WO2004029094A1 - Monoclonal antibody specific for human mitochondrial adenylate kinase isozyme 3 - Google Patents

Abstract

Disclosed are monoclonal antibodies specific for human mitochondrial adenylate kinase isozyme 3 (AK3), a composition and a kit comprising the monoclonal antibodies for the diagnosis of heart disease, and a method of detecting a heart disease marker AK3 using such a composition or kit.

Description

MONOCLONAL ANTIBODY SPECIFIC FOR HUMAN MITOCHONDRIAL ADENYLATE KINASE ISOZYME 3

TECHNICAL FIELD

The present invention relates to monoclonal antibodies specific for human mitochondrial adenylate kinase isozyme 3 (AK3), which have high specificity and affinity to AK3.

PRIOR ART

Heart diseases including acute myocardial infarction often develop in adults after age 40, the rate of mortality from the disease is on an increasing trend. The monthly mean number of diagnosis for heart disease in general and university hospitals in Korea is over 200. In America, each year millions visit emergency rooms with chest pain.

Electrocardiogram (ECG) patterns have been generally employed for diagnosis of heart disease patients suffering from chest pain. With this method, myocardial infarction (MI) is diagnosed by Q waves and abnormal ST-T segment wave changes on the ECG. However, over 50% of the patients presented to the emergency department for acute MI were misdiagnosed. Of them, 5% were misdiagnosed as not having myocardial infarction, and 16% of the misdiagnosed patients eventually died. In order to solve these problems, biomarkers for myocardial infarction have been developed. An ideal biomarker for MI should have the following features: first, the biomarker should be present in only myocardial cells and released into the blood after myocardial injury; second, the rapid release of the biomarker into the blood soon after a myocardial injury depends on its intracellular location and molecular weight; third, the degree of myocardial injury should have a linear relationship with the released amount of the biomarker; fourth, particular training or techniques should be not required upon diagnosis with the biomarker, and reagents used for the diagnosis should be economical and stable; fifth, the biomarker should be elevated in patient's serum or plasma after the onset of chest pain; and sixth, the released biomarker should be rapidly removed in order to evaluate subsequent myocardial infarction. However, there is yet no a satisfactory biomarker with all of these ideal conditions.

Currently, creatine kinase (CK) mass assay and troponin test is available for the detection of myocardial damage. Creatine kinase (CK) is found primarily in muscle and brain tissue, and exists as three dimeric isoenzymes: CK-MM, CK- MB, and CK-BB. CK-MM is predominant in skeletal muscle. CK-BB is present primarily in brain tissue, and is the only CK form found in the cerebrospinal cord. The CK-MB heterodimer is enriched in cardiac muscle, and is present at low concentrations in skeletal muscle. Due to their tissue-specific expression, the CK isoenzymes are used as biomarkers for the detection of tissue damage or cancer, where serum levels of the CK isoenzymes are evaluated. In particular, CK-MB is known as an enzyme reflecting the developed states of acute myocardial infarction, and its blood level fluctuates in a range of about 5% in all kinds of muscle diseases ranging from burn and trauma to heart and skeletal muscle diseases. However, CK-MB has some disadvantages, as follows. CK-MB remains elevated only for 2-3 days after the onset of chest pain, and can be measured in the blood after skeletal-muscle injuries, thus limiting its use as a diagnostic tool. Also, in case of using CK-MB in the diagnosis of myocardial infarction, about 20% false positive results occur. The criteria set by World Health Organization (WHO) for AMI diagnosis requires two of three conditions, as follows; (1) typical chest pain; (2) ECG changes such as Q waves; and (3) 2-fold or higher blood levels of the aforementioned enzymes (e.g., CK-MB) than their normal maximum levels by the aforementioned enzyme assay. On the other hand, in order to reduce misdiagnosis upon use of CK-MB as a cardiac failure marker, Boyce N. et al. developed cardiac troponin T (cTnT) test (Katus, H.A. et al.,(1989) J. Mol. Cell. Cardiol. 21:1349-1353). The cTnT test has been approved from the Food and Drug Administration in the U.S. A for use in the detection of myocardial infarction, commercialized by Boehringer Manheim Diagnostics (Manheim, Germany), and used in America since November 1996.

However, the cTnT test was identified as having cross-reactivity with troponin T originated in skeletal muscle. For this reason, the cardiac troponin I (cTnl) test is preferred.

However, since cTnT and cTnl levels do not rise for 6 hrs after the onset of chest pain, the measurement of the troponin levels should be repeated 8 to 10 hrs after the onset of chest pain (Eisenbrey et al. (1995) The Journal of American Medical Association, 74, 1343-1344). Therefore, the cardiac-specific troponin assays are unsatisfactory in the detection of myocardial damage, and thus should be carried out in conjunction with the CK-MB activity or mass assay. In addition, all of the aforementioned methods for the diagnosis of myocardial damage are disadvantageous in terms of requiring expensive testing devices, specialists in heart disease (cardiac disease) and highly trained personnel, resulting in very high test costs and thereby limiting their availability to a small number of either university or general hospitals. On the other hand, as disclosed in Korean Pat. Application No. 2000-

0005808 filed by the present applicants, in immunohistochemistry of various tissues, human adenylate kinase isozyme 1 (hAKl) was detected in all tested tissues, while human AK2 (hAK2) was found in liver, brain, cardiac muscle, heart and alveolar macrophages in lung, and hAK3 was found in liver, cardiac muscle, heart and alveolar macrophages in lung. In addition, in Western blotting of cardiac muscle and skeletal muscle, AK1 was found to express in all tested tissues, while AK2 and AK3 were found to not be detected in skeletal muscle but showed cardiac-specific expression. Based on the finding, the present applicants described in the publication that the problems encountered in the conventional biomarkers for the detection of heart disease with respect to accuracy and convenience can be substantially solved by employing antibodies specific for mitochondrial adenylate kinase isozyme 3.

The conventional diagnostic kits for myocardial injuries can give false positive results, causing medical doctors to make an incorrect diagnosis that may result in patient's death. In this situation, lawsuits may be filed against medical doctors or manufacturers of the diagnostic kits. Therefore, there is an urgent need for development of monoclonal antibodies available as highly reliable diagnostic markers for myocardial infarction with improved specificity and affinity to mitochondrial adenylate kinase isozyme 3. In this regard, leading to the present invention, the present inventors established monoclonal antibodies specifically bind to mitochondrial adenylate kinase isozyme 3 (AK3), and found that the antibodies with high specificity and affinity to mitochondrial AK3 accurately detect a heart disease marker, AK3.

DISCLOSURE OF THE INVENTION

The present invention relates to a monoclonal antibody specific to adenylate kinase isozyme 3 (AK3), comprising 4 or more of 6 CDRs (Complementarity Determining Region) of a group selected from (a) SEQ ID Nos: 41 to 46, (b) SEQ ID Nos: 47 to 52, (c) SEQ ID Nos: 53 to 58, (d) SEQ ID Nos: 59 to 64, (e) SEQ ID Nos: 65 to 70, (f) SEQ ID Nos: 71 to 76, (g) SEQ ID Nos: 77 to 82, (h) SEQ ID Nos: 83 to 88, (i) SEQ ID Nos: 89 to 94 and (j) SEQ ID Nos: 95 to

100.

Preferably, the monoclonal antibody has 6 CDRs of the group selected from (a) to (j). More preferably, the monoclonal antibody is produced by a hybridoma selected from the group consisting of accession numbers KCLRF-BP- 00058 to KCLRF-BP-00066.

In addition, the present invention relates to a monoclonal antibody specific to mitochondrial adenylate kinase isozyme 3 (AK3), recognizing peptides of SEQ ID Nos: 102 to 106. Further, the present invention relates to a monoclonal antibody specific to mitochondrial AK3, recognizing a peptide of SEQ ID NO: 107.

Still further, the present invention relates to a monoclonal antibody with an epitopic specificity identical to a monoclonal antibody produced by a hybridoma cell selected from the group consisting of accession numbers KCLRF-BP-00058 to

KCLRF-BP-00066.

Still further, the present invention relates to a composition for detecting a heart disease marker comprising the monoclonal antibody.

