KR20040085749A - Monoclonal antibody recognizing resistin, production method and use thereof - Google Patents

Abstract

PURPOSE: A monoclonal antibody recognizing resistin, a method for production of the same antibody and use thereof are provided, thereby detecting resistin concentration in blood, so that it can be useful for diagnosis of diabetes and obesity. CONSTITUTION: The monoclonal antibody recognizing human or mouse resistin is provided, wherein the monoclonal antibody recognizing resistin comprises a detection marker being capable of forming detectable signal; the detection marker is selected from enzyme, fluorescence material, luminous material and radioactive material; the monoclonal antibody recognizing resistin is produced from hybridoma(KCTC 10428BP, KCTC 10429BP or KCTC 10430BP). A kit for detecting resistin comprises the monoclonal antibody recognizing resistin, the detection marker, a vessel containing carrier solution, dissolving solution and detergent, and a description.

Description

Monoclonal antibody against resistin, preparation method thereof, and use {Monoclonal antibody recognizing resistin, production method and use}

The present invention relates to monoclonal antibodies that recognize resistin, methods for their preparation and uses. The present invention also relates to monoclonal antibodies that recognize resistin and the use of such monoclonal antibodies for the quantification of resistin in a sample and the diagnosis of diabetes and obesity.

Lack of energy homeostasis results in excess or deficiency of adipose tissue. Adipose tissue plays an important role in energy storage, fatty acid metabolism, and sugar homeostasis. Studies in humans and animals have reported that excess fat tissue in obese states and adipose tissue deficiency in lipodystrophic states are associated with insulin resistance and type 2 diabetes (non-insulin dependent diabetes mellitus, NIDDM). have. Fat production and storage are controlled by insulin. For example, insulin promotes the delivery of sugar to cells, which are metabolized to alpha-glycerophosphate and used for esterification of fatty acids and stored as triglycerides. Recent endocrine studies have shown that adipocytes not only function as energy stores but also secrete several cytokines called adipokines, and thus signaling between adipocytes regulates insulin signaling to Demonstrates maintaining homeostasis. TNF-a, adipsin, adiponectin, resistin, leptin, and the like have been reported for such a type of adipocaine.

Resistin was discovered by various teams on its own under various names. As an example, resistin has been identified as a gene that is expressed when differentiating from mast cells to mast cells. For example, its mRNA expression was found to be significantly increased when mast cells proliferate into mast cells. However, its induced expression on mast precursor cells, for example, 3T3-L1, has been shown to inhibit the differentiation of mast cells in mast cells, and as a differentiation inhibitory factor to mast cells. Known. This opposing action of resistin suggests that resistin can act as a sensor for the nutritional status of animals, and its inhibitory effect on the differentiation of adipocytes into adipocytes is a feedback regulator of adipogenesis. It may be to suggest its function as [Kim et al., J. Biol. Chem. (2001) 276: 11252-11256. In addition, resistin has been discovered by differential display technology as a gene whose expression level is lowered when treatment of differentiated mast cells with rosiglitazone, an insulin-sensitive drug, results in resistance to insulin sensitivity. The name 'resistin' was named as 'resistin' when it was found that the actual recombinant resistin was a cytokine with the function of inducing insulin resistance by blocking the signaling of insulin and preventing the influx of sugar into the cells [Steppan et al. . Nature 2001. 409: 307-312. It was also found to be a gene expressed in differentiated mouse mast cells. Resistin has also been termed a cysteine-rich adipose tissue-specific secretory factor (ADSF) with a molecular weight of 12.5 kDa, which comprises 10% of the total cysteine residue in the protein's amino acid sequence. In addition, when insulin resistance appears, its intracellular mRNA amount is increased by about 20 to 30 times and blood concentration is also known to increase rapidly [Sul et. al. 2001. J. Bio. Chem. 276: 11252-11256.

Increasing the plasma concentration of resistin increases insulin resistance, blocks the migration of Glut4 to the cell surface, and reports that the use of resistin's polyclonal neutralizing antibodies restores insulin signaling in animal experiments [Steppan et al. Nature 2001: 307-312, and resistin is the cytokine that has received the most attention since leptin. These two cytokines have been recently reported as genetically and clinically the cause of type 2 diabetes. Resistin is one of the most noticeable edipocaines, and genetic, physiological, and epidemiological studies have shown that increased levels of resistin blood are the causes and consequences of obesity and type 2 diabetes.

Because of the clinical significance of resistin, it is important to measure resistin plasma concentrations as an analytical system for the prognosis and treatment development of the treatment of obesity and type 2 diabetes. The development of ELISA kits is an analytical system for pharmaceutical companies that develop hospitals and diabetes drugs, and anticipates that the demand is high, and several diagnostic companies in the US are rushing to develop them. However, it is still very difficult to secure monoclonal antibodies. No test methods have been developed for clinical use.

It is an object of the present invention to provide monoclonal antibodies that recognize resistin and methods for their preparation.

It is still another object of the present invention to provide hybridoma cells that produce monoclonal antibodies that recognize resistin.

It is still another object of the present invention to provide a method for detecting or quantifying resistin in blood using a monoclonal antibody that recognizes resistin.

It is another object of the present invention to provide a quantitative kit for the diagnosis of obesity, and type 2 diabetes using monoclonal antibodies that recognize resistin.

1 is a diagram showing the structure of a resistin.

Fig. 2 shows the comparison of amino acid sequences of the resistin proteins of humans, mice and rats.

Figure 3 is a Western blot photograph of the dilution step for the recombinant protein in order to confirm the sensitivity of HRES 106, a monoclonal antibody according to the present invention.

4 is a Western blot photograph of the recombinant proteins of MRES 06 and MRES 18, which are monoclonal antibodies according to the present invention.

Figure 5 is a Western blot photograph showing the cross-reaction with other recombinant proteins of the monoclonal antibodies MRES 06 and MRES 18 according to the present invention.

