JP2001056338A - Method for detecting possibility of coronary restenosis after percutaneous coronary angioplasty - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a method for detecting the possibility of coronary rastenosis after percutaneous coronary angioplasty. SOLUTION: By an immunologically analyzing method through the use of anti-CD36 monoclonal antibodies which specifically react to human CD36 and of which especially the reactivity to human wash blood platelet membrane CD36 is suppressed dependently on the concentration of human plasma or through the use of antibody fragments of the anti-CD36 monoclonal antibodies, the affinity of monoclonal antibodies to human blood platelet CD36 in the presence of human plasma in each sample to be tested collacted before an operation of percutaneous coronary angioplasty and after the operation is analyzed to compare the preoperative affinity with the postoperative affinity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting the possibility of coronary restenosis after percutaneous coronary angioplasty, that is, for predicting the presence or absence of restenosis.

[0002]

2. Description of the Related Art Cardiovascular diseases account for a large proportion of deaths not only in Europe and the United States but also in Japan. The main causes of atherosclerosis, myocardial and cerebral infarction account for the majority of these deaths. In recent years, medical and
The development of surgical treatment is remarkable. In ischemic heart disease, ie, angina and myocardial infarction, usually is based on intracoronary stenosis due to coronary atherosclerosis. Coronary angiography is performed as a definitive diagnosis and severity determination, and three types of treatments are used for treatment: medical treatment and percutaneous translumina with a balloon catheter (percutaneous translumina).
l coronary angioplasty: PT
CA) and the surgical treatment of coronary artery bypass grafting (co
ronary arterybypass graph
t: CABG) is taken into account.

[0003] Patients who cannot be controlled by medical treatment with medication or have poor load-tolerance are treated with PT
Invasive treatment of CA or CABG is selected. In particular, PTCA is useful as a therapeutic method because it is relatively invasive. However, the limitations of PTCA include (1) the presence of lesion forms that result in inadequate dilation, (2) thrombus formation due to coronary artery dissection after treatment, and acute coronary artery occlusion, (3). Approximately 40% of patients suffer from restenosis within 6 months after treatment. In recent years, after dilating the stenosis with a balloon,
A method of deploying a stent (a mesh or coiled metal support) has been developed and all of the above problems have been reduced.

[0004] However, it has been reported that restenosis occurs within 6 months after treatment in still 20-30% of patients. The onset of restenosis is caused by the activation of platelets derived from neutrophils that adhere to the adherent platelets via P-selectin, or activated platelets adhere to the stent placement site. This is considered to be caused by the effect of the above that smooth muscle cells proliferate in the stent placement portion. Thus, since the involvement of platelets in restenosis is important, antiplatelet drugs (eg, aspirin, ticlopidine, cilostazol, anti-GPIIb / IIIa antibodies, etc.) are used to prevent restenosis, and the effect is Although elevated, serious side effects such as agranulocytosis and bleeding complications have also been recognized. In other words, it is very clinically important to be able to predict restenosis, and it is considered that this leads to an appropriate prescription of antiplatelet drugs.

[0005] As described above, since the involvement of platelets in the onset of restenosis is important, attempts have been made to measure platelet membrane P-selectin as an index of platelet activation and to use it for diagnosis of prognosis. I have. However, when preparing platelets as a test sample, measurement errors due to platelet activation may become a problem.
As is well known, dozens of platelet membrane glycoproteins have been identified so far. As its structure and function became increasingly clear, it was suggested that examining its expression status could provide an indication of platelet status.

[0006] CD3, one of the platelet membrane glycoproteins
6 is also known as GPIV or GPIIIb. It is a glycoprotein expressed on the membrane surface of monocytes, endothelial cells, or mammary epithelial cells as well as platelets, and is considered as a receptor for thrombospondin, collagen, oxidized LDL, and fatty acids. Heretofore, there have been reports on the relationship between CD36 deficiency cases and ischemic heart disease, but there has been no report on the prediction of restenosis by immunologically measuring CD36 on the platelet membrane surface.

[0007]

DISCLOSURE OF THE INVENTION The present inventor aimed at understanding the state of platelets by flow cytometry using various monoclonal antibodies against CD36 by using a platelet membrane surface in patients who underwent PTCA. When the binding of each monoclonal antibody to the above CD36 was examined, it was surprisingly found that there was a significant difference in the binding between the restenosis group and the non-restenosis group only for a specific monoclonal antibody. The present invention is based on such findings. It is therefore an object of the present invention to provide a method for detecting the possibility of coronary restenosis after percutaneous coronary angioplasty.

[0008]

SUMMARY OF THE INVENTION The object of the present invention is to provide a method for detecting the possibility of coronary restenosis after percutaneous coronary angioplasty according to the present invention, said method being specific for human CD36, An anti-CD36 monoclonal antibody whose reactivity with human washed platelet membrane CD36 is suppressed in a concentration-dependent manner in human plasma (hereinafter referred to as a human plasma-sensitive anti-CD36 monoclonal antibody) or an immunological analysis method using an antibody fragment thereof. In each of the test samples collected before and after the percutaneous coronary angioplasty, the presence of human plasma on human platelet membrane CD36 (including human washed platelet membrane CD36 and human unwashed platelet membrane CD36) Analyzing the binding of the monoclonal antibody, and comparing the binding before and after surgery,
This can be achieved by the detection method.

The test sample in the above detection method is not particularly limited as long as it is a human biological sample containing human platelets and human plasma at the same time.
Or platelet-rich plasma.

[0010] The present invention also provides an immobilized first antibody in which a first antibody or an antibody fragment thereof that specifically reacts with human CD36 is immobilized on an insoluble carrier, and a known amount of purified CD.
36 is contacted with a test sample to which 36 has been added in advance, and subsequently, a second antibody or an antibody fragment thereof which specifically reacts with human CD36 with an epitope different from the first antibody is labeled. The second antibody is brought into contact with the second antibody to analyze the signal derived from the label of the labeled second antibody bound to the first antibody-CD36 complex, or the first antibody-CD36 complex is immobilized. Analyze the signal derived from the label of the labeled second antibody that did not bind with [provided that either the first antibody or the second antibody is
An anti-CD36 monoclonal antibody that specifically reacts with human CD36 and suppresses the reactivity to human washed platelet membrane CD36 in a human plasma concentration-dependent manner (ie, a human plasma-sensitive anti-CD36 monoclonal antibody). Characterized in that it is contained in the test sample,
The present invention relates to an immunological analysis method for a substance that suppresses the binding between the human plasma-sensitive anti-CD36 monoclonal antibody and the purified CD36.