Still further, the present invention relates to a diagnostic kit for detecting a heart disease marker comprising the monoclonal antibody.

Still further, the present invention relates to a method of detecting a heart disease marker, comprising the steps of treating a biological sample with monoclonal antibody and detecting antigen-antibody complex formation.

In the method, the antigen-antibody complex formation is preferably detected by ELISA, and more preferably, by sandwich ELISA.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 shows the intracellular location of AK3;

Fig. 2 is a photograph showing PCR product for AK3, separated on an agarose gel by electrophoresis;

Fig. 3 shows a pCR2.1-AK3 vector construct;

Fig. 4 is a photograph showing a restriction enzyme mapping pattern of pCR2.1-AK3;

Fig. 5 is a photograph showing a result of a restriction enzyme mapping pattern of ρQE30-AK3;

Fig. 6 shows a pQE30-AK3 vector construct; and Fig. 7 is a graph showing cross-reactivity of an anti-AK3 monoclonal antibody to AK isozymes.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention provides monoclonal antibodies with high specificity and affinity to mitochondrial adenylate kinase isozyme 3 (AK3).

The monoclonal antibodies of the present invention are different from the other antibodies against conventional biochemical markers for the diagnosis of myocardial infarction, the MB isoenzyme of creatine kinase (CK-MB), troponin T and troponin I, in terms of having a very weak cross-reactivity to the known adenylate kinase isozymes 1 and 2, as well as having a highly selective binding affinity to the cardiac muscle-specific mitochondrial AK3. For these reasons, the monoclonal antibodies are suitable for the diagnosis of heart disease.

The term "mitochondrial adenylate kinase isozyme 3 (AK3)", as used herein, includes mitochondrial AK3 itself, recombinant proteins thereof by genetic engineering, and artificial variants and mutants thereof, as well as the wild type of AK3 and functional equivalents thereof.

Adenylate kinase, with reference to Reaction Formula 1, below, rapidly maintains dynamic equilibrium between adenine nucleotides within a cell by catalyzing the reversible transfer of phosphate groups from XTP to AMP, releasing XDP and ADP.

[Reaction Formula 1]

XTP + AMP XDP + ADP

Adenylate kinase is one of enzymes essential for phosphorylation associated with cellular metabolism and signal transduction. and known to participate in energy metabolism, apoptosis and tumorigenesis. About 40 different kinds of adenylate kinase were reported in the biological system (Matsuura, S., Igarashi, M., Tanizawa, Y., Yamada, M., Kishi, F., Kajii, T., Fujii, H., Miwa, S., Sakurai, M., & Nakazawa, A. (1989) J. Biol. Chem. 264, 10148- 10152). In vertebrate animals, as shown in Fig. 1, there are three different isozymes: AK1 (EC 2.7.4.3), which is present in the cytosol; AK2(EC 2.7.4.3), which is located in the intermembrane space of mitochondria; and AK3 (EC

2.7.4.10), which is located in the mitochondrial matrix (Kuby, S. A., Palmieri, R. H., Frischat, A., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2392-2399; Sachsenheimer, W., & Schulz, G. E. (1977) J. Mol. Biol. 114, 23-36; Egner, U., Tomasselli, A. G., & Schulz, G. E. (1987) J. Mol. Biol. 195, 649-658). Human AK3 consists of 223 amino acids, and also called nucleoside triphosphte-adenylate phosphotransferase . Its cDNA sequence and primary amino acid sequence were reported by Xu et al. (Xu, G., O'Connell, P., Stevens, J. and White, R. (1992) Characterization of human adenylate kinase 3 (AK3) cDNA and mapping of the AK3 pseudogene to an intron of the NF1 gene. Genomics 13: 537-542). Amino acid sequence of human AK3 is disclosed in

SEQ ID NO: 101.

AK3 catalysts the reaction as shown in Reaction Formula 2, below (Chiga, M., Rogers, A.E., Paut, G.W.E. (1961) J. Biol. Chem., 236, 1800; Albrecht, G.J., (1970) Biochemistry, 9:2426).

[Reaction Formula 2]

GTP + AMP GDP + ADP

Human andenylate kinase deficiency was reported to be associated with hereditary hemolytic anemia (Matsuura, S., Igarashi, M., Tanizawa, Y., Yamada,

M., Kishi, F., Kajii, T., Fujii, H., Miwa, S., Sakurai, M. and Nakazawa, A. (1989) J. Biol. Chem. 264, 10148-10152). Also, in muscle and brain tissue, synthesis of thiamine triphosphate is catalyzed by creatine kinase and adenylate kinase.

Compared with other enzymes, AK3 has been not seriously investigated. In this situation, the present applicants disclosed the tissue specificity of AK3 in human in Korean Pat. Application No. 2000-0005808.

As described in the Korean Pat. Application, the present inventors found first that AK3 is expressed in cardiac muscle but not or under the detectable range of concentration in skeletal muscle, wherein the heart-specific AK3 can be used as a heart disease marker for the detection of diseases of the circulatory system associated with myocardial injury.

As used herein, the term "monoclonal antibody", which is well known in the art, refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against multiple different epitopes (antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies are advantageous in term of improving selection and specificity of diagnosis and analytic assays using antigen-antibody binding, as well as being not contaminated with other immunoglobulins because of being produced by hybridoma cell culture.

Monoclonal antibodies specific to AK3 may be produced by a fusion method well known in the art (see, Kohler and Milstein (1976) European Journal of Immunology 6:511-519). In order to establish "hybridomas" secreting the monoclonal antibodies, one of two different cells to be fused is obtained from immunologically suitable host animals such as mice immunized with adenylate kinase isoenzymes, and the other cells are tumor or myeloma cells. The two different cells are fused by a method known in the art, for example, employing polyethylene glycol, and the resulting antibody-producing cells are proliferated by a standard tissue culture method. Homogeneous cell populations are obtained by subcloning using a limited dilution technique. Then, hybridomas producing monoclonal antibodies specific to AK3 are cultivated on a large scale in vitro or in vivo according to a standard technique. Monoclonal antibodies produced by the hybridomas can be used in an unpurified state. Preferably, the monoclonal antibodies are obtained at a high purity (for example, over 95% purity) according to a method known in the art. Examples of the purification method include gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and using the method, the antibodies may be purified from culture supernatants or ascitic fluids.

The monoclonal antibody-producing hybridomas according to the present invention, SJB3-31 (KCLRF-BP-00066), SJB3-32 (KCLRF-BP-00058), SJB3-33 (KCLRF-BP-00059), SJB3-34 (KCLRF-BP-00060), SJB3-35 (KCLRF-BP- 00065), SJB3-36 (KCLRF-BP-00061), SJB3-37 (KCLRF-BP-00062), SJB3-38

(KCLRF-BP-00063), SJB3-39 (KCLRF-BP-00064) and SJA3-86 (KCLRF-BP- 00030) were deposited at KCLRF (Korean Cell Line Research Foundation), where SJA3-86 were deposited on May 19, 2000 and the rest on Oct. 7, 2002.

In the present invention, "monoclonal antibodies produced by the hybridoma SJB3-31" was designated as "SJB3-31". Also, monoclonal antibodies produced by other hydridomas were designated in the same manner.

The present inventors designed single-chain variable fragments (scFv) of anti-AK3 monoclonal antibodies produced by the hybridomas, carried out out sequence analysis of light chain (LC) and heavy chain (HC) variable regions, and determined CDR (complementarity determining region) using the Kabat

Numbering Scheme (http://www.bioinf.org.uk/abs). The obtained nucleotide and amino acid sequences of the variable regions are disclosed in SEQ ID Nos: 1 to 40. In addition, amino acid sequences of CDR of the variable regions are disclosed in SEQ ID Nos: 41 to 100, and this relation of SEQ ID Nos with CDR is summarized in Table 1 , below. TABLE 1

The term "variable" refers to the fact that certain portions of hyper- variable domains in immunoglobulin variable regions (or domains) differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. ' However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called CDRs (Complementarity Determining Regions) both in the light chain and the heavy chain variable domains. The CDRs are responsible for recognition of a particular antigen, especially in this patent, AK3, forming antigen-antibody complexes. CDRs in each of the monoclonal antibodies of the present invention have characteristic amino acid sequences. A portion or all of the six CDRs in each monoclonal antibody are held together and contribute to the formation of the antigen-binding site of the antibody, thereby allowing the antibody to recognize its particular epitope.