FIG. 6 is a graph showing ELISA data obtained by performing enzyme-immune reaction of dilute step HRES 106, a monoclonal antibody according to the present invention, with respect to human resistin recombinant protein. FIG.

Figure 7 is a graph showing the ELISA data that the enzyme immunoreactivity of the mouse monoclonal antibody MRES 06 and MRES 18 according to the present invention in the recombinant recombinant protein.

8 is a graph showing a sandwich ELISA standard curve for quantifying resistin protein in human plasma using the monoclonal antibody HRES 106 and the polyclonal antibody according to the present invention.

9 is a graph showing sandwich ELISA data for quantifying resistin in normal human plasma and type 2 diabetic patients using HRES 106 and polyclonal antibodies according to the present invention.

10 is a graph showing a sandwich ELISA standard curve for quantifying resistin protein in mouse plasma by using a monoclonal antibody MRES 06 and a polyclonal antibody according to the present invention.

FIG. 11 is a graph showing a sandwich ELISA standard curve for quantifying resistin protein in mouse plasma using a monoclonal antibody MRES 18 and a polyclonal antibody according to the present invention.

In the following description of the invention, many terms used in recombinant DNA and immunology are used extensively. In order to provide a clearer and more consistent understanding of the present specification and claims, the following definitions are provided.

The term "antibody" is a term known in the art and means a molecule or active fragment of a molecule that binds a known antigen. Examples of active fragments that bind known antigens include Fab and F (ab) ₂ fragments. Such active fragments can be derived from the antibodies of the invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved by enzymes such as pepsin and filtered by HPLC gel. Suitable fractions containing Fab fragments can be collected and concentrated by membrane filtration and the like. A description of general techniques for active fragment separation of antibodies can be found in, for example, Khaw, BA et al., J. Nucl. Med. 23: 1011-1019 (1982). The term “antibody” also includes bispecific and chimeric antibodies.

The term “monoclonal antibody” is a term known in the art and is a highly specific antibody directed against a single antigenic site. Unlike polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies of the invention can be prepared using conventional cloning and cell fusion techniques. For example, an immunogen of interest (antigen) can be administered to wild type or breeding mice (eg BALB / c) to produce natural or human monoclonal antibodies. Such antigens can be administered alone, in admixture with an adjuvant, can be expressed from a vector and can elicit an immune response as a DNA or fusion protein. Fusion proteins include carrier proteins coupled with peptides intended for an immune response, such as β-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH), and bovine serum albumin, Carrier proteins are not limited thereto. In this case the peptide acts as a hapten for the carrier protein. The method for producing monoclonal antibody is briefly described as follows. After boosting the animal, the spleen is removed and the spleen cells are extracted by methods known in the art [Kohler and Milstein, Nature 256: 495-497 (1975); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)]. The hybrid cells obtained are cloned by conventional methods, for example by limiting dilution, and the resulting clones are cultured to produce the desired monoclonal antibody. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and because they are synthesized by hybridoma culture, they also have another advantage that they are not contaminated by other immunoglobulins. Have

The term “epitope” is a term known in the art and generally refers to the region of the antigen that interacts with the antibody. Epitopes of peptide or protein antigens may be formed from consecutive or discontinuous amino acid sequences of the antigen. Many proteins can contain many epitopes. Epitopes recognized by the antibodies of the invention form an aspect of the invention. Antibodies that recognize specific epitopes can be used in immunoaffinity columns to purify specific epitopes. Since antigenic epitopes are reported to be exhibited by as few as five amino acid residues, terminal cleavage of certain epitopes may also be possible.

The term “hybridoma” is known in the art and refers to a cell formed by fusion of antibody producing cells with immortal cells, such as myeloma cells. The hybrid cell can continuously supply the antibody.

As used herein, the term "forming an immunological complex" is intended to include identifying the presence or absence of a resistin antigen in a sample. The presence or absence of the resistin antigen can be detected using immunoassays. Numerous immunoassays used to detect and / or quantify antigens are known to those of ordinary skill in the art and are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988, 556-612. ].

As used herein, the term "detection labeling agent" means a label for detection of an immunocomplex, and specific examples are radioisotope labels, enzymes, chemoluminescent compounds, fluorescein, picobilis Fluorescent materials such as proteins, rare earth chelates, rhodamines, enzyme cofactors, biotin, and the like, but are not necessarily limited thereto.

The term "(biological) sample" is intended to include biological material, including cells, tissues or biological fluids.

The present invention relates to monoclonal antibodies (HRES 106) that recognize human and recombinant resistins made from hybridomas prepared by fusion of myeloma cells with cells capable of producing antibodies against human resistin protein antigens. The present invention also relates to mice made from hybridomas prepared by fusing cells capable of producing monoclonal antibodies against mouse resistin and to monoclonal antibodies (MRES 06 and MRES 16) that recognize recombinant resistins.

The present invention also relates to a quantitative kit for quantifying resistin in a sample comprising an ELISA assay kit using the monoclonal antibody against human resistin.

The present invention also relates to a method for quantifying resistin in a sample by enzyme immunoassay using the monoclonal antibody against the mouse resistin.

Hereinafter, the present invention is described in more detail.

The present invention provides antibodies that are immunologically reactive to the resistin protein or epitope thereof. Antibodies provided herein can be generated in an animal by inoculating cells expressing the resistin protein or epitope thereof by methods known in the art. The protein can be isolated from the cells at various levels of homogeneity by conventional biochemical methods. Synthetic peptides prepared by synthetic methods established in vitro and optionally conjugated to heterologous amino acid sequences are also included in the antibody preparation methods of the invention. Animals used for the inoculation include cattle, sheep, pigs, mice, rats, rabbits, hamsters, goats and primates. Preferred animals for inoculation include rodents such as mice, rats, hamsters. The most preferred animal is a mouse.