The test sample used in the above-mentioned analysis method comprises a human plasma-sensitive anti-CD36 monoclonal antibody and purified CD36.
It is not particularly limited as long as it is a human biological sample that may contain a binding inhibitory substance with, but is substantially free of platelets, or a human biological sample with a low platelet content. Preferably, for example, platelet poor plasma can be mentioned.

As used herein, "human CD36" is expressed on the membrane surface of platelets, monocytes, endothelial cells, or mammary epithelial cells, and is capable of receiving thrombospondin, collagen, oxidized LDL, or fatty acids. It is a glycoprotein with a molecular weight of about 88,000 which is considered as a body.
The “CD36” includes various CD36s having different states of existence, for example, “purified CD36”, “washed platelet membrane CD36” (that is, CD36 present on the cell membrane of washed platelets described later), or “platelet membrane”. CD36 "(ie, CD36 present on the cell membrane of platelets) and the like.

[0013] In the present specification, "human plasma" refers to a portion obtained by removing blood cells from blood, and includes, for example, platelet poor plasma obtained from blood supplemented with sodium citrate. Further, in the present specification, “platelet-rich plasma” is plasma containing a large amount of platelets, and “platelet-poor plasma” is plasma having a small amount of platelets. Further, in the present specification, “washed platelets” refers to platelets separated from sodium citrate-supplemented blood, HEPES-Tyrod.
Platelets washed with e's buffer to remove plasma components. The “unwashed platelets” are platelets that have not been subjected to the above-mentioned washing operation.

Further, in the present specification, “percutaneous coronary angioplasty” refers to percutaneous coronary angioplasty (percutaneous coronary angioplasty) using a balloon catheter for coronary artery disease.
transluminal coronary an
gioplasty (PTCA).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The human plasma-sensitive anti-CD36 monoclonal antibody which can be used in the present invention is human CD
The antibody is not particularly limited as long as it is an antibody that specifically reacts with G.36 and the antibody whose reactivity with human washed platelet membrane CD36 is suppressed in a concentration-dependent manner in human plasma. The human plasma-sensitive anti-CD36 monoclonal antibody includes the 162nd to 1st human CD36.
A monoclonal antibody capable of recognizing a partial polypeptide consisting of the 83rd amino acid residue (for example, the monoclonal antibody CD36-
1) can be mentioned. The first of human CD36
The amino acid sequence from position 62 to position 183 is as follows: Ile Asn Lys Ser Lys Ser Ser Met Phe Gln
Val Arg Thr Leu Arg Glu Leu LeuTrp Gly Tyr Arg

The human plasma-sensitive anti-CD36 monoclonal antibody that can be used in the present invention is, for example, a Glanzmann thrombasthenia type deficient in one of the platelet membrane glycoproteins GPIIb / IIIa (also called αIIb / β3). I (Glanzmann's Thromba
Washed platelets derived from patients of S. stenia type I) can be used as antigens for immunization and screening, and can be obtained by a monoclonal antibody production method using hybridomas produced by a cell fusion method, which has recently been carried out in various fields. This Glanzmann thrombasthenia type I
By using the washed platelets derived from the above-mentioned patient, it is possible to avoid formation of antibodies against GPIIb / IIIa, which are usually present in large amounts on the platelet membrane surface.

The monoclonal antibody is produced by culturing the hybridoma (for example, mouse hybridoma) capable of producing the monoclonal antibody in, for example, an appropriate medium or intraperitoneally of a mammal (for example, mouse). be able to. The hybridoma may be a mammalian (eg, mouse) or avian spleen cell and a mammalian (eg, mouse) myeloma cell immunized with washed platelets from a patient with Glanzmann's thrombasthenia type I, as described above. (Myeloma cells) and the basic method of Koehler and Milstein [Natu
re, vol. 256, p. 495 (1975)]. For details,
It can be produced by the method described in the following examples.

By culturing the above hybridoma,
As a culture medium, a medium suitable for hybridoma culture
And preferably a bovine embryo in RPMI 1640 medium.
Infant serum, L-glutamine, L-pyruvic acid, and antibiotics
Medium containing the qualities (penicillin G and streptomycin)
Used. The culture of the hybridoma is performed in a medium.
When performing, for example, 5% CO _TwoConcentration and 37 ° C
Perform under conditions for about 3 days. Alternatively, in the mouse intraperitoneally
When performing, for example, about 14 days.

The monoclonal antibody can be separated and purified from the thus-prepared culture solution or mammalian ascites by, for example, a method generally used for isolating and purifying proteins. is there. Such methods include, for example, ammonium sulfate salting out, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel, affinity column chromatography using protein A binding polysaccharide, dialysis, or freezing. Drying etc. can be mentioned.

The antibody fragment that can be used in the present invention is not particularly limited as long as it is a fragment of the above-mentioned monoclonal antibody and has the same reaction specificity as the original monoclonal antibody. . That is, the reactivity of the antibody fragment that can be used in the present invention with the washed platelet membrane CD36 is suppressed depending on the concentration of human plasma.

The antibody fragment includes, for example, Fa
b, Fab ', F (ab') ₂ , Fv and the like. These fragments can be obtained, for example, by digesting the monoclonal antibody with a proteolytic enzyme by a conventional method, and then separating and purifying the protein by a conventional method.

In the detection method of the present invention, the binding of the monoclonal antibody to human platelet membrane CD36 before percutaneous coronary angioplasty is determined by immunological analysis using the monoclonal antibody or an antibody fragment thereof. Platelet membrane CD3 after percutaneous coronary angioplasty
The binding of said monoclonal antibody to 6 is analyzed respectively. The immunological analysis method is not particularly limited, and examples thereof include an immunological analysis method using a flow cytometer, a sandwich method, and a latex agglutination method.