In an aspect, the present invention provides a monoclonal antibody specific to adenylate kinase isozyme 3 (AK3), comprising 4, 5 or 6 of 6 CDRs (Complementarity Determining Region) of a group selected from (a) SEQ ID Nos: 41 to 46, (b) SEQ ID Nos: 47 to 52, (c) SEQ ID Nos: 53 to 58, (d) SEQ ID Nos: 59 to 64, (e) SEQ ID Nos: 65 to 70, (f) SEQ ID Nos: 71 to 76, (g) SEQ ID Nos: 77 to 82, (h) SEQ ID Nos: 83 to 88, (i) SEQ ID Nos: 89 to 94 and (j) SEQ ID Nos: 95 to l00. In a preferred aspect, the monoclonal antibody specific to AK3 comprises

6 CDRs of the group selected from (a) to (j).

In a more preferred aspect, the monoclonal antibody specific to AK3 is produced by a hybridoma selected from the group consisting of SJB3-31 to SJB3- 39 (accession Nos KCLRF-BP-00058 to KCLRF-BP-00066). The present invention provides a monoclonal antibody with an epitopic specificity identical to a monoclonal antibody produced by a hybridoma cell selected from the group consisting of accession Nos KCLRF-BP-00058 to KCLRF- BP-00066.

The use of monoclonal antibodies in the detection of the antigen has an advantage of having particular interaction with the antigen by recognizing a single epitope on the antigen. Epitope mapping in order to identify antigenic regions participating in the interaction between an antigen and its antibody can be approached by various methods, as follows: biopanning using phage display peptide libraries; determination of fragments including epitope sequences through a screening process comprising immunoblotting antigenic polypeptide fragments produced by protease digestion; screening a peptide array immobilized on a solid phase such as an activated membrane or polyethylene pin; and competitive ELISA using soluble peptides to determine the importance of each amino acid residue in an epitope. In order to identify epitopes on AK3, recognized by the monoclonal antibodies of the present invention, the present inventors employed phage display peptide libraries, and found that the monoclonal antibodies specific to AK3 recognize epitopes having the peptide sequences listed in Table 2, below.

TABLE 2

As shown in Table 2, the epitope designated as SEQ ID NO 102 may be recognized by the monoclonal antibodies SJB3-31, SJB3-34 and SJB3-39. The epitope designated as SEQ ID NO 103 may be recognized by the monoclonal antibodies SJB3-33, SJB3-36 and SJB3-35.

In another aspect, the present invention provides monoclonal antibodies specific to AK3, recognizing the peptides of SEQ ID Nos: 102 to 106.

Based on the epitopes identified by using a phage display library kit, a linear epitope was determined, which has an amino acid sequence KSLLNPDH of SEQ ID NO 107, corresponding to residues 60 to 68 of SEQ ID NO 101 representing the amino acid sequence of AK3. In a further aspect, the present invention provides a monoclonal antibody specific to AK3, recognizing the peptide of SEQ ID NO 107.

Binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard's method described by Munson et al., Anal Biochem.,

107:220, 1980. Binding affinities of the monoclonal antibodies of the present invention are given in Table 3, below. As shown in Table 3, the monoclonal antibodies SJB3-31 to SJB3-39 have binding affinities over 10-fold higher than that of the SJA3-86 monoclonal antibody, and over 100-fold higher than that of the AK3-specific SJA3-1-17 monoclonal antibody. Also, heavy-chain and light-chain isotypes, pi, heavy-chain and light-chain gene loci (excluding CDRs) of the monoclonal antibodies are listed in Table 3.

TABLE 3

In a still further aspect, the present invention provides a composition for detecting a heart disease marker, comprising the aforementioned monoclonal antibody. In a still further aspect, the present invention provides a method of detecting a heart disease marker, comprising the steps of treating a biological sample with monoclonal antibody and detecting antigen-antibody complex formation.

The term "heart disease marker', as used herein, refers to a substance expressed in heart-associated tissues and cardiac muscle and thus is capable of detecting abnormalities in heart-associated tissues. With respect to the objects of the present inventions, in a composition, kit or method comprising or using the monoclonal antibodies of the present invention, the heart disease marker means mitochondrial AK3. The term "heart disease", as used herein, refers to disease of the circulatory system, associated with myocardial injury, and includes myocardial infarction, angina pectoris, coronary artery disease and cardiac failure.

The term "biological sample", as used herein, refers to a body fluid or a portion thereof, and includes urine, blood, serum and plasma isolated from the body.

The term "antigen-antibody complexes", as used herein, refers to binding products of AK3 to a monoclonal antibody recognizing the enzyme in order to detect the presence or absence of AK3 in a biological sample. In the present invention, "detection label" means a label for detecting antigen-antibody complexes. Non-limiting examples of the label include enzymes, fluorophores, ligands, luminescent materials, microparticles and radioisotopes.

Examples of the enzymes used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-lactamase. Examples of the fluorophores include fluorescein, Eu³⁺, Eu³⁺ chelate and cryptate. Examples of the ligands include biotin derivatives. Examples of the luminescent materials include acridium ester and isoluminol derivatives, and examples of the microparticles include colloidal metal and colored latex. Examples of the radioisotopes include ⁵⁷Co, ³H, ¹²⁵I and ¹²⁵I-

Bolton and Hunter reagents.

The formation of the antigen-antibody complexes may be detected by a method, but not limited to this, selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, luminometry, a particle counting method, visual assessment and a scintillation counting method.

Preferably, the antigen-antibody complexes are detected by ELISA (enzyme-linked immunosorbent assay). Examples of ELISA include direct ELISA using a labeled antibody recognizing an antigen immobilized on a solid support; indirect ELISA using a labeled antibody recognizing an antibody forming complexes with an antigen immobilized on a solid support; direct sandwich ELISA using a labeled antibody recognizing an antigen bound to a capture antibody immobilized on a solid support; and indirect sandwich ELISA, where the captured antigen bound to a capture antibody immobilized on a solid support is detected by first adding an antigen-specific antibody, and then another labeled antibody which binds the antigen-specific antibody. More preferably, the antigen-antibody complexes are detected by sandwich ELISA, where a serum sample reacts with an antibody immobilized on a solid support, and the resulting antigen-antibody complexes are detected by an labeled antibody specific for the antigen, followed by enzymatic development, or by first adding an antigen-specific antibody and then a secondary labeled antibody which binds to the antigen-specific antibody, followed by enzymatic development.

According to the ELISA detection method, a biological sample contacts with the monoclonal antibody of the present invention, which have been coated in a solid support, for example, a microtiter plate, a membrane or a test strip. In an embodiment, a microtiter plate well is coated with the monoclonal antibody of the present invention, and the nonoccupied binding region is blocked with, for example, BSA. The coated well is incubated in a sample, and then the presence of antigen-antibody complexes is determined. The presence of the antigen-antibody complexes may be detected by using an antibody specific to the antigen of the antigen-antibody complexes, for example, a monoclonal or polyclonal antibody specifically binding to AK3. The monoclonal or polyclonal antibody may have a detection label. If not having a detection label, the monoclonal or polyclonal antibody may be detected by another antibody capable of detecting the same.

In the sandwich ELISA detection method of the present invention, an antibody recognizing an antigen bound to the monoclonal antibody (capture antibody) selectively recognizing AK3 is preferably the monoclonal antibody (detection antibody), which recognize specifically AK3 as the capture antibody but recognize a different epitope from the capture antibody.

The capture antibody suitable for the sandwich ELISA detection method includes SJB3-34, SJB3-38, and the like, and the monoclonal antibody is used alone or in combination with any of other monoclonal antibodies. In addition, the detection antibody includes SJB3-31, SJB3-35, SJB3-37, SJB3-36, SJB3-39, SJB3- 32, SJB3-33, and the like. The preferred detection antibody is SJB3-32 or SJB3- 36. The detection antibody may be used alone or in combination with any of other monoclonal antibodies. Most preferably, the capture antibody is SJB3-34, and the detection antibody is SJB3-32 or SJB3-36.