The present invention also provides monoclonal antibodies immunologically reactive with the resistin proteins of the invention present on the surface of the aforementioned cells. The antibody is prepared using methods and techniques known to those skilled in the art.

Monoclonal antibodies provided by the present invention are also produced by recombinant genetic engineering methods known to those skilled in the art, and the present invention relates to antibodies by the above methods, which are immunologically reactive with epitopes of the resistin proteins of the present invention. Include. The invention also includes fragments of such antibodies, such as, but not limited to, F (ab) and F (ab) ₂ . The fragments are prepared by any method, including but not limited to the preparation of such fragments by proteolytic cleavage, chemical synthesis or genetic engineering methods. The present invention may also include single-chain antibodies immunologically reactive with resistin proteins, prepared by methods known to those skilled in the art. The invention also includes chimeric antibodies comprising light and heavy chain peptides that are immunologically reactive with the resistin induced epitopes according to the invention. Chimeric antibodies provided herein include natural antibodies as well as chimeric antibodies prepared by methods of genetic engineering techniques known to those skilled in the art.

It is to be understood that the monoclonal antibody according to the present invention includes all antibodies which recognize the resistin as an antibody prepared from human or recombinant resistin. In a specific embodiment of the present invention, the human resistin antigen is amplified by PCR by a specific primer from cDNA isolated from human blood cells (HL-60, ATCC CRL-240) and the FC domain of the immunoglobulin in the pCEP4 expression vector. It can be prepared by linking together and transferring to HEK-293 cells and expressing them. Cells that can be used herein are any cells or cell lines capable of expressing the natural or genetically engineered resistin protein provided by the present invention. Cells that can be used in the present invention include, but are not limited to, mammalian cells, HEK-293 cells.

According to the present invention, glycosylation, which is not found in Escherichia coli, expresses a mammalian cell because the Fc region of the heavy chain of immunoglobulin was fused with a resistin gene for expression in a mammalian cell (HEK-293 cell). By using it, it is possible to produce structures and properties that are almost identical to those of real human blood registers and to produce antigens capable of producing more powerful antibodies.

Preferably, according to the present invention, the resistin recognition monoclonal antibody, HRES 106 is human, MRES 06 and MRES 18 is a monoclonal antibody expressed in mice and rats using mouse full length resistin, mouse recombinant protein, mouse This can be confirmed by performing Western blot analysis on plasma and recombinant proteins.

The production of monoclonal antibodies against the resistin of the present invention is substantially the same as a conventional monoclonal antibody production method, except that positive clones are identified in the primary hybridoma cells prepared, and the recognition and characteristics of the antibodies produced by each clone are produced. As a reference, hybridoma cells producing monoclonal antibodies were selected.

Monoclonal antibodies provided in the present invention are produced by hybridoma cell lines, which are also provided by the present invention and prepared by methods known in the art. See Harlow and Lane, Antibodies: A Laboratory. Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988). Hybridoma cell lines immunize each cell of a myeloma cell line with a homogenizing agent of resistin protein, including the resistin protein itself or a heterologous or fusion protein construct, a membrane comprising the same, a cell encoding the protein, the protein Prepared by fusion with splenocytes derived from animals. Myeloma cell lines used in the present invention include cell lines derived from myeloma of mice, rats, hamsters, primates and humans. As myeloma cells, for example, P3X63Ag8, p3-U1, NS-1, MPC-11, SP-2 / 0, F0, P3x63 Ag8. Mice derived from mice such as V653 and S194 can be used. In addition, cell lines such as R-210 which are rat-derived cells can be used. Preferred myeloma cell lines for use in the present invention include myeloma cells SP2 / 0-Ag14 (ATCC CRL-1581) and P3X63Ag8.653 (ATCC CRL-1580).

Preferred animals from which spleen can be obtained after immunization include rats, mice and hamsters, preferably mice. Splenocytes and myeloma cells can be fused using a number of methods known in the art, including, but not limited to, inoculation with Sendai virus and inoculation with polyethylene glycol (PEG), and the like. . Monoclonal antibodies produced by hybridoma cell lines can be recovered from cell culture supernatants via in vitro cell growth, or hybridoma cells can be injected subcutaneously and / or intraperitoneally in animals, most preferably mice, Monoclonal antibodies are obtained from blood or ascite.

Hybridoma cells producing monoclonal antibodies against resistin according to the present invention produce antibodies against myeloma cells SP2 / 0-Ag14 cells (ATCC CRL-1581) and P3X63Ag8.653 cells (ATCC CRL-1580) and resistin A hybrid cell line fused to the spleen cells (mouse spleen cells (HRES 106) and rat spleen cells (MRES 06 and 106)), preferably producing the monoclonal antibodies HRES 106, MRES 06 and MRES18. to be. Each hybridoma cell that produces monoclonal antibodies against these resistins is a hybridoma that produces a monoclonal antibody HRES 106 against human resistin at a depositary institution (Bank of Biotechnology Research Institute, Eoeun-dong, Yuseong-gu, Daejeon, Korea). For hybridoma cells producing the monoclonal antibody MRES 18 against the mouse resistin as accession number KCTC 10428BP for the cells, monoclonal antibody MRES 06 against the mouse's resistin, and for the hybridoma cells producing the monoclonal antibody MRES 18 against the mouse resistin. Accession No. KCTC 10430BP was deposited on February 21, 2003. The invention also provides a method for diagnosing diabetes and obesity by detecting the expression of resistin protein in an animal, preferably a human. Diagnostic reagents for use in the method include antibodies, most preferably monoclonal antibodies of the invention. The antibody can be, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), western blot assay, immunological titer assay, immunological diffusion assay, and other methods known to those skilled in the art. It can be used in conventional immunological techniques, including but not limited to these.