In the detection method of the present invention, the binding of the monoclonal antibody to human platelet membrane CD36 before the operation and the binding of the monoclonal antibody to human platelet membrane CD36 after the operation, which are analyzed as described above, are determined. By comparison, the possibility of post-operative coronary restenosis is detected. Specifically, if the post-operative connectivity is lower than the pre-operative connectivity (preferably reduced to 80% or less), the possibility of coronary restenosis within 6 months after the operation is low. In other cases, for example, when the connectivity before the operation and the connectivity after the operation do not change, it is determined that the possibility of coronary restenosis within 6 months after the operation is high.

In the detection method of the present invention, when an immunological analysis method using a flow cytometer is used, the monoclonal antibody or its antibody fragment is directly or indirectly labeled with a fluorescent substance. For example, a suitable fluorescent substance can be bound to the monoclonal antibody or the antibody fragment thereof, or an appropriate fluorescent substance can be bound to the antibody or the antibody fragment capable of binding to the monoclonal antibody or the antibody fragment thereof. They can also be combined. The fluorescent substance is not particularly limited, and may be, for example, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, tetra-N-cyclopropylrhodamine isothiocyanate, Texas Red, phycoerythrin, or a combination thereof. it can.

In the detection method of the present invention, when an immunological analysis method using a flow cytometer is used, for example, a monoclonal antibody or an antibody fragment thereof labeled with a fluorescent substance may be collected before and after surgery. After bringing the test sample into contact with the test sample, and applying it to a flow cytometer, the binding of the monoclonal antibody to human platelet membrane CD36 before and after the operation can be analyzed as the fluorescence intensity.

In the analysis method of the present invention, a substance inhibiting binding between human plasma-sensitive anti-CD36 monoclonal antibody and purified CD36 can be analyzed by the sandwich method. In the analysis method of the present invention, a latex agglutination method can be used instead of the sandwich method.

When the sandwich method is used in the analysis method of the present invention, for example, a human plasma-sensitive anti-CD36 monoclonal antibody and purified CD36
Can be analyzed. That is,
Anti-CD36 antibody or antibody fragment thereof (first antibody)
Is immobilized on a suitable insoluble carrier. Next, in order to avoid non-specific binding between the insoluble carrier and the test sample, a suitable blocking agent [for example, bovine serum albumin (BSA)]
Or gelatin or the like] to cover the surface of the insoluble carrier. Subsequently, a test sample to which a known amount of purified CD36 has been added is added, and the sample is brought into contact for a fixed time (for example, 5 minutes to 3 hours) and a constant temperature (for example, 4 ° C to 40 ° C, preferably around room temperature), Reaction (primary reaction). Subsequently, an anti-CD that binds to human CD36 at an epitope different from the first antibody
36 antibody or its antibody fragment (second antibody) to which a labeled antibody is added is added for a certain time (for example, 5 minutes to 3 hours) and a certain temperature (for example, 4 ° C to 40 ° C, preferably around room temperature). ) And react (secondary reaction). This is washed with an appropriate washing solution (for example, a physiological saline solution containing BSA), and then a signal derived from the label of the labeled antibody present on the insoluble carrier is analyzed. The signal from the label of the labeled antibody present is analyzed. From the value, a substance that inhibits binding between human plasma-sensitive anti-CD36 monoclonal antibody and purified CD36 can be analyzed.

In the analysis method of the present invention, when the sandwich method is used, either the first antibody or the second antibody specifically reacts with a human plasma-sensitive anti-CD36 monoclonal antibody, ie, human CD36. In addition, an anti-CD36 monoclonal antibody whose reactivity with human washed platelet membrane CD36 is suppressed in a concentration-dependent manner in human plasma is used. The remaining other antibody may be, for example, an anti-CD36 polyclonal antibody, or an anti-C36 antibody.
A D36 monoclonal antibody or the like can be used.

In the analysis method of the present invention, when the sandwich method is used, the insoluble carrier is not particularly limited. For example, polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, Crosslinked dextran, polymers such as polysaccharides, other nitrocellulose,
Examples include paper, agarose, and combinations thereof. Further, it is advantageous to use, for example, an enzyme, a fluorescent substance, or a luminescent substance as the labeling substance of the second antibody. Examples of the enzyme include, for example, alkaline phosphatase, peroxidase, and β-D-galactosidase.Examples of the fluorescent substance include fluorescein isothiocyanate, and examples of the luminescent substance include acridinium ester and luciferin. Can be used. Further, as the purified CD36, for example, a sample purified from human platelets, or
Recombinant purified CD applying genetic recombination technology
36 can be used.

By using the analysis method of the present invention, the binding inhibitor between human plasma-sensitive anti-CD36 monoclonal antibody and purified CD36 in each test sample collected before and after percutaneous coronary angioplasty is determined. Analyze each,
By comparing the amount of the binding inhibitor before and after surgery, the possibility of coronary restenosis after percutaneous coronary angioplasty can be detected. That is, an immobilized first antibody in which a first antibody or an antibody fragment thereof specifically reacting with human CD36 is immobilized on an insoluble carrier is brought into contact with a test sample to which a known amount of purified CD36 has been added in advance. The human CD36 with an epitope different from that of the first antibody.
A second antibody or an antibody fragment thereof that specifically reacts with a labeled second antibody that has been labeled, and contacts the labeled second antibody bound to the immobilized first antibody-CD36 complex. Analyzing the signal from the label, or
The signal derived from the label of the labeled second antibody that did not bind to the immobilized first antibody-CD36 complex is analyzed [provided that either the first antibody or the second antibody is human. Is a plasma-sensitive anti-CD36 monoclonal antibody], whereby human platelet membrane CD36 in each test sample collected before and after the percutaneous coronary angioplasty is obtained.
Analyzes the binding of the human plasma-sensitive anti-CD36 monoclonal antibody to lipoproteins and detects the likelihood of coronary restenosis after percutaneous coronary angioplasty by comparing the binding before and after surgery be able to.