In an additional embodiment, a capture monoclonal antibody immobilized on a solid support reacts with a biological sample, and the captured antigen is then detected by a detection monoclonal antibody specifically binding to AK3 and additionally by a secondary labeled antibody producing a detectable signal if the detection antibody is not labeled, followed by visualization and/or quantitation of the labeled antibody.

In a still further aspect, the present invention provides a diagnostic kit for the diagnosis of heart disease, comprising the aforementioned monoclonal antibody.

The monoclonal antibody used in the kit for the diagnosis of heart disease is available as a fragment, if the antibody is able to recognize selectively AK3. The antibody fragment may include F(ab')2, Fab, Fab' and Fv fragments.

The kit may include the monoclonal antibody selectively recognizing AK3 or its fragments, and instruments/reagents used in the immunoassay.

Examples of the instruments/reagents used in the immunoassay include suitable carriers, chemiluminophores capable of producing detectable signals, dissolving agents and washing agents. If the chemiluminophore is an enzyme, the instruments/reagents may additionally include substrates allowing the measurement of the activity of the enzyme and reagents terminating the enzymatic reaction.

Non-limiting suitable carriers include soluble carriers, for example, physiologically acceptable buffer known in the art, e.g., PBS, insoluble carriers, for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, cross-linked dextran, polysaccharides, polymers such as magnetic microparticles made of latex coated with a metal, other paper, glass, metals, agarose, and mixtures thereof.

Non-limiting examples of the assay system useful in the detection method and diagnosis kit of the present invention include ELISA plates, dip-stick devices, immunochromatography test strips and radial partition immunoassy devices, and flow-through devices.

The present invention will be explained in more detail with reference to the following examples in conjunction with the accompanying drawings. However, it will be apparent to one skilled in the art that the following examples are provided only to illustrate the present invention, and the present invention is not limited to the example.

EXAMPLE 1: Cloning of AK3 gene

Total RNA isolation from human skeletal muscle

In order to isolate total RNA, human skeletal muscle of 100 mg was homogenized in 1 ml of RNAzol (4M guanidine thiocyanate, 25 mM sodium citrate, 0.5% salcosyl, 0.1 M 2-mercaptoefhanol), and supplemented with 100 μl of chloroform and vortexed for 15 sec. After being placing on ice for 15 min, the homogenate was centrifuged at 12,000 xg for 15 min. The aqueous phase was transferred to a new tube, and mixed with an equal volume of isopropanol, followed by incubation at -70°C for 15 min. Subsequently, the mixture with isopropanol was centrifuged at 12,000 xg at 4°C for 15 min. The resulting RNA pellet was washed with 75% ethanol, dried, and dissolved in 100 μl of DEPC-treated water.

Synthesis of AK3 cDNA by RT-PCR

10.5 μl of the total RNA as prepared above was mixed with 1 μl of oligo(dT) (500 ng/μl), 1.5 μl of 2.5 mM dNTPs, 1 μl of 100 mM DTT, 1 μl of

MMLV reverse transcriptase (200 units/μl) and 6 μl of 5χ reverse transcriptase buffer, and total volume of the mixture was adjusted to 30 μl using 9 μl of DEPC- treated water. Then, the mixture was incubated at 42°C for 30 min, and then at 75°C for 30 min to inactivate the enzyme, thus yielding a cDNA sample.

The cDNA sample was subjected to PCR, where the synthesized cDNA was used as a template. A PCR reaction mixture was prepared by mixing the cDNA sample with 8 μl of 2.5 mM dNTPs, 1 μl of Ex Taq DNA polymerase (5 units/μl), 10 μl of lOx DNA polymerase buffer, and each 1 μl of sense primer and antisense primer (100 pmol/μl), and total volume of the mixture was adjusted to 100 μl using distilled water. PCR was carried out under conditions of denaturation at 98°C for 10 sec, annealing at 55°C for 30 sec and extension at 72°C for 40 sec, followed by soaking at 4°C. The used primer set consists of 5'- GGATCCGCAATGGCTTCCAAACTCTGC-3' (sense, SEO ID No: 108) and 5'- CAGGGTCAATATGCTTCTTTGG-3' (antisense, SEO ID No: 109). The PCR product was identified as AK3 cDNA by separation on a 1% agarose gel (Fig. 2).

Construction of AK3 cloning vector (pCR2.1-AK3)

The AK3 PCR product of 680 bp was purified from the agarose gel using a gel extraction kit, and cloned using a pCR 2.1 PCR cloning kit (Invitrogen) (Fig. 3). A ligation mixture was prepared by mixing 1 μl of linearized pCR 2.1 vector of 25 ng, 5 μl of the PCR product, 1 μl of T4 DNA ligase (4 units/μl), 1 μl of lOx ligase buffer and 2 μl of distilled water, resulting in a total volume of 10 μl, and incubated at 16°C for 12 hrs. Transformation was carried out by using an E. coli strain, JM 109 as a host cell, as follows. 2 μl of the ligation mixture was added to 50 μl of JM 109 competent cells, followed by incubation on ice for 30 min. After heat shock at 42°C for 45 sec, the competent cells were placed on ice for 2 min, and incubated in 250 μl of SOC medium at 37°C for 1 hr with agitation at 225 rpm. Then, 100 μl of the transformed cells were plated in LB/ampicillin plates containing X-gal and IPTG, and cultured at 37°C overnight. Among the formed white colonies, 10 white colonies were picked and cultured in LB medium containing amipicllin of 50 μg/ml at 37°C overnight. Thereafter, plasmid DNA was isolated from the cultured cells using a plasmid Prep kit, and evaluated for carrying AK3 cDNA. The plasmid DNA was digested with EcoRI for 1 hr in a mixture of 5 μl of the plasmid DNA, 1 μl of EcoRI, 1 μl of EcoRI buffer and 3 μl of distilled water. The digested plasmid DNA was electrophoresed on a 1% agarose gel. As a result, the plasmid DNA was identified to carry AK3 cDNA (Fig. 4), thus giving pCR2.1-AK3. The cloned AK3 cDNA was confirmed by DNA sequence analysis using an automatic DNA sequencing analyzer, where the used sequencing primers were a Ml 3 reverse primer and a T7 forward primer.

EXAMPLE 2: Construction of AK3 expression vector and purification of recombinant AK3