Reagents used in immunological assays include suitable carriers, detection labels capable of producing detectable signals, solubilizers, cleaning agents. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers (eg PBS) or insoluble carriers known in the art such as polystyrene, polyethylene, polypropylene, polyesters, polyacrylonitrile , Fluorine resins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated metals on lactex, other papers, glass, metals, agarose and combinations thereof.

Antibodies of the invention may be coupled with a detectable substance, ie a detection labeling agent, to facilitate detection. Examples of detection markers include various enzymes, prosthesis molecules, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, and the like. Detection markers can be directly coupled or coupled to the resistin monoclonal antibodies of the invention using techniques known in the art or indirectly coupled or bound through intermediates (eg, linkers known in the art). Or may be coupled or coupled to another polyclonal antibody that recognizes a resistin protein or a secondary antibody that is an antibody that recognizes a resistin antibody. Examples of suitable enzymes are horseradish peroxidase, acetylcholine esterase, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, malate dehydrogenase, glucose-6-phosphate Dehydrogenase, invertase, and the like; Examples of suitable submolecular complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin or picobiliprotein, and the like; Examples of luminescent materials include luminol, isoleucinol, lucigenin; Examples of bioluminescent materials include luciferase, luciferin and aequorin; Examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ¹¹¹ In, ⁹⁹ Tc ¹⁴ C, ³ H, and the like.

In addition, the present invention is applied to an immunoassay or immunoassay kit for detecting the formation of an immunological complex with a monoclonal antibody using the monoclonal antibody against the resistin, an assay method or assay kit for detection or quantification of resistin in a sample To provide. As mentioned above, immunoassays and kits for immunoassays using monoclonal antibodies known in the art, such as ELISA assays and radioimmunoassays, are all applicable to the present invention. For example, the kits used for the quantitative analysis of the resistin of the present invention may comprise a register containing a resistin monoclonal antibody, a suitable carrier solution, a detection label capable of producing a detectable signal, a dissolving agent, a cleaning agent, and instructions for use. It may include. In addition, when the labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. In addition, the detection labeling agent may further comprise another secondary antibody directed against the resistin bound to or directly through a linker to the resistin monoclonal antibody according to the invention.

In a preferred embodiment of the present invention, by adding a biological sample to a microtiter plate coated with a monoclonal antibody according to the present invention, reacting the enzyme-antibody conjugate and then treating the substrate of the enzyme to measure the absorbance, A method of quantifying resistin is provided that quantifies resistin present in a biological sample by comparing absorbance with a standard curve. Herein, biotin may be combined with an antibody in the enzyme-antibody conjugate. In addition, the enzyme may be horse radish peroxidase (hereinafter referred to as HRP), but is not limited thereto, and all enzymes and substrates used in the ELISA assay may be applied to the present invention.

ELISA analysis is an analysis method that can quantify the presence or absence of antigen present in a sample by using antibody and antigen reaction. There are about two or three antibodies required for one ELISA kit. ELISA kits using monoclonal antibodies that recognize resistin consist of the three antibodies mentioned above. The composition and principle are as follows.

First, HRES 106 monoclonal antibody (primary antibody) is coated in a 96-well plate (when quantitating resistin in human plasma), and a sample containing resistin is added to the well to react, and an enzyme-labeled polyclonal antibody (2 Secondary antibody) and the substrate capable of reacting with the enzyme label, the enzyme-substrate reaction can be detected and the resistin present in the sample can be quantified compared to the standard curve. In a specific example of the present invention, enzymes and substrates may use, but are not limited to, biotin and streptavidin conjugate HRP.

When comparing the concentration of resistin in the plasma of a normal person with type 2 diabetic patients, the plasma of a normal person may contain lower resistin. Therefore, obesity and type 2 diabetes can be diagnosed by quantifying the resistin in the sample according to the present invention. The present invention can examine the actual in vivo variation of the subject, the distribution of blood resistin in normal and diabetic patients, the sensitivity and specificity of the resistin level that can distinguish between normal and diabetic patients.

Resistin is present in serum as a double complex. The concentration of resistin in serum of normal subjects is ng / ml, which indicates a significant increase in the concentration of resistin in plasma for obesity and type 2 diabetes, indicating that the resistin has or is associated with metabolic action on insulin resistance. Diagnosis of obesity and type 2 diabetes conditions will be possible by comparatively quantifying the resistin concentration of a biological sample using a quantitative method and quantification kit of the present invention.

EXAMPLE

Example 1: Antigen Preparation

1-1: Human Resistin Antigen Preparation

PCR was performed using primer pairs having cDNA (HL-60, ATCC CRL-240) of human blood cells as a template, NheI restriction enzyme recognition site at the 5 'end and Bgl II recognition site at the 3' end. 35 minutes of human resistin DNA was amplified after setting 2 minutes at ℃, 30 seconds at 94 ° C as denaturation, 30 seconds at 55 ° C as annealing process, and 1 minute at 72 ° C as chain extension process (MJ Research, minicycler PTC150 model).

The human resistin Fc fusion protein primer pairs used were as follows:

Forward primer: 5'-CCT AGC TAG CTG TGC CGG ATT TGG-3 '

Reverse primer: 5'-GAA AGA TCT GGA GGG CTG CAC ACG ACA GCA GCG CG-3 '.

The amplified fragment was inserted into a pCEP4 mammalian expression vector (Invitrogen, cat # V044-50) containing Fc (Fc portion of immunoglobulin) to prepare a recombinant vector, and transformed HEK 293-EBNA cells with the recombinant vector. Cultured in DMEM medium containing antibiotic G418 and hygromycin (invitrogen 10687010).