Specifically, when the amount of the binding inhibitor after the operation is greater than the amount of the binding inhibitor before the operation [ie, the binding after the operation is higher than the binding before the operation] Is decreased], it is determined that the possibility of coronary restenosis within 6 months after the operation is low. In other cases, for example, the amount of the binding inhibitor before the operation and the amount of the binding inhibitor after the operation are determined. When the amount does not change (ie, when the connectivity before and after the operation does not change), it is determined that the possibility of coronary restenosis within 6 months after the operation is high. it can.

[0032]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << Preparation of washed platelets for immunization and washed platelets for screening >> Glanzmann platelet asthenia type I
(Glanzmann's Thrombasthen
ia Type I) Blood collected from a patient's elbow vein using a syringe containing 3.8% sodium citrate (1/10 volume of the collected blood volume) was dispensed into a silicon-treated glass centrifuge tube (5 ml). /tube). 120
The mixture was centrifuged at room temperature for 15 minutes at × g to obtain platelet-rich plasma.

[0033] 1M citric acid was added to the platelet-rich plasma thus obtained to adjust the pH to 6.8.
The mixture was centrifuged at room temperature for 15 minutes at room temperature. Platelets obtained as a precipitate were added to Philips buffer (96.5 mM
-NaCl, 85.7 mM glucose, 5 mM-EDT
A, and 8.57 mM Tris-HCl;
4) was added, and washed twice by centrifugation. The platelets after washing thus obtained are suspended in physiological saline,
It was measured with a Coulter counter (Beckman Coulter, Inc.) and adjusted to a concentration of 1 × 10 ⁷ cells / ml. Washed platelets from Glanzmann's thrombasthenia type I patient thus prepared were used as platelets for immunization.

As screening platelets, CD36
CD36-expressing washed platelets derived from healthy persons were prepared from healthy persons not deficient in, and CD36-deficient washed platelets were prepared from CD36-deficient persons by the same method.
However, the final suspended concentration of platelets was 1 × 10 ⁸ / ml.

[0035]

Example 2 << Preparation of Washed Platelets for Examining Antibody Characteristics >> 3.8% of 1/10 volume of collected blood from healthy elbow vein
Blood collected with a syringe containing sodium citrate,
The mixture was dispensed into a silicon-treated glass centrifuge tube (5 ml / tube). Centrifugation was performed at 120 xg for 15 minutes at room temperature to obtain platelet-rich plasma.

The thus obtained platelet-rich plasma was added with 1M citric acid to adjust the pH to 6.8, and then was added to 1,200 ×
The mixture was centrifuged at room temperature for 15 minutes at room temperature. 1 unit / ml of apyrase (apyr) was added to platelets obtained as a precipitate.
HEPES-Tyr containing ASE (manufactured by Sigma)
mode's buffer (137 mM-NaCl, 2.7 mM
_{-KCl, 0.42mM-NaH 2 PO 4} , 4.2mM-
HEPES, 1mM-MgCl _2, 5.5mM dextrose, and 0.35% bovine serum albumin; pH 6.
5) was added, and the mixture was washed twice by centrifugation. The washed platelets obtained in this manner were converted to apyrase-free HE.
PES-Tyrode's buffer (137 mM-NaC
1, 2.7 mM KCl, 0.42 mM NaH ₂ P
O ₄ , 4.2 mM-HEPES, 1 mM-MgCl ₂ ,
The cells were suspended in 5.5 mM dextrose and 0.35% bovine serum albumin (pH 7.4), counted using a Coulter counter (Beckman Coulter, Inc.), and measured at 5 × 1.
0 were prepared at a concentration of ⁸ / ml. The washed platelets thus prepared were used as platelets for examining antibody characteristics.

[0037]

Example 3 << Preparation of platelet-poor plasma >> Platelet-rich plasma prepared in the same manner as in Example 2 was further subjected to 2,000 ×
The supernatant centrifuged at room temperature for 15 minutes at g was used as platelet poor plasma.

[0038]

Example 4 << Preparation of Hybridoma >> (a) Preparation of Immunized Spleen Cells Washed platelets (1 × 10 ⁷ cells / ml) of Glanzmann's platelet asthenia type I patient obtained in Example 1 were subjected to BAL.
1 ml each intraperitoneally or subcutaneously on the back of B / c mice.
Immunization was performed by injecting eight times every other day. Three days after the final immunization, the spleen was aseptically removed from the mouse and used for the following steps.

(B) Cell fusion The spleen aseptically removed was placed in a petri dish containing 5 ml of RPMI 1640 medium containing 10% fetal bovine serum. Next, the spleen was subjected to RPM containing 10% fetal bovine serum.
After the spleen cells were drained by refluxing with about 15 ml of I1640 medium, the spleen cell suspension was passed through a nylon mesh.
The spleen cells are collected in a 50 ml centrifuge tube and 500 × g
For 10 minutes. A hemolyzing solution (155 mM-NH ₄ Cl, 10 mM-KH) was added to the pellet thus obtained.
CO ₃ , and 1 mM-Na ₂ EDTA; pH 7.0) 5 m
1 was added and suspended. When left at 0 ° C. for 5 minutes, the red blood cells in the suspension were destroyed. After adding 15 ml of RPMI1640 medium containing 10% fetal bovine serum, the mixture was centrifuged. The cell pellet thus obtained was washed with a DME medium by a centrifugation method, and the number of living spleen cells was measured.

On the other hand, mouse myeloma cells (myeloma cells) P3-NS1-1-Ag4-1 previously cultured
The spleen cells (1 × 10 ⁸ cells) were added to (about 2 × 10 ⁷ cells), mixed well in RPMI1640 medium, and centrifuged (500 × g, 10 minutes). The supernatant is aspirated, the pellet is well loosened, and 0.5 m of a 40% polyethylene glycol 4000 solution kept at 38 ° C.
The polyethylene glycol solution was mixed with the cell pellet by gently rotating the centrifuge tube by hand for 1 minute. Next, 1 ml of RPMI1640 medium kept at 38 ° C. was added every 30 seconds, and the tube was gently rotated. After this operation was repeated 10 times, 20 ml of RPMI1640 medium containing 10% fetal bovine serum was added, centrifuged (500 × g, 10 minutes), and the supernatant was removed.
HAT medium containing 0% fetal bovine serum (RPMI1640
In the medium, aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 ×
10 ^-5 M and hypoxanthine 1 × 10 ^-4 M), and washed twice by centrifugation.
The cells were suspended in 40 ml of the HAT medium.