The pCR2.1-AK3 vector carrying AK3 cDNA was digested with BamHI and Xhol, and the resulting AK3 cDNA fragment was eluted from an agrose gel after electrohoresis. In order to construct an AK3 expression vector, pQE 30 (Quiagene) was digested with BamHI and Sail, and the resulting large fragment was eluted from an agarose gel. A ligation mixture was prepared by mixing 3 μl of the eluted large fragment of the digested pQE 30 plasmid, 5 μl of the excised AK3 cDNA fragment, 1 μl of T4 DNA ligase and 1 μl of 1 Ox ligase buffer, and incubated at 16°C overnight. Transformation was carried out by using an E. coli strain, Ml 5, as a host cell. The transformed cells were plated in LB plates containing ampicillin and kanamycin. Each of formed colonies was cultured in LB medium containing amipicllin of 100 μg/ml overnight. Thereafter, plasmid DNA was isolated from the cultured cells, and was digested with EcoRI. As a result, the AK3 cDNA fragment was found to be successfully inserted into the pQE 30 plasmid (Figs. 5 and 6). One of the confirmed colonies was precultured overnight in a 50 ml LB medium containing amipicllin of 100 μg/ml and kanamycin of 25 μg/ml. The 50 ml culture was then inoculated in a 1 L LB medium, and incubation was carried out at 37°C for 1 hr, where OD at 600 nm was about 0.5 to 0.7. In this state, IPTG was added to the medium at a final concentration of 1 mM, and the cells were further cultured for 4 hrs in order to induce AK3 expression. Thereafter, the medium was centrifuged at 4,000xg for 20 min. The cell pellet was suspended in 50 ml of a binding buffer (20 mM Tris/HCl containing 5 mM imidazole, 0.5 M NaCI and 0.1% Tween 20, pH 7.9) and incubated at -20°C overnight. After thawing the frozen cells on ice, the cells were lysed by treating five times with ultrasonication for 30 sec at intervals of 1 min, and centrifuged at 10,000xg at 4°C for 30 min. The supernatant was recovered, thus giving a crude extract containing soluble proteins. The expressed AK3 was purified using a chelate resin (Pharmacia), as follows. A column was packed with the chelate resin up to a bed volume of 5 ml, and washed with a five column volume of water to remove ethanol. After charging the chelate resin with Ni²⁺ using a five column volume of charge buffer (50 mM NiSO₄ containing 0.1% Tween 20), the chelate resin was washed with a three column volume of water and equilibrated with a five column volume of the binding buffer. After loading the prepared crude protein extract onto the column, the column was washed with a ten column volume of the binding buffer and then with a five column volume of a washing buffer (20 mM Tris/HCl containing 60 mM imidazole, 0.5 M NaCI and 0.1% Tween 20, pH 7.9) to eliminate nonspecific binding, and eluted with a five column volume of an elution buffer (10 mM Tris/HCl containing 0.5 M NaCI, IM imidazole and 0.1% Tween 20, pH 7.9). Herein, all of the steps were carried out a flow rate of 1 ml/min. To remove salt from the elute, the eluted AK3 fraction was dialyzed in a dialysis buffer (0.1% Tween 20-containing 10 mM Tris/HCl, pH 7.9) for 12 hrs, where the dialysis buffer was exchanged to a new one three times. The resulting AK3 fraction was then concentrated using PEG 8000. The protein concentration was measured using a BCA protein assay kit, and purification yield was also calculated (Table 4). TABLE 4 Purification of recombinant AK3

EXAMPLE 3: Development of hybridoma cells producing anti-AK3 monoclonal antibodies

After emulsifying 0.16 ml of the recombinant AK3 protein solution of 200 μg, prepared in Example 2, with an equal volume of complete Freund's adjuvant, the resulting emulsion was intraperitoneally injected three times into 6 week-old BALB/c mice (H-2^d haplotype) in intervals of 3 weeks. In the case of second and third injection, incomplete Freund's adjuvant was used. The final boosting was carried out by intravenous injection on the tail of the mice.

After the mice were examined to produce antibodies specific to the injected antigen AK3 by ELISA test using mouse serum, the spleen was aseptically excised from the immunized mice, and dispersed in DMEM (Dulbecco's Modified Eagle's Medium, Gibco BRL) medium containing fetal bovine serum (Gibco BRL) and 0.5% gentamycin (Gibco BRL), thus giving a splenocyte dispersion solution. The splenocytes were fused with the already prepared SP2/0-Agl4 myeloma cells from the same mice strain by using PEG, as follows. The splenocytes were mixed with SP2/0-Agl4 myeloma cells at a ratio of 1. TO in a 50 ml centrifugal tube, and the mixed cells were centrifuged at 1200 rpm, thereby forming a tight pellet.

After completely removing the supernatant, the cell pellet was completely dispersed by tapping, and well mixed with 1 ml of PEG1500 warmed to 37°C. Then, the cells were diluted gradually by addition of 1 ml of serum-free medium warmed to 37°C for 1 min and then 18 ml of the medium for 4 min, while not inducing cell lysis.

In order to wash the fused cells, the diluted cells were centrifuged at 500 rpm. After completely removing the supernatant, the cell pellet was sufficiently dispersed with culture medium, aliquotted to 96-well tissue culture plates at a density of l.lxlO⁵ cells/150 μl/well, and incubated in a 5% CO₂ incubator at 37°C for 1 week, thus giving hybridoma cells.

EXAMPLE 4: Preparation of monoclonal antibodies

Selection of hybridoma cells

Hybridoma cells were selected by their viability in HAT (Hypoxanthine- Aminopterin-Thymidine) medium. One week after cell fusion, 70 μl of the HAT medium was added to each well. From 2 days after HAT medium addition, the cells were observed for colony formation, and culture supernatants were collected. The culture supernatants were subjected to ELISA using 96-well microtiter plates of which wells had been coated with AK3 (100 ng/well). Absorbance was measured at 490 nm. After selecting wells showing of absorbance of over 0.5, the colonies in the ELISA-positive wells were transferred to 0.5 ml-scale culture. As a result, among 1440 wells in total, colonies were formed in 1263 wells. Of the colony-containing wells, 49 wells were used for cloning of hybridoma cells.

Cloning of hybridoma Cloning of hybridoma cells was carried out by a limiting dilution method.

Cells obtained from ELISA-positive wells were serially diluted in order to give a density of 0.5 cells/well in two 96-well plates containing DMEM medium supplemented with fetal bovine serum, gentamycin and a HAT supplement.

Finally, the diluted cells were added to a cloning medium of 30 ml to give a density of 0.5 cells/well, and 150 μl of the finally diluted cells were aliquotted to each well. After 5 days, the HAT medium was added to each well. After 5 more days, wells displaying good hybridoma culture were selected. By ELISA, hybridoma cells secreting anti-AK3 monoclonal antibodies were selected.

The cloning process was carried out three times. At the first cloning step, 26 clones were selected by ELISA, which have an optical density of over 1.0 at 490 nm. The second cloning was carried out according to the same method as in the first cloning step. The 23 clones selected in the second cloning, which also have an optical density of over 1.0 at 490 nm, were subjected to the third cloning. Finally, 19 clones showing stable reactivity to AK3 were selected.

After the third cloning, culture supernatants from wells containing hybridoma cells producing monoclonal antibodies were identified by ELISA to produce and secrete monoclonal antibodies. After excluding clones exhibiting cross-reactivity with AK1 and AK2, the hybridoma cells specifically recognizing only AK3, SJA3-86, SJA3-1-17, and SJB3-31 to SJB3-39 were selected. The hybridomas SJA3-86 and SJB3-31 to SJB3-39 were deposited at KCLRF(Korean Cell Line Research Foundation), where SJA3-86 (KCLRF-BP-00030) was deposited on May 19, 2000, and SJB3-31 (KCLRF-BP-00066), SJB3-32(KCLRF-

BP-00058), SJB3-33 (KCLRF-BP-00059), SJB3-34 (KCLRF-BP-00060), SJB3-35 (KCLRF-BP-00065), SJB3-36 (KCLRF-BP-00061), SJB3-37 (KCLRF-BP-00062), SJB3-38 (KCLRF-BP-00063) and SJB3-39 (KCLRF-BP-00064) on Oct. 7, 2002.

Finally, the selected hybridomas were proliferated in from 48-well plates, 12-well plates to 175 cm²-T flasks.

Production of monoclonal antibodies

Monoclonal antibodies from the selected clones were purified from culture supernatants of fused cells cultured in fetal bovine serum-containing DMEM medium under 5% CO₂. Separately, by affinity column chromatography, monoclonal antibodies were isolated from ascitic fluids obtained from BALB/C mice injected with 0.5 ml of pristine and intraperitoneally injected with l lO⁷ hybridoma cells in a good state to induce tumor in their abdominal cavity, thus giving a yield of 1 mg antibody/ml. Purification of monoclonal antibodies using protein A BALB/C mice were intraperitoneally injected with 0.5 ml of pristane, and within 1 to 2 weeks, intraperitoneally injected with lxlO⁷ hybridoma cells/ml/mouse. After one week, ascitic fluids were collected from the swollen abdominal cavities of the mice. A column was filled with 10 ml of Protein A Fast Flow Resin and washed with distilled water. Then, phosphate buffered saline(PBS) flew through the column until a base line absorbance at 280 nm of 0.1 was achieved.

Thereafter, the ascitic fluids were loaded onto the column. The column was equilibrated with lx PBS, and eluted with an elution buffer (0.1 M Glycin-

HC1, pH 3.5). The eluted fractions were neutralized with a 0.25 volume of 3 M

Tris-HCl (pH 7.5), concentrated using Centriprep 30. After dialysis, protein concentration was measured by BCA assay.