The culture supernatant was collected in a pipette when the culture medium of the transformant was changed every three days. Protein A affinity column (Amersham Bioscience, HiTrap rProteinA FF) was homogenized with 10-fold amount of DPBS at a rate of 1 ml / min and passed through the cell culture collection. At this time, the rate was passed at 1 ml / min by the AKTA purifier (Amersham Bioscience), eluted with 100 mM glycine, pH 3.0 and neutralized with 1 / 10-fold 2M Tris, pH 8.0. At this time, the dissolution rate was 1 ml / min to isolate the resistin-Fc fusion protein. The isolated resistin-Fc fusion protein was used as an antigen for antibody preparation in later experiments. Antibodies for producing HRES 106 were prepared from antigens prepared according to the methods of this example.

1-2: Mouse Resistin Antigen Preparation

After differentiating mouse adipocytes, cDNA (3T3-L1, ATCC CL-173) of mouse cells was used as a template, and it had a Nhe I restriction enzyme recognition site at the 5 'end and a Bgl II recognition site at the 3' end. Using primer pairs, two minutes at 95 ° C by PCR, and 30 seconds at 94 ° C by denaturation, 30 seconds at 55 ° C by annealing, and one minute at 72 ° C by chain extension were performed. Resistin DNA was amplified (MJ Research, minicycler PTC150 model).

The mouse resistin Fc fusion protein primer pairs used were as follows:

Forward primer: 5'-CCT AGC TAG CTA CCC AGA ACT GAG TT-3 '

Reverse primer: 5'-GAA AGA TCT GGA AGC GAC CTG CAG CTT A-3 '

The amplified fragment was inserted into a pCEP4 mammalian expression vector (Invitrogen, cat V044-50) containing Fc (Fc portion of immunoglobulin) to prepare a recombinant vector, and transformed HEK 293-EBNA cells with the recombinant vector, followed by antibiotics. Cultured in DMEM medium containing G418 and hygromycin (invitrogen 10687010).

The culture supernatant was collected in a pipette when the culture medium of the transformant was changed every three days. Protein A affinity column (Amersham Bioscience, HiTrap rProteinA FF) was homogenized with 10-fold amount of DPBS at a rate of 1 ml / min and passed through the cell culture collection. At this time, the rate was passed at 1 ml / min by an AKTA purifier (Amersham Bioscience), eluted with 100 mM glycine, pH 3.0 and neutralized with 1/10 fold 2M Tris, pH 8.0. At this time, the dissolution rate was 1 ml / min to isolate the resistin-Fc fusion protein. The isolated resistin-Fc fusion protein was used as an antigen for antibody preparation in later experiments. Antibodies for producing MRES 06 and MRES 18 were both prepared from one and the same antigen prepared according to the method of this example.

Example 2: Hybridoma Cell Preparation

2-1: Antibody Producing Cells (HRES 106)

The resistin-Fc fusion protein prepared in Example 1-1 was dissolved in phosphate-buffered saline (PBS) and electrophoresed with polyacrylamide gel electrophoresis (SDS-PAGE), and the same volume of Freund's complete adjuvant An emulsion containing (Freund's complete adjuvant) was prepared. The emulsion was injected intraperitoneally in five 7 week old female BALB / C mice. 20 μg of antigen was injected per animal, with a total volume of 400 μl (primary immunization). After 14 days the antigen (Fc fusion protein) was mixed with Freund's Incomplete adjuvant and injected into the abdominal cavity (secondary immunization). After 28 days, antibody production was induced by injecting an Fc fusion protein mixed with antigen and PBS (tertiary immunization). After the completion of the third immunization, blood was collected from the mouse to confirm whether the antibody was produced and confirmed whether the antibody was produced by ELISA and Western blot analysis. High antibody titers were induced by injecting an antigen (10 μg) dissolved in PBS into the tail vein of the mouse 3 days before the cell fusion was confirmed. All mice were raised in selected areas to prevent contamination.

2-2: Antibody Producing Cells (MRES 06 and 18)

The resistin-Fc fusion protein prepared in Example 1-2 was dissolved in phosphate-buffered saline (PBS) and electrophoresed with polyacrylamide gel electrophoresis (SDS-PAGE), and the same volume of Freund's complete adjuvant An emulsion containing (Freund's complete adjuvant) was prepared. The emulsion was injected intraperitoneally in four female week-old Lewis rats. 50 μg of antigen was injected per animal, with a total volume of 500 μl (primary immunization). After 14 days, the antigen (Fc fusion protein) was mixed with Freund's Incomplete adjuvant and injected intraperitoneally (secondary immunization). After 28 days, antibody production was induced by injecting an Fc fusion protein mixed with antigen and PBS (tertiary immunization). After the completion of the third immunization, blood was collected from the mice to confirm whether the antibody was produced and the antibody was confirmed by ELISA and Western blot analysis. Three days prior to cell fusion, rats with antibody production confirmed were injected with antigen (20 µg) dissolved in PBS into the tail vein of the rat to induce high antibody titers. All mice were raised in selected areas to prevent contamination.

2-3: Identification and Screening of Antibody Producing Cells

In the immunized rats, blood samples were obtained from an eye ball and placed in a 1.5 ml centrifuge tube to separate serum (1800 rpm, 10 minutes centrifugation) to test whether antibodies were produced. Store at -20 ° C until.

After confirming the production of antibodies by the ELISA method with human resistin protein and mouse resistin protein, fusion to antibody producing cells was started. In order to confirm the production of antibodies as described below, another recombinant protein was prepared and subjected to complementary assays.