This cell suspension was dispensed in a volume of 200 μl into each well of a 96-well cell culture plate, and culture was started in a 37 ° C. carbon dioxide incubator containing 5% carbon dioxide. During culture, about 100 μl of the medium in each well was removed at intervals of 2 to 3 days, and 100 μl of the HAT medium was newly added to select hybridomas that grow in the HAT medium. HT medium containing 10% fetal bovine serum (R)
Thymidine 1.6 × 10 ⁻⁵ M and hypoxanthine 1 × 10 ⁻⁴ M added to PMI1640 medium)
And observe the growth of the hybridoma,
On about day 10, hybridomas producing the desired monoclonal antibody were screened by the ELISA method described below.

(C) Establishment of Hybridoma The presence or absence of the produced antibody in the culture supernatant of the hybridoma was measured by platelet immobilization ELISA. 96-well ELISA
To each well of the plate for A (Immulon II; Japan Dynatech Co., Ltd.), 100 μl (1 × 10 6
⁷ ) and centrifuged with a plate centrifuge.
PBS (8.1mM-Na ₂ HPO ₄ containing 2% paraformaldehyde without removing the supernatant, 1.5MM-
_{KH 2 PO 4, 2.7mM-KCl} , and 137 mM-N
aCl; pH 7.4) was added to each well. After fixing at room temperature for 30 minutes, the plate was washed with PBS.

Then, after blocking with 5% bovine serum albumin and washing with PBS, 100 μl of the culture supernatant from each well of the hybridoma culture plate was added, and the mixture was added at 25 ° C.
For 1 hour. After washing the plate with PBS,
100 μl of peroxidase-conjugated anti-mouse immunoglobulin rabbit IgG antibody (Dako, Denmark) diluted 200-fold with PBS was added to each well. After the reaction, PB
Each well was washed three times with S, and an enzyme substrate solution [0.4 mg
/ Ml citrate-phosphate buffer (pH 5.0) containing orthophenyldiamine and 0.005% hydrogen peroxide solution]
200 μl was added to each well, and after a certain time, 3
The reaction was stopped by adding 50 μl of M sulfuric acid. 4 in each well
The absorbance at 92 nm was measured.

The above operation was repeated using CD36-deficient washed platelets instead of CD36-expressing washed platelets derived from healthy persons. As a result of performing the screening in this manner, the cells reacted with the washed platelet
Two wells were identified as wells that did not react with 36-deficient washed platelets. 24 hybridomas in the two wells
HT containing 10% fetal bovine serum
The cells were cultured in the medium for 4 to 5 days. After that, the presence or absence of the production of the monoclonal antibody was again confirmed by the platelet immobilization ELISA method described above, and then cloned by the limiting dilution method.

Ten days later, hybridoma clones producing the desired monoclonal antibody were screened by platelet immobilization ELISA. As a result, 20 to 40 antibody-producing clones were obtained for each hybridoma. From these clones, two clones (clone No. 1 and clone No. 2) having good growth properties, high antibody secretion ability and being stable were selected.
The reactivity of the antibodies produced from these clones was further examined by binding experiments using flow cytometry shown below.

The platelet-poor plasma prepared in Example 3 was added to 5 μl of the washed platelets for characteristic evaluation prepared in Example 2 above using a HEPES-Tyrode's buffer (137 mM-N
aCl, 2.7 mM-KCl, 0.42 mM-NaH ₂
PO ₄ , 4.2 mM-HEPES, 1 mM-MgCl ₂ ,
Diluted to various concentrations with 5.5 mM dextrose and 0.35% bovine serum albumin; pH 7.4) 30
was added to 5 μl of the hybridoma culture supernatant.
The reaction was performed at 10 ° C. for 10 minutes. Next, 1 μl of a fluorescein isothiocyanate (FITC) -labeled anti-mouse immunoglobulin rabbit antibody (one that does not react with human or bovine immunoglobulin; DAKO) was added, and reacted at room temperature and in a dark place for 30 minutes. After 50 μl of PBS containing 1% paraformaldehyde was added and reacted at room temperature for 30 minutes, 500 μl of PBS was further added, and a flow cytometer (FACS Calibur; Becton-Di) was added.
kinkson Immunocytometry S
systems).

The binding of the antibody is represented by the average fluorescence intensity, and the average fluorescence intensity when the proportion of platelet poor plasma is 0% is 10%.
FIG. 1 shows the result in the case of 0%. The polygonal line a indicates the clone No. The results obtained when the antibody (No. 1) produced from No. 1 was used are shown. 2 shows the results obtained when the antibody (No. 2) produced from Antibodies No. 2 was used. The line c indicates the commercially available anti-CD36 monoclonal antibody OKM5 (OrthoDiagnostic Syst.).
The results when em) were used are shown. Clone No. Antibody (No. 1) produced from Clone No. 1 has a lower reactivity depending on the abundance of platelet poor plasma, but clone No. 1 2
Antibody (No. 2) produced by the method and a commercially available anti-CD36
The reactivity of the monoclonal antibody OKM5 hardly decreased depending on the abundance ratio of platelet poor plasma. Normal,
When analyzing the protein on the platelet membrane surface by flow cytometry, it is inconvenient in analysis that the reactivity is greatly affected by the presence of the plasma component. Because, as a platelet-containing sample, whole blood, platelet-rich plasma,
This is because the obtained result differs when washed platelets are used.

The inventor of the present invention has proposed clone No. 1 which is normally considered as negative reactivity. The reactivity of the antibody produced from the clone No. 1 was determined by the limiting dilution method. 1 was re-cloned to establish a monoclonal antibody-producing hybridoma CD36-1.
Similarly, clone No. was obtained by limiting dilution. Recloning 2 was also performed to establish a monoclonal antibody-producing hybridoma CD36-2. The washed platelets solubilized with a surfactant were fractionated by SDS-polyacrylamide electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, subjected to immunoblot, and hybridoma CD36-1 and hybridoma CD36-
Each monoclonal antibody secreted from 2 has a molecular weight of 8
Specific reaction with 8,000 CD36 proteins was confirmed.