Assay for cross-reactivity of monoclonal antibodies with AK1 and AK2 100 μl of AK1, AK2 and AK3 was aliquotted to 96-well microtiter plates at an amount 100 ng per well, followed by incubation at 4°C for 24 hrs to allow coating of the wells with the proteins. In this coating step, 0.05 M bicarbonate buffer of pH 9.6 was used. Then, each well was washed with PBS containing 0.05% Tween 20. After washing, 300 μl of PBS containing 0.5% casein, 0.02% sodium azide and 0.05% Tween 20 was added to each well, and the plate was incubated at 37°C for 2 hrs. After blocking, each well was washed with PBS containing 0.05% Tween 20.

100 μl of hybridomas, transferred to 48-well plates and cultured in 1 ml of HAT medium therein for 1 day, added to each well coated with AK1, AK2 or AK3. After incubation at 37°C for 2 hrs, each well was washed with PBS containing 0.05% Tween 20.

100 μl of a 1:1000 dilution of a goat anti-mouse IgG(Fc specific)-HRP- conjugated antibody (PIERCE #31439) in PBS was added to each well, followed by incubation at 37°C for 1 hr 30 min. After washing, absorbance at 490 nm was measured using a substrate, OPD. The results for cross-reactivity of the monoclonal antibodies with AK1, AK2 and AK3 are given in Table 5, below (see, Fig. 7). Compared with the monoclonal antibody SJA3-86, the monoclonal antibodies SJA3-31 to SJA3-39 were found to have no cross-reactivity with AK1 and AK2 and specifically recognize AK3. Therefore, SJA3-31 to SJA3-39 are useful as a marker of the diagnosis of myocardial infarction.

TABLE 5

Measurement of affinity of monoclonal antibodies to AK3 Determination of dissociation constant The dissociation constants (Kd) of the monoclonal antibodies were determined according to solid-phase immunoassay. In order to obtain a degree of dissociation, a saturation concentration and a low range concentration of an antigen were determined. First, to determine a suitable antigen concentration, 100 μl of an antigen of various concentrations was aliquotted to plates, and the plates were incubated at 4°C overnight. The plates coated with the antigen were washed with

PBST (PBS-0.05% Tween 20) three times, treated with 200 μl of a blocking buffer (0.5% Casein-PBS) at 37°C for 2 hr, and washed with PBST. The monoclonal antibodies were added to the plated at a fixed concentration. After incubation at 37°C for 1 hr, the plates were washed and treated with a 1:1,000 dilution of a secondary antibody (goat anti-mouse HRP-conjugated antibody). Assay was carried out using the substrate OPD, and absorbance was measured at 450 nm. For saturation concentration (plate I) measurement of the antigen and affinity assay, a low concentration (plate II) of the antigen was determined from an optical absorbance versus antigen concentrations. The low concentration must be minimum 20-fold lower than the saturation concentration. Based on the result, the plates I and II were coated with an antigen. The monoclonal antibodies were serially diluted to twice from a concentration of 1.5 μg/ml (=10,000 pmol), and added to the plates. Subsequently, the plates were treated with a 1:1000 dilution of the secondary antibody. Development was carried out using the OPD substrate, and absorbance was measured at 450 nm. A standard curve for known antibody concentrations in an antigen saturation state was obtained, and an equation expressing the standard curve was obtained. The concentration (X) of the bound antibody was calculated from the absorbance of the plate II using the linear region of the standard curve. Free antibody (Ab) concentration (d) was calculated by d= B-X, wherein B is a total concentration of an antibody added to the plate II. The bound Ab region (nX) was calculated from the X and n values, wherein n is an Ab valence, and n values for a Fab fragment, IgG and IgM are 1, 2 and 10, respectively. An X/d ratio was depicted against nX, and Keq was calculated by Keq= -a(slope)/pmol.

From a plot of an X/d ratio against nX, Keq and Kd values were calculated, and the results are given in Table 6, below.

TABLE 6

Measurement of binding affinity bv serial dilution

SJB3-31, SJB3-32, SJB3-33, SJB3-34, SJB-3-39 and SJA3-86 were serially diluted, and evaluated for binding affinity to AK3 by ELISA according to the same procedure as described above. The results are given in Table 7, below. ^■

TABLE 7

As shown in Tables 6 and 7, when degree of antibody dilution was increased and thus antibody concentration was reduced, the absorbance measured in SJA3-86 was much lower than that in SJB3-31, SJB3-32, SJB3-33, SJB3-34 and SJB-3-39, and SJB3-31, SJB3-32, SJB3-33, SJB3-34 and SJB-3-39 displayed about 10-fold higher binding affinity to AK3 (based on the Kd values) than SJB3-89. These results indicates that the monoclonal antibodies except SJB3-89 are suitable as a marker for the diagnosis of myocardial infarction.

EXAMPLE 5: Sequence analysis of variable regions of antibodies and determination of CDR

Isolation of total RNA from hybridoma cells

Total RNA was prepared using an RNeasy Mini Kit (Qiagen: Cat. No. 74104). All plastic means used in this experiment were treated with 0.1% DEPC (diethyl pyrocarbonate) water to inhibit RNase activity. The cultured hybridoma cells producing monoclonal antibodies were harvested, and treated with 600 μl of RLT buffer per 5xl0¹⁰ cell. The cell suspension was completely lysed by primarily pipetting or vortexing and passing through 19 to 22-gauge needles. 600 μl of 70% ethanol was added to the cell lysate, followed by mixing by pipetting. Then, 600 μl of the lysed cells was loaded onto spin columns contained in the kit, and the spin columns were centrifuged at over 10,000 rpm for 25 sec. After discarding the flow-through, this step was repeated until all the lysate was loaded. 700 μl of RW1 buffer was added to each column, and the column was incubated for 5 min and centrifuged at over 10,000 rpm for 25 sec. After discarding the flow-through, the retentate was transferred to a new 2 ml collection tube contained in the kit. 500 μl of RPE buffer was loaded onto the column, followed by centrifugation at over 10,000 rpm for 25 sec. After loading 500 μl of RPE buffer onto the column again, the column was centrifuged at over 10,000 rpm for about 3 min to dry the spin-column membrane. After discarding the flow-through, the column was transferred to a new 1.5 ml collection tube. 50 μl of RNase-free water was directly added to the spin-column membrane. After letting the column stand for 1 min, RNA was eluted. RNA concentration was measured using Genequant II (Pharmarcia Biotech), and successful RNA preparation was confirmed by separating 1 μl of the isolated RNA on a 1% agarose gel (0.5% TAE).

cDNA synthesis using total RNA

Synthesis of cDNA was carried out using a cDNA cycle™ kit (Invitrogen). 400 ng of the total RNA isolated from the hybridoma cells was added to a PCR tube, and the final volume was adjusted to 11.5 μl using DEPC water. Then, 1 μl of an oligo dT primer was added to the PCR tube. After well mixing, the PCR tube was incubated for 10 min in a water bath at 65 °C and then for 2 min at room temperature. After adding 1 μl of RNase inhibitor, 4 μl of 5x RT buffer, 1 μl of 100 mM dNTPs, 1 μl of 80 mM sodium pyrophosphate and 0.5 μl of AMN reverse transcriptase and gently mixing, the tube was incubated for 1 hour in a water bath at 42°C and then for 2 min at 95°C . The synthesis of cDNA was confirmed by electrophoresis on a 1% agarose gel.

Amplication of variable regions of antibodies by PCR

The variable regions of antibodies were amplified using DNA Thermal cycler 480 (Perkinelmer). To amplify heavy chain and light chain of antibodies, two PCR reaction mixtures were prepared, as follows. 6 μl of lOx PCR buffer, 8 μl of dNTP, 1 μg of the prepared cD A and 1 μl of Taq DNA polymerase

(Takara) were added to each of two PCR tubes. One of the two PCR reaction mixtures was supplemented with 1 μl each of heavy chain primers 1 and 2, and the other with 1 μl of a light chain primer mixture. The final volume of each mixture was adjusted to 60 μl. PCR was carried out under conditions of predenaturation at 94°C for 5 min, 30 cycles of denaturation at 98°C for 10 sec, annealing at 55°C for 30 sec and extension at 72°C for 40 sec, followed by final extension at 72°C for 5 min. The PCR products were separated on a 1.5% agarose gel (0.5% TAE buffer). About 350 bp bands were excised from the gel, and the 350-bp fragments were purified using a MlPSpin™ Gel kit (Genotein).