First of all, this. The preparation of the resistin expressed in E. coli (E. coli) was cloned into the bacterial protein expression vector (pET-21a) and the human resistin gene (10.6 kDa protein formation) and the mouse resistin gene (11.06 kDa protein formation). Six histidine tags are linked to the C-terminus. This cloned vector. E. coli BL21 strain was transformed. The transformed bacteria were incubated at 20 ° C. for 16 hours in LB medium to which 1 mM IPTG was added to induce the expression of the desired protein. The bacteria were treated with 100 μg / ml of Lysozyme for 30 minutes at R.T. and then sonicated and centrifuged at 15000 rpm, 4 ° C. for 30 minutes. The supernatant was passed through a nickel column. After washing with wash buffer (60 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl), eluted with elution buffer (1 M imidazole, 500 mM NaCl, 20 mM Tris-HCl). Purified protein was dialyzed in 2L PBS for 16 hours in a Slide-A-Lyzer dialysis cassettes (PIERCE, Cat. 66370). Above. Resistin expressed in E. coli was coated on a 96-well plate at 0.5 ug / well and then reacted overnight. After the third wash with PBST (PBS buffer, 0.05% Tween 20), the reaction was terminated with 1% BSA. After washing again with PBST 3 times, the serum was diluted 1: 100 to 1: 10000 and treated for 1 hour. After a third wash with PBST, anti-mouse IgG conjugated HRP was treated and reacted for 1 hour. After washing 3 times with PBST, and treated with 100 ul OPD (Sigma), the color was developed for 20 minutes. After addition of 15 µl reaction termination solution (8N sulfuric acid) to obtain a value at 492 nm wavelength.

2-4: Hybridoma Cell Preparation (HRES 106 Antibody Production)

After antibody production was confirmed, the mice were sacrificed to isolate antibody producing cells (splenocytes), and myeloma cells SP2 / 0-Ag14 (ATCC CRL-1581) were isolated from known methods [Cesar Milstein and Georges Kohler's methods]. Modified method derived from [Method in enzymology, vol. 73, p3. Academic Press, New York]. That is, myeloma cells (myeloma cell) SP2 / 0-Ag14 cells were maintained in the culture plate to reach the optimal growth period using DMEM medium supplemented with 10% FBS. After washing twice with serum free medium before fusion, the concentration was adjusted to 1 x 10 ⁷ cells. Mice were sacrificed by cervical dislocation to obtain a spleen and placed in a mesh container to separate cells one by one. At this time, all the medium was used as serum-free. Completely isolated antibody producing cells were centrifuged at 400 × g for 5 minutes, washed twice with serum free medium and suspended in 10 ml of medium. Spleen lymph cells were counted with a haemocytomer to adjust the number of lymphocytes to 1 x 10 ⁸ , mixed SP2 cells with lymphocytes (1:10), and centrifuged at 400 xg for 5 minutes for washing with serum-free medium. It was. 50%, PEG 4000 (Sigma) solution pretreated at 37 ° C. was mixed with 1 ml slowly dropping over 1 minute. The resulting fusion mixture was centrifuged at 200 × g for 3 minutes, then diluted with DMEM over 4ml / 3 minutes and 30ml / 10 minutes, centrifuged at 200 × g for 5 minutes, and the cells suspended in 35 ml of HAT selection medium. . 100 μl of the suspension was dispensed into 96-well plates coated with feeder cells (macrophage isolated from PBS from rat abdominal cavity) one day before incubating for 10-14 days in a 37 ° C., 5% CO ₂ incubator.

2-5: Hybridoma Cell Preparation (MRES 06 and MRES 18 Production)

After antibody production was confirmed, rats were sacrificed to isolate antibody producing cells (splenocytes), and myeloma cells P3X63Ag8.653 (ATCC, CRL-1580) were isolated from known methods [Cesar Milstein and Georges Kohler's. Modified methods derived from methods [Method in enzymology, vol. 73, p3. Academic Press, New York]. That is, myeloma cells (myeloma cell) P3X63Ag8.653 (ATCC, CRL-1580) cells were maintained in the culture plate to reach the optimal growth period using DMEM medium supplemented with 10% FBS. After washing twice with serum free medium before fusion, the concentration was adjusted to 1 x 10 ⁷ cells. Mice were sacrificed by cervical dislocation to obtain a spleen and placed in a mesh container to separate cells one by one. At this time, all the medium was used as serum-free. Completely isolated antibody producing cells were centrifuged at 400 × g for 5 minutes, washed twice with serum free medium and suspended in 10 ml of medium. Spleen lymphocytes were counted with a haemocytomer to adjust the lymphocyte count to 1x10 ⁸ , followed by mixing (1:10) P3X63Ag8.653 (ATCC, CRL-1580) cells with lymphocytes and washing them with serum-free medium. Centrifuge at 400 xg for 5 minutes. 50%, PEG 4000 (Sigma) solution pretreated at 37 ° C. was mixed with 1 ml slowly dropping over 1 minute. The resulting fusion mixture was centrifuged at 200 × g for 3 minutes, then diluted with DMEM over 4ml / 3 minutes and 30ml / 10 minutes, centrifuged at 200 × g for 5 minutes, and the cells were suspended in 35 ml of HAT selective medium. . 100 μl of the suspension was dispensed into 96-well plates coated with feeder cells (macrophage isolated from PBS from rat abdominal cavity) one day before incubating for 10-14 days in a 37 ° C., 5% CO ₂ incubator.

2-6: Screening and Isolation of Hybridomas Producing Each Antibody

Culture supernatants were obtained from 2-4 and 2-5, and positive clones for resistin were selected by the direct ELISA method of 2-3, and then confirmed as positive clones by Western blot for confirmation. Here, among the hybridomas, one having a high antibody activity against resistin was selected, and one hybridoma cell was obtained for human resistin and two for mouse resistin.

The positive clones thus identified were diluted in a series of procedures, in which one fusion cell was divided into 0.5 cells per well of a 96-well plate, incubated for 10 days, and then again verified by ELISA. The final positive clone was identified as HRES 106, MRES 06 and MRES 18 according to the antibody properties and recognition sites. Here, finally verified fusion cells were transferred to 24 wells to grow in size and finally transferred to T25 flasks and cultured in isolation.