[0049]

Example 5 << Production of Monoclonal Antibody >> (a) In Vitro Method Mouse hybridoma CD36 obtained in Example 4 above
-1 in RPMI 1640 medium containing 10% fetal calf serum at 37 ° C. in a 5% carbon dioxide atmosphere.
The cells were cultured for 96 hours. Centrifuge the culture (10,000 ×
g, 10 minutes), and 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, left to stand for 60 minutes, and centrifuged (10,000 × g, 10 minutes). The obtained precipitate was dissolved in a small amount of 10 mM Tris-HCl buffer (pH 8.0) and dialyzed against a 1000-fold amount of 10 mM Tris-HCl buffer.

The dialysate was applied to a column of Q-Sepharose strong ion exchanger which had been pre-equilibrated with 10 mM Tris-HCl buffer. Elution of monoclonal antibody is 10m
M Tris-HCl buffer (pH 8.0) and 0.5M-NaC
This was carried out by a concentration gradient method with 10 mM Tris-HCl buffer (pH 8.0) containing 1 l. The eluted monoclonal antibody was concentrated by an ultrafiltration method, and dialyzed against a 0.1 M phosphate buffer (pH 8.0). The dialysate was passed through a column of goat anti-bovine serum IgG Sepharose 4B to remove bovine serum IgG. Next, the flow-through was applied to a protein A Sepharose 4B column equilibrated with a 0.1 M phosphate buffer (pH 8.0). The column was adjusted to pH 3.
Elution with the buffer solution No. 5 yielded a purified monoclonal antibody CD36-1. Monoclonal antibody CD
36-2 was obtained in the same manner. In this specification, the name of the hybridoma is also used as the name of the monoclonal antibody produced from the hybridoma.

(B) In vivo method 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to BALB / C mice of 10 to 12 weeks of age, and 14 to 20 after administration. Hybridoma CD grown in vitro in the mouse intraperitoneal cavity on day
36-1 was inoculated at 2 × 10 ⁶ cells per mouse. About 10-15 ml of ascites was obtained from one mouse. The antibody concentration was 5-10 mg / ml. Purification of the monoclonal antibody in the ascites was performed in the same manner as in the in vitro method described above. However, an operation of passing through a column of goat anti-bovine serum IgG Sepharose 4B was not performed. Monoclonal antibody CD36-2 was obtained in a similar manner.

[0052]

Example 6 << Identification of Immunoglobulin Class of Monoclonal Antibody >> The immunoglobulin class of monoclonal antibody CD36-1 and monoclonal antibody CD36-2 was identified using a mouse monoclonal antibody typing kit (Amersham Pharmacia Biotech). Immunoglobulin classes of the monoclonal antibodies CD36-1 and monoclonal antibodies CD36-2 were both IgG _1.

[0053]

Example 7 << Preparation of FITC-Labeled Monoclonal Antibody >> Purified Monoclonal Antibody C Obtained in Example 5
Labeling of D36-1 with FITC was performed in accordance with the introduction to monoclonal antibody experimental procedures (second edition, page 209, edited by Kodansha Scientific). That is, monoclonal antibody CD36-1 (1.33 mg / ml) 1.5 dialyzed against 0.1 M sodium bicarbonate buffer (pH 8.5)
50 μl of FITC (10 mg / ml; Sigma) dissolved in dimethyl sulfoxide (DMSO)
Is gradually added under stirring and under light shielding, and further under light shielding,
The reaction was performed at 4 ° C. for 15 hours. It was applied to a Sephadex G-25 column (PD-10; manufactured by Pharmacia) previously equilibrated with PBS to remove unreacted FITC. The eluted fractions of the monoclonal antibody were collected and dialyzed sufficiently against PBS. The dialyzed monoclonal antibody solution was passed through a membrane filter having a pore size of 0.22 μm, added with an equal amount of glycerol, and stored frozen at −20 ° C. until use.

[0054]

Example 8 << Measurement of Platelet Membrane CD36 in Blood of Patient Who Underwent Percutaneous Coronary Angioplasty >> In this example, except for using sodium citrate-containing blood derived from the patient as a platelet sample, The measurement was performed by the flow cytometry described in Example 4 above. That is, 10 μl of sodium citrate-added blood and modified HEPES-
Tyrode's buffer (137 mM NaCl, 2.
_{68mM-KCl, 3.75mM-NaH 2} PO 4, 3.
_{78mM-HEPES, 0.99mM-MgCl 2} ,
FITC-labeled anti-CD36 monoclonal antibody diluted to a concentration of 0.8 μg / ml with 0.28 mM glucose and 0.35% bovine serum albumin (pH 7.35) (that is, the FITC-labeled monoclonal antibody CD36- prepared in Example 7 above). 1 or 40 μl of a commercially available FITC-labeled monoclonal antibody OKM5) solution, and allowed to react at room temperature under light shielding for 30 minutes. Then, 50 μl of PBS containing 1% paraformaldehyde was added, and the mixture was added at room temperature for 3 hours.
After reacting for 0 minutes, 500 μl of PBS was further added,
Flow cytometer (FACS Calibur; B
ecton-Dickinson Immunocyt
OmetrySystems).

The results are shown assuming that the reactivity with platelets before percutaneous coronary angioplasty (mean fluorescence intensity) is 100%, and the reactivity with platelets on 1 to 5 days after percutaneous coronary angioplasty (mean mean). The ratio of the fluorescent intensity is shown in FIGS. The results shown in FIG. 2 show that after performing percutaneous coronary angioplasty,
FIG. 3 shows the measurement results for patients who did not develop restenosis within 6 months. The results shown in FIG. 3 are the measurement results for patients who developed restenosis within 6 months after percutaneous coronary angioplasty. is there. 2 and 3, the polygonal line a is
The results are shown for the monoclonal antibody CD36-1, and the line b shows the results for the commercially available anti-CD36 monoclonal antibody OKM5.