The heavy chain and light chain primers used in the PCR reaction originate from a portion of FR1 and the conserved regions of the currently identified mouse antibodies (mouse ScFv module, Product Code 27-9400-01, Amersham Pharmacia Biotech).

Cloning of the variable regions of antibodies into pCLTAl 7 μl of each of the obtained PCR products was mixed with 0.5 μl of pCLTAl DNA, 1 μl of T4 DNA ligase (MBI Fermentas), and the final volume of the mixture was adjusted to 20 μl. Ligation was carried out at 16°C overnight.

Transformation and culture

The ligation mixture was mixed with 200 μl of JM 109 competent cells. After gently mixing, the cells were placed on ice for 30 min, and heat-shocked at 42°C for 1 min, followed by incubation on ice for 1 min. Then, 800 μl of LB medium was added to the cells, and the cells were incubated at 37°C for 1 hr with agitation at 200 rpm. The tube containing the transformed cells was spin-downed. After discarding the supernatant, the cell pellet was suspended in 100 μl of LB medium and smeared in an LB/Amp⁺ plate, followed by incubation at 37°C overnight.

To obtain JM109 cells transformed with the plasmid carrying the variable region, single colonies were picked from the plate, seeded in 10 ml of LB medium containing Ampicillin medium, and cultured at 37°C overnight.

Identification of transformed cells using alkali lysis method Plasmid DNA was isolated from the cultured cells by an alkali lysis method, as follows. Total 3ml(1.5ml x 2) of cultured medium was added to a 1.5 ml tube, and the tube was centrifuged at 14,000 rpm for 5 min at room temperature. After the supernatant, the cell pellet was resuspended in 100 μl of Solution I (50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA (pH 8.0)) by vortexing, and placed on ice for 10 min. 200 μl of Solution II (0.2N NaOH, 1% SDS) was added to the suspended cells, and the tube was gently inverted up to about five times to assist cell lysis and placed on ice for about 10 min. Subsequently, 150 μl of Solution III (3 M potassium acetate, 5M acetic acid) was added to the tube, and the tube was inverted up to about five times and placed on ice for about 10 min. The tube was centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant was transferred to a new tube, and mixed with an equal volume of phenol: chloroform by vortexing. After centrifugation at 14,000 rpm for 15 min at 4°C, the supernatant was transferred to a new tube, and mixed with a two volume of cold absolute ethanol, followed by centrifugation at 14,000 rpm for 15 min at 4°C. After completely discarding the supernatant, the DNA pellet was washed with 1 ml of cold 70% ethanol by inverting the tube up to about five times, followed by centrifugation at 14,000 rpm for 15 min at 4°C. After completely discarding the supernatant, the DNA pellet was dried at room temperature for about 5 min to remove the residual ethanol, dissolved in 20 μl of TER buffer (Tris EDTA pH 8.0/Ribonucliase A), and stored at -20°C.

Identification of variable region DNA using restriction enzymes

10 μl of the isolated plasmid DNA was mixed with 2 μl of lOx H buffer,

0.2 μl of EcoRI (Takara), 0.2 μl of Xhol (Takara) and 7.6 μl of distilled water. After adjusting the final volume to 20 μl, the reaction mixture was incubated at

37°C for 2 hrs. The digested plasmid DNA was separated on a 1.5% agarose gel

(0.1% TAE). The variable region DNA fragments with correct restriction enzyme mapping results were further analyzed by DNA sequencing using the Sanger method. The resulting DNA sequences of the heavy chain and light chain variable regions and amino acid sequences corresponding to the DNA sequences were disclosed in SEQ ID Nos: 1 to 40. From the DNA and amino acid sequences of the variable regions, CDRs were determined using Kabat Numbering Scheme, and their amino acid sequences were disclosed in SEQ ID Nos: 41 to 100.

EXAMPLE 6: Epitope mapping using phage display peptide library

Using the random peptide library (Ph.D.-12™ Phage display library kit,

NEB) derived from recombinant M13mpl9 carrying a minor coat protein pill gene modified to have an additional 12 amino acids in its transcript by genetic engineering, epitope mapping was carried out by selecting phages having a high affinity to the monoclonal antibodies to adenylate kinase isozyme, known as biopanning technique. Biopanning was performed according to the following procedure, in which further rounds of biopanning were carried out up to a minimum four times.

Biopanning

The monoclonal antibody (mAb) was added to a petri dish (diameter: 35 mm, Falcon) containing 1 ml of 0.1 M NaHCO₃ (pH8.6) up to 100 μg, and incubated at 4°C for 12 hrs under humid condition. The petri dish was fully filled with a blocking solution. After incubation at 4°C for 2 hrs under humid condition with agitation, the dish was sufficiently washed with TBS/Tween (0.1% Tween 20). 10 μl of phages of 1.5xl0^π pfu/ml was diluted in 990 μl of TBS/Tween (0.1% Tween 20) and added to the mAb-coated petri dish. The petri dish was incubated at room temperature for 1 hr with agitation to allow phages having affinity to the mAb binding the mAb. After washing the petri dish with TBS/Tween (0.1% Tween 20) ten times, the bound phages were eluted for 5 min with 1 ml of 0.2 M glycine-HCl (pH 2.2) with agitation. The eluted M13mpl9 pharges were then immediately neutralized with 150 μl of 1 M-Tris HCl (pH9.1). The eluted phages were inoculated 20 ml of E. coli ΕR2738 cells grown to the absorbance of 0.2 at 600nm, and cultured at 37°C for 4.5 hrs with agitation. After centrifugation of the culture medium, 80% of the supernatant was recovered and mixed with a 1/6 volume of PEG8000/NaCl. After letting it at 4°C for 15 min, centrifugation was carried out at 10,000 rpm at 4°C for 15 min. After discarding the supernatant, the pellet was suspended in 1 ml of TBS, and centrifuged again at

14,000 rpm at 4°C for 5 min. The supernatant was mixed with a 1/6 volume of PEG8000/NaCl, and incubated on ice for 1 hr. After centrifugation at 14,000 rpm at 4°C for 10 min, the pellet was suspended in 200 μl of TBS containing 0.02% NaN₃, thereby completing the first round of biopanning. The second, third, forth and fifth rounds of biopanning were carried out using the eluted pharges prepared in the prior round as a starting material in the next round according to the same procedure as in the first round except for use of TBS/Tween (0.5% Tween 20) at a higher concentration than the first round.

Determination of amino acid sequences of mimetic peptides by DNA ^' sequencing

The selected phages by biopanning using the AK3 -specific monoclonal antibodies were diluted and plated in solid agar plates. Each of the formed plaques was proliferated in 3 ml of LB medium. Single-stranded (ss) DNA was isolated from the cultured phages by PEG precipitation, and analyzed for DNA sequence. By the Sanger method, DNA sequencing was carried out using the isolated ssDNA as a template and an oligonucleotide as a sequencing primer, where the oligonucleotide (5'-CCCTCATAGTTAGCGTAACG-3', SEQ ID No: 110) synthesized by BioNex, Korea is complementary to the anti-sense strand present 96 bases downstream from an insert in M13mpl9. The DNA sequencing was performed by a service company Macrogen, Korea. As a result, mimetic peptides as listed in Table 2, above, were found. The monoclonal antibodies SJB3-31, SJB3-34 and SJB3-39 recognized an amino acid sequence EHQTREL designated as SEQ ID No: 102. The SJB3-33 and SJB3-36 monoclonal antibodies recognized an amino acid sequence KSLSRHDH designated as SEQ ID No: 103.

