as constituents. 2. The method according to claim 1, wherein the anti-hh-FABP antibody is a monoclonal antibody.