As shown in FIG. 2, in this patient who did not develop restenosis within 6 months after percutaneous coronary angioplasty, the monoclonal antibody The binding of CD36-1 (line a) was reduced. However, a commercially available anti-CD36 monoclonal antibody OK
The binding of M5 (line b) did not change. That is, the expression level of CD36 present on the platelet membrane surface in the patient's blood does not change before and after percutaneous coronary angioplasty. Some substance that inhibits or competes with the binding to platelet membrane CD36 is thought to have arisen in the blood. Considering the properties of the monoclonal antibody CD36-1 shown in Example 4 above, this substance is naturally present in the blood of healthy individuals, but in patients who do not develop restenosis, percutaneous coronary angioplasty can be used. It is thought that the blood concentration will increase later.

On the other hand, in this patient who developed restenosis within 6 months after percutaneous coronary angioplasty, as shown in FIG. The binding (line a) did not change. In addition, a commercially available anti-CD36 monoclonal antibody O
The binding property of KM5 (line b) did not change.

Similarly, for 38 patients who underwent percutaneous coronary angioplasty, the binding of monoclonal antibody CD36-1 to platelet membrane CD36 on day 1 postoperatively before percutaneous coronary angioplasty The relationship between the ratio and the presence or absence of restenosis within 6 months after the operation was examined. Table 1 shows the results
Shown in In addition, when the binding to the platelet membrane CD36 after the operation was 80% of the binding to the platelet membrane CD36 before the operation, the cutoff value (disease state discrimination value) was determined. Platelet membrane CD3
The discriminating ability of the disease state by measuring the binding ratio of the monoclonal antibody CD36-1 to No. 6 was calculated from the results in Table 1 (Japanese clinical practice, Vol. 53, pp. 9-28, 1995 extra edition). Sensitivity (discriminating power for disease group) is 0.750, and specificity (discriminating power for non-diseased group) is 0.909.
And the likelihood ratio (total discriminability) was 8.251. These results are clearly superior to discriminating the presence or absence of restenosis within 6 months after the operation, as compared with the ability to discriminate the disease state by measuring the expression level of P-selectin on the platelet membrane surface, which will be described later. That is, the method of the present invention for examining the binding ratio of the monoclonal antibody CD36-1 to the platelet membrane CD36 on the first postoperative day before percutaneous coronary angioplasty can accurately predict restenosis within 6 months after the operation. Method was confirmed.

<< Table 1 >> Binding ratio of monoclonal restenosis antibody CD36-1 after percutaneous coronary angioplasty Yes No 80% or more 12 cases 2 cases Less than 80% 4 cases 20 cases

The above FITC-labeled monoclonal antibody C
Instead of D36-1, a FITC-labeled anti-P-selectin monoclonal antibody (antibody name: monoclonal antibody 2T
60; Tagami et al., Platelets, Vol. 4, 31-
39, 1993) and 40 patients who underwent percutaneous coronary angioplasty (including 38 patients who participated to obtain the results shown in Table 1) after percutaneous coronary angioplasty The relationship between the expression level of P-selectin on the platelet membrane surface and the presence or absence of restenosis within 6 months after the operation was examined. Table 2 shows the results. In addition, the expression level of P-selectin on the platelet membrane surface after the operation was represented by an average fluorescence intensity measured by a flow cytometer, and 6.0 was set as a cutoff value (disease state discrimination value). When the disease state discrimination ability by measuring the expression level of P-selectin on the platelet membrane surface was similarly calculated from the results in Table 2, the sensitivity was 0.714, the specificity was 0.808, and the likelihood was high. The degree ratio was 3.713.

<< Table 2 >> Expression of P-selectin on the surface of restenosis of platelet membrane after percutaneous coronary angioplasty Yes None 6.0 or more 10 cases 5 cases Less than 6.0 4 cases 21 cases

[0062]

Example 9 << Preparation of purified CD36 antigen >>
Known methods (McGregor, JL et al., Journal)
al of Biological Chemist
ry, 264, 501-506, 1989)
Performed according to. That is, 5 × 10 ¹¹ washed human platelets were prepared, and a solution for preparing CD36 (10 mM-Tris-HC) was adjusted to 5 × 10 ⁹ / ml.
1, 0.1 mM leupeptin, and 5 mM EDTA;
After suspending at pH 7.4), sonication was performed at 4 ° C. for 20 minutes. This treatment solution was centrifuged at 9,000 × g for 20 minutes, and the resulting supernatant was collected.
The precipitate was collected by centrifugation at 000 × g for 1 hour. To this precipitate, a CD36 preparation solution containing 1% Triton X-114 was added, and CD36 was extracted into the Triton X-114 fraction.

Triton X-114 containing CD36
Fractions were separated by Q-Sepharose Fast Flow (Sepha
It was applied to a rose fast flow column (Pharmacia Biotech) and ion exchange chromatography was performed. CD36 is 0.15 M NaCl
Collected in the eluted fraction. This eluted fraction is further treated with Mono-Q
(Mono-Q) column (Pharmacia Biotech)
Was purified by ion exchange chromatography using HPLC and gel filtration using a Superose 12 column (Pharmacia Biotech) to obtain purified CD36. The purified CD36 thus obtained was used in the following Examples.

[0064]

Example 10 << Measurement of Binding Inhibitory Substance between Human Plasma Sensitive Anti-CD36 Monoclonal Antibody and Purified CD36 by Sandwich Method >> In this example, within 6 months after percutaneous coronary angioplasty as a test sample Platelet-poor plasma was used from a patient who did not develop restenosis and a patient who developed restenosis within 6 months after percutaneous coronary angioplasty. Monoclonal antibody CD36-1 at 10 μg /
100 μl of 50 mM sodium bicarbonate buffer (pH 9.5) containing a
A microtiter plate (Immulon II; Nippon Dynatech Co., Ltd.) for each well
For 2 hours. The plate was washed with 1% BSA and 0.
The plate was washed three times with a 50 mM sodium phosphate buffer (pH 7.0) containing 15 M-NaCl (hereinafter referred to as PBS-BSA). A sample (platelet-poor plasma as a test sample) was added with 1 μg / m of the purified CD36 prepared in Example 9 in the wells of the plate sensitized with the antibody in this manner.
1), reacted at 25 ° C. for 30 minutes, and diluted 10-fold with PBS-BSA.
1 was added and reacted at 25 ° C. for 30 minutes.