The SJB3-35 monoclonal antibody recognized two amino acid sequences KSLSRHDH of SEQ ID No: 103 and SPMLQLMTLLSR of SEQ ID No: 104. The SJB3-38 monoclonal antibody recognized two amino acid sequences GHIHSMRHHRPT of SEQ ID No: 105 and DNANSSIRSHTY of SEQ ID No: 106. In particular, the amino acid sequence of SEQ ID No: 103 was strongly recognized by many antibodies during cloning of hybridomas. When compared with the amino acid sequence of human AK3, the amino acid sequence of SEQ ID No: 103 was found to have a high homology with an amino acid sequence KSLLNPDH of SEQ ID No: 107, which was known to be present on the surface of spherical yeast isozyme AKy (Egner, U., and Tomasselli, A.G., Schultz, G.E.

(1987) J. Mol. Biol. 195: 649-658). Based on these results, the amino acid sequence of SEQ ID No: 103 was determined as a dominant epitope because of facilitating antibody binding to AK3.

INDUSTRIAL APPLICABILITY

As described hereinbefore, due to their high specificity and binding affinity to AK3, the monoclonal antibodies of the present invention are very useful as a marker for the diagnosis of heart disease, and can largely reduce false positive results of the conventional biochemical markers, thereby reducing the risk to the patients 's lives and medical suit costs of pharmaceutical manufacturers and doctors.

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D DESIGNATED STATES FOR WHICH INDICATIONS ARE MAOE the indications are notfo ll designated States)

E SEPARATE FURNISHING OF INDICATIONS(7eαve blank if not applicable)

The indications listed below will be submitted to the International Bureau lateφpeajy the general nature of the indications e q , "Accession Number of Deposit")

For receiving Office use only For international Bureau use only

D This sheet was received with the international □ This sheet was received by the International Bureau application on

Authorized officer Authorized officer

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

A The indications made below relate to the deposited microorganism or other biological mateπal referred to in the description on page 9 . line 8-12

B. IDENTIFICATION OF DEPOSIT Further deposits are on an additional sheetD

Name of depositary institution

Korean Cell Line Research Foundation (KCLRF)

Address of depositary mstιtutιon(ιncludmg postal code and country)

Cancer Research Institute

Seαul National University College of Medicine

Date of deposit Accession Number 07/08/2002 KCLRF-BP-00063

C ADDITIONAL INDICATIONS < a,e This information is continued on an additional sheet D

D DESIGNATED STATES FOR WHICH INDICATIONS ARE MAOEffthe indications are notfor all designated States)

E SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau \a\e,τ(specιjy the geneial nature of the indications e q , "Accession Number of Deposit")

For receiving Office use only For international Bureau use only

D This sheet was received with the international D This sheet was received by the International Bureau application on

Authorized officer Authorized officer

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

A The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 9 . line 8-12

B. IDENTIFICATION OF DEPOSIT Further deposits are on an additional sheetD

Name of depositary institution

Korean Cell Line Research Foundation (KCLRF)

Address of depositary mstύutιon(mcludιng postal code and country)

Date of deposit Accession Number 07/08/2002 KCLRF-BP-00064

C ADDITIONAL IHDlCATlONS aebbnkfnctq≠cabk) This information is continued on an additional sheet

D

D DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE(fι t!ιe indications are notfor all designated States)

E SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau laterfipecyj' the general nature of the indications e q , "Accession Number of Deposit")

For receiving Office use only For international Bureau use only

D This sheet was received with the international D This sheet was received by the International Bureau application on

Authorized officer Authorized officer

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

For receiving Office use only For international Bureau use only

D This sheet was received with the international D This sheet was received by the International Bureau application on

Authorized officer Authorized officer

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

A The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 9 . line 8-12

B. IDENTIFICATION OF DEPOSIT Further deposits are on an additional sheetD

Name of depositary institution

Korean Cell Line Research Foundation (KCLRF)

Address of depositary ιnstιtutιon( including postal code and country)

Cancer Research Institute

Sgqul National University College of Medicine

Date of deposit Accession Number 07/08/2002 KCLRF-BP-00066

C ADDITIONAL rNDIC TIO Sfta-W ntf-jjfa&J?) This information is continued on an additional sheet

D

D DESIGNATED STATES FOR WHICH INDICATIONS ARE MADEffthe indications are notfor all designatedStates)

E SEPARATE FURNISHING OF INDICATIONS(7eαve blank if not applicable)

The indications listed below will be submitted to the International Bureau laterfspecry the general nature of the indications e q , "Accession Number of Deposit ")

For receiving Office use only For international Bureau use only

D This sheet was received with the international D This sheet was received by the International Bureau application on

Authorized officer Authorized officer

Claims

1. A monoclonal antibody specific to adenylate kinase isozyme 3, comprising 4 or more of 6 complementarity determining regions (CDRs) of a group selected from (a) SEQ ID Nos: 41 to 46, (b) SEQ ID Nos: 47 to 52, (c) SEQ ID Nos: 53 to 58, (d) SEQ ID Nos: 59 to 64, (e) SEQ ID Nos: 65 to 70, (f) SEQ ID Nos: 71 to 76, (g) SEQ ID Nos: 77 to 82, (h) SEQ ID Nos: 83 to 88, (i) SEQ ID Nos: 89 to 94 and (j) SEQ ID Nos: 95 to 100.

2. The monoclonal antibody as set forth in claim 1, wherein the monoclonal antibody comprises 6 CDRs of the group selected from (a) to (j).

3. The monoclonal antibody as set forth in claim 2, wherein the monoclonal antibody is produced by a hybridoma selected from the group consisting of accession numbers KCLRF-BP-00058 to KCLRF-BP-00066.

4. A composition comprising the monoclonal antibody of the claim 1 for detecting a heart disease marker AK3.

5. A kit comprising the monoclonal antibody of the claim 1 for the diagnosis of heart disease.

6. A method of detecting a heart disease marker AK3, comprising the steps of treating a biological sample with the monoclonal antibody of the claim 1 and detecting antigen-antibody complex formation.

7. The method as set forth in claim 6, wherein the antigen-antibody complex formation is detected by ELISA.

8. The method as set forth in claim 7, wherein the antigen-antibody complex formation is detected by sandwich ELISA.

9. A monoclonal antibody specific to mitochondrial adenylate kinase isozyme 3, recognizing peptides of SEQ ID Nos: 102 to 106.

10. A composition comprising the monoclonal antibody of the claim 9 for detecting a heart disease marker AK3.

11. A kit comprising the monoclonal antibody of the claim 9 for the diagnosis of heart disease.

12. A method of detecting a heart disease marker, comprising the steps of treating a biological sample with the monoclonal antibody of the claim 9 and detecting antigen-antibody complex formation.

13. The method as set forth in claim 12, wherein the antigen-antibody complex formation is detected by ELISA.

14. The method as set forth in claim 13, wherein the antigen-antibody complex formation is detected by sandwich ELISA.

15. A monoclonal antibody specific to mitochondrial adenylate kinase isozyme 3, recognizing a peptide of SEQ ID No: 107.

16. A composition comprising the monoclonal antibody of the claim 15 for detecting a heart disease marker AK3.

17. A kit comprising the monoclonal antibody of the claim 15 for the diagnosis of heart disease.

18. A method of detecting a heart disease marker AK3, comprising the steps of treating a biological sample with the monoclonal antibody of the claim 15 and detecting antigen-antibody complex formation.

19. The method as set forth in claim 18, wherein the antigen-antibody complex formation is detected by ELISA.

20. The method as set forth in claim 19, wherein the antigen-antibody complex formation is detected by sandwich ELISA.

21. A monoclonal antibody with an epitopic specificity identical to a monoclonal antibody produced by a hybridoma cell selected from the group consisting of accession numbers KCLRF-BP-00058 to KCLRF-BP-00066.

22. A composition comprising the monoclonal antibody of the claim 21 for detecting a heart disease marker AK3.

23. A kit comprising the monoclonal antibody of the claim 21 for the diagnosis of heart disease.

24. A method of detecting a heart disease marker AK3, comprising the steps of treating a biological sample with the monoclonal antibody of the claim 21 and detecting antigen-antibody complex formation.

25. The method as set forth in claim 24, wherein the antigen-antibody complex formation is detected by ELISA.

26. The method as set forth in claim 25, wherein the antigen-antibody complex formation is detected by sandwich ELISA.