Each hybridoma cell producing a monoclonal antibody against resistin is a hybridoma cell producing a monoclonal antibody HRES 106 against human resistin at a depository institution (Bank of Biotechnology Research Institute, Eoeun-dong, Yuseong-gu, Daejeon, Korea). As for Accession No. KCTC 10428BP, for the hybridoma cells producing the monoclonal antibody MRES 06 against the mouse's resistin, Access for Accession No. KCTC 10429BP for the hybridoma cells producing the monoclonal antibody MRES 18 for the mouse resistin. Deposited No. KCTC 10430BP, dated February 21, 2003.

Example 3: Antibody Production and Isolation

Mouse intraperitoneal cultures were prepared using the positive clones for each antibody obtained in Example 2, and antibodies were separated therefrom. Only the production hybridoma cells were different for each antibody, and the method of preparing and separating the intraperitoneal culture was performed in the same manner.

In order to obtain three kinds of antibody intraperitoneal cultures (Ascites) distinguished above in Example 2, prestin (positive clone) 3x10 ⁶ cells which generate three monoclonal antibodies identified by ELISA method and Western blot method, respectively Intraperitoneal injection of BALB / C mice (HRES 106) and BALB / c-nu mice (MRES 06 or MRES 18) treated beforehand. The plural was extracted after 12-15 days. All three monoclonal antibodies, HRES 106, MRES 06 and MRES 18, were prepared in the same way, except that they were divided into individual antibodies in the final validation step, and the intraperitoneal cultures were obtained independently to make each antibody and intraperitoneally containing each antibody. Separation of the antibody from the culture was carried out in the following manner. The obtained ascites was purified through a Protein A affinity column (Amersham Bioscience, HiTrap rProtein A FF). That is, the Protein A column was homogenized with a 10-fold volume of DPBS at a rate of 1 ml / min and then passed through ascites. At this time, the rate was passed at 1 ml / min by the AKTA purifier (Amersham Bioscience) and the elution was made to 100 mM glycine, pH 3.0 and then neutralized with 1 / 10-fold 2M Tris, pH 8.0. At this time, the elution rate was 1 ml / min, and the antibody was purified and separated, respectively.

Example 4 ELISA Assay for Differentiating Antibody Isotypes

In order to distinguish the isotypes of the HRES 106, MRES 06 and MRES 18 antibodies prepared in Example 3, HRES 106 used an ELISA kit purchased from Pierce Inc. and MRES 06 and MRES 18 were manufactured by Zymed Laboratory. Inc.), an ELISA kit was used. The method used to distinguish the isotype was used in the test book provided by the kit company and the results are shown in Table 1.

characteristic Monoclonal Antibody (People) Monoclonal antibody (mouse) HRES 106 MRES 06 MRES 18 ELISA using recombinant resistin O O O Western blot using recombinant resistin O O 0 Human Plasma (Sandwich ELISA) 0 X X Mouse Plasma (Sandwich ELISA) X 0 0 Iso type IgG2a / Kappa IgG1 / kappa IgG1 / kappa

Example 5: Western Blot Analysis for Antibody Distinction

5-1: Antibody Binding Specificity for Human and Mouse Plasma Resistin

The binding specificity of each antibody was examined for the residues of human and mouse plasma and their recombinant proteins, and the experimental results are shown in FIGS. 3 and 4. Human Resistin recombinant protein was electrophoresed at the same concentration (100 ng) and then reacted with HRES 106 antibody by concentration (dilution step) to confirm the specificity and titer of the antibody (FIG. 3). MRES 06 and MRES produced by hybridomas 18 antibody was electrophoresed with mouse plasma protein (1 μl) and recombinant protein at 12% PAGE, and the plasma protein was transferred to nitrocellulose membrane for 12 hours (FIG. 4). The membrane was blocked with 5% scheme milk and washed five times with PBST. The membrane was treated with the hybridoma-producing monoclonal antibodies HRES 106, MRES 06 and MRES 18, incubated for 1 hour, washed with PBST, and treated with anti-IgG-HRP diluted 1: 5,000 in mice for 1 hour. Incubated. ECL (Pierce Inc.) reagents using chemifluorescence were mixed 1: 1, and then 4 ml were evenly sprayed on the membrane and left for 2 minutes. Thereafter, the ECL reagent was removed, and then, the color was confirmed by the ECL detector (FUJIFILM LAS-1000) to identify the antibody that reacts to the resist. In addition, the recombinant protein was electrophoresed under the same conditions as shown in FIG. 3 to confirm reaction specificity in the same manner.

As shown in FIG. 3, HRES 106 can recognize human recombinant protein resist and reacts even at high dilution folds to reveal high antibody titers. ELISA analysis confirmed the reaction specificity.

As shown in FIG. 4, MRES 06 and MRES 18 were able to recognize mouse plasma resistin as a result of Western blot and also recognize recombinant protein.

In addition, mouse resistin specificity of the mouse resistin monoclonal antibodies MRES 06 and MRES 18 was tested. As shown in FIG. 5, MRES 06 and MRES 18 showed specific responsiveness to recombinant mouse resistin, mouse recombinant resistin expressed in the pET system, and mouse recombinant resistin in the form of an Fc fusion, whereas recombinant human resistin was not recognized. This confirmed that the mouse resistin monoclonal antibodies MRES 06 and MRES 18 are specific for mouse resistin and its recombinant protein.

5-2: Recombinant Resistin ELISA Analysis According to Antibody Concentration

this. ELISA analysis was performed using human Resistin and HRES 106 antibodies expressed in E. coli, and the results are shown in FIG. 6.

7 is a graph showing the binding specificity of MRES 06 and MRES 18 to recombinant resistin by ELISA method. In addition, it can be seen from FIG. 7 that the mouse resistin monoclonal antibodies MRES 06 and MES 18 show specificity with respect to mouse recombinant resistin compared to human recombinant resistin. As shown in FIGS. 6 and 7, both the human resistin monoclonal antibody HRES 106 and the mouse resistin monoclonal antibodies MRES 06 and MRES 18 were detectable by binding to recombinant resistin.