After washing the plate three times with PBS-BSA, the horseradish peroxidase-labeled monoclonal antibody C diluted to 1 μg / ml with PBS-BSA was used.
D36-2 (100 μl) was added and reacted at 25 ° C. for 30 minutes. Labeling of monoclonal antibody CD36-2 with horseradish peroxidase was carried out according to the method of Nakane and Kawaoi (Journal of Histochem).
istry and Cytochemistry, 2nd
2, 1084-1091, 1974). After washing the plate three times with PBS-BSA, 200 μl of an enzyme substrate solution [citrate-phosphate buffer (pH 5.0) containing 0.4 mg / ml orthophenyldiamine and 0.005% hydrogen peroxide solution] was added. After a certain period of time, the reaction was stopped by adding 50 μl of 3 M sulfuric acid. The absorbance at 492 nm of each well was measured.

FIG. 4 shows the results of measurement using platelet-poor plasma one day after surgery for patients who did not develop restenosis within 6 months after percutaneous coronary angioplasty. The absorbance obtained using platelet-poor plasma before the operation was defined as 100%, and the relative absorbance was shown. In this patient who did not develop restenosis, the measured absorbance of platelet-poor plasma on the first day after operation was reduced to about 50% as compared to before percutaneous coronary angioplasty. On the other hand, for patients who developed restenosis within 6 months after percutaneous coronary angioplasty, the results of measurement using platelet poor plasma on day 1 after the operation are shown in FIG. As in FIG. 4, the absorbance obtained using preoperative platelet poor plasma was 1
The relative absorbance was set to 00%. For this patient who developed restenosis, the measured absorbance of platelet-poor plasma on the 1st postoperative day compared to before percutaneous coronary angioplasty was:
Hardly changed.

[0067]

EFFECT OF THE INVENTION The human platelet membrane protein CD36 is useful as a predictive marker of restenosis after percutaneous coronary angioplasty with a balloon catheter for coronary artery disease.
According to the detection method of the present invention, the possibility of coronary restenosis after percutaneous coronary angioplasty can be detected, that is, the presence or absence of restenosis can be predicted.

[Brief description of the drawings]

FIG. 1 is a graph showing that the binding of monoclonal antibody CD36-1 to washed platelet membrane CD36 is inhibited in a concentration-dependent manner in healthy human plasma.

FIG. 2. For patients who did not develop restenosis within 6 months after percutaneous coronary angioplasty,
Monoclonal antibody CD36- against platelets on day 5
1 is a graph showing the results of measurement of the binding property of 1 or a commercially available anti-CD36 monoclonal antibody OKM5 by flow cytometry.

FIG. 3: Monoclonal antibody CD36 of the invention against platelets 1-5 days after percutaneous coronary angioplasty for patients who developed restenosis within 6 months after percutaneous coronary angioplasty
-1 or commercially available anti-CD36 monoclonal antibody OKM5
4 is a graph showing the results obtained by measuring the binding property of a sample by flow cytometry.

FIG. 4. Monoclonal antibody CD36 present in platelet poor plasma before and one day after percutaneous coronary angioplasty for patients who did not develop restenosis within 6 months after percutaneous coronary angioplasty. 1 is a graph showing the change in the amount of a binding inhibitor between purified -1 and purified CD36 as a relative absorbance.

FIG. 5: For patients who developed restenosis within 6 months after percutaneous coronary angioplasty, before and after percutaneous coronary angioplasty 1
It is the graph which represented fluctuation | variation of the quantity of the binding inhibitor of monoclonal antibody CD36-1 and purified CD36 which exists in the platelet poor plasma on the day as a relative absorbance.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/08 C12N 15/00 C (72) Inventor Noriko Akamatsu 4-25 Minamikase, Saiwai-ku, Kawasaki-shi, Kanagawa -6 (72) Inventor Hideki Suzuki 596-1 Tsu, Kamakura City, Kanagawa Prefecture (72) Inventor Atsumi Yamaguchi 5-10-5, Oizumi Gakuen-cho, Nerima-ku, Tokyo (72) Inventor Masamasa Yamamoto, Asama-cho, Omiya-shi, Saitama 2-56-11 (72) Inventor Kenjiro Tagami 1030 Kashiwada-cho, Ushiku-shi, Ibaraki Pref. (72) Inventor Hiroshi Kasanuki 1-13-1 Todoroki, Setagaya-ku, Tokyo (72) Inventor Kaori Murasaki 3 Nukii, Nerima-ku, Tokyo −26−6 (72) Inventor Masatoshi Kawana 1-22-11 Komaba, Meguro-ku, Tokyo (72) Inventor Masahiko Aosaki 2-7-22-203, Tamagawa, Ota-ku, Tokyo (72) Inventor Isao Kawano, Chiyoda, Tokyo 1-11 Higashi Kanda, Ward No. Co., Ltd. Yatoron in the F-term (reference) 4B024 AA15 BA44 DA02 GA03 GA18 GA27 HA15 4B064 AG27 CA10 CA20 CC24 CE04 CE06 CE11 CE12 DA13 4H045 AA30 BA17 BA53 BA70 CA42 DA50 DA76 DA86 EA50 FA72 GA06 GA10 GA15 GA22 GA23 GA26 HA06

Claims (2)

[Claims] 1. A method for detecting the possibility of restenosis of a coronary artery after percutaneous coronary angioplasty, wherein the method specifically reacts with human CD36 and reacts with human washed platelet membrane CD36 with human plasma. -CD3 suppressed in a concentration-dependent manner
6 by the immunological analysis method using the monoclonal antibody or its antibody fragment, the human platelet membrane CD36 in the presence of human plasma in each test sample collected before and after the percutaneous coronary angioplasty The above-mentioned detection method, wherein the binding properties of the monoclonal antibodies are respectively analyzed, and the binding properties before and after the operation are compared. 2. The method according to claim 1, wherein the immunological analysis method is an immunological analysis method using a flow cytometer.
The detection method described in 1.