Example 6: Sandwich Enzyme Immunoassay of Resistin in Clinical Blood

HRES 106, MRES 06 and MRES 18 antibodies are used in the same manner as using the recombinant resistin. The labeling antibody was a biotin-bound polyclonal antibody and the color reaction was streptavidin coupled with horseradish peroxidase (Sigma, hereinafter abbreviated as HRP) (Figs. 8, 9, 10 and 11).

6-1: Preparation of Biotin Labeled Antibody

For the production of polyclonal antibodies, polyclonal antibodies are inserted into the pET-21a vector (Novagen) by gene recombination. this. Transferred to coli. Here, IPTG was added to induce expression, followed by expression of resistin at 20 ° C. The thus expressed resistin was isolated and purified using a His-tag column. 100 ug of purified resistin was mixed with Freund's complete adjuvant in a 1: 1 ratio to make 1 ml of the suspension solution, and then injected into the rabbit's dorsal subcutaneous 5 to 7 spots. Once every two weeks, 100 ug of resistin was mixed 1: 1 with Freund's incomplete adjuvant in the same way to amplify the formation of the antibody. Collect the After centrifugation, only serum was isolated and polyclonal antibodies were purified by passing serum through a Protein-A column.

In order to bind the biotin labeling to the polyclonal antibody, 4.5 ul of biotin-7-NHS solution (20 mg / ml) dissolved in DMSO was mixed with purified resin in a 1 mg / 1 ml Protein-A column for 2 hours at 20 ° C. Reacted. A solution of a polyclonal antibody and biotin-7-NHS was added to a Sephadex G25 column saturated with 3.5 ml of PBS solution, and 3.5 ml of PBS was added thereto to fractionate 0.5 ml of the Eppendorf tube. The polyclonal antibodies and biotin thus obtained were identified by O.D. The amount eluted was measured by reading at a wavelength of 280 nm.

6-2: black

For enzymatic immunoassay (step 2), HRES 106, MRES 06 and MRES 18 monoclonal antibodies (1 mg / ml) were diluted to 5 ug / ml concentration in carbonate buffer (96 well in 100 ul (final 0.5 ug). / Well) and allowed to react overnight. After washing three times with PBST, 100ul of PBST (blocking solution) containing 1% BSA was added to each well and reacted at 37 ° C for 1 hour. Human serum samples were prepared as follows. Plasma was diluted 10-fold in PBST containing 0.1% BSA and then 100ul was added to each well of the blocking plate and reacted at 37 ° C for 1 hour.

For the determination of enzyme activity, the plates were washed three times with PBST (PBS buffer, 0.05% Tween20) and the reaction was terminated with 1% BSA. After washing three times with PBST again, the polyclonal antibody labeled with biotin was diluted 1: 5,000 and treated for 1 hour. After washing three times with PBST treated with streptavidin conjugate HRP and reacted for 1 hour. After washing three times with PBST, and treated with 100 ul OPD (Sigma), the color was developed for 20 minutes. Values were obtained at 492 nm wavelength after addition of 15 ul reaction termination solution (8 N sulfuric acid).

6-3: standard curve

In order to quantify blood resistin through O.D. obtained in ELISA, the recombinant resistin was serially diluted according to the concentration, followed by ELISA method, and a standard curve between O.D. Therefore, O.D. obtained from the resistin in the sample by this equation. By substituting the values, the resistin in the sample could be quantified (FIG. 8).

As shown in Figure 9, HRES 106 was confirmed by the ELISA method has the ability to recognize the resistin in the blood.

The present invention relates to a monoclonal antibody that recognizes a resistin protein, a method for preparing the same, and a method and a quantification kit for resistin using the same. By measuring the concentration of resistin in the blood using a monoclonal antibody that recognizes the resistin protein according to the present invention, it can be usefully used for diagnosis of diabetes and obesity.

Claims (11)

A monoclonal antibody or active fragment thereof that specifically recognizes a resistin protein. The monoclonal antibody or active fragment thereof according to claim 1, which recognizes human or mouse resistin protein. The monoclonal antibody or active fragment according to claim 1 or 2, wherein the detection label is bound to a detectable signal capable of producing a detectable signal. 4. The monoclonal antibody or active fragment of claim 3, wherein the detection labeling agent capable of producing a detectable signal is an enzyme, fluorescent substance, luminescent substance or radioactive substance. The monoclonal antibody according to claim 1, which is produced by hybridoma cells of Accession No. KCTC 10428BP, KCTC 10429BP, or KCTC 10430BP. A hybridoma cell producing the monoclonal antibody of claim 1. The hybridoma cell of claim 6, having accession number KCTC 10428BP, KCTC 10429BP, or KCTC 10430BP. A monoclonal antibody according to any one of claims 1 to 5, comprising a detection label capable of generating a detectable signal bound to the monoclonal antibody, a carrier solution, a dissolving agent, a container comprising a cleaning agent and instructions for use. A quantitative kit of resistin in a sample, characterized by the above-mentioned. The kit of claim 8, further comprising another secondary antibody that recognizes a monoclonal antibody, wherein the detection label is bound to the secondary antibody. The kit of claim 8, wherein the detection labeling agent is an enzyme, further comprising a substrate capable of measuring enzymatic activity and a reaction terminator. i) contacting the biological sample with the resistin monoclonal antibody according to any one of claims 1 to 5 combined with a detection labeling agent, ii) quantitatively detecting the immunological complex obtained in step i), iii ) Quantifying the immunological complex obtained by contacting the purified resistin protein with the resistin monoclonal antibody according to any one of claims 1 to 5 combined with a detection marker to obtain a standard curve, iv) step ii Quantifying the resistin present in the biological sample by comparing the amount of detected protein to the standard curve obtained in step iii). Way.