WO2004022596A1 - Monoclonal antibody against adiponectin, preparation method and use thereof - Google Patents

Monoclonal antibody against adiponectin, preparation method and use thereof Download PDF

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Publication number
WO2004022596A1
WO2004022596A1 PCT/KR2003/001213 KR0301213W WO2004022596A1 WO 2004022596 A1 WO2004022596 A1 WO 2004022596A1 KR 0301213 W KR0301213 W KR 0301213W WO 2004022596 A1 WO2004022596 A1 WO 2004022596A1
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Prior art keywords
adiponectin
monoclonal antibody
human
sample
antibody
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PCT/KR2003/001213
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French (fr)
Inventor
Byung-Soo Youn
Young-Soo Yang
Nam-Seok Lee
Kang-Yeol Yu
Moon-Yeon YOUN
Hong-Je PARK
Sung-Shik MIN
Jae-Jun JEOUNG
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Komed Co., Ltd.
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Priority claimed from KR1020030038880A external-priority patent/KR20040022384A/en
Application filed by Komed Co., Ltd. filed Critical Komed Co., Ltd.
Priority to AU2003244237A priority Critical patent/AU2003244237A1/en
Publication of WO2004022596A1 publication Critical patent/WO2004022596A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a monoclonal antibody that recognizes adiponectin, and a preparation method and use thereof, and more particularly to a monoclonal antibody that recognizes adiponectin, quantification of adiponectin in a sample, and a use of the monoclonal antibody for diagnosis of diabetes and obesity.
  • Fat tissue plays an important role in energy storage, fatty acid metabolism, sugar homeostasis, etc. According to studies on humans and animals, it is reported that an excess of fat tissue in an obese condition and a lack of fat tissue in a lipodystrophic condition are related to insulin-resistant diabetes, also called Type II diabetes and insulin-independent diabetes.
  • adipocytes secrete various cytokines called adipokines, as well as functioning for energy storage and, signal transfer between adipocytes which controls insulin signal transfer to maintain blood sugar homeostasis.
  • adipokines TNF- ⁇ , adipsin, adiponectin, leptin, etc. have been reported.
  • Adiponectin which is an adipocyte-specific secretion protein initially called Acrp30, is now called AdipoQ, apM1 , GBP28, adiponectin, etc.
  • Adiponectin is one of the adipokines that is attracting the most attention, and it is a polypeptide consisting of 247 amino acids expressed by an adipocyte.
  • adiponectin as shown in Fig. 1 , consists of an N-terminal signal sequence, a 27 amino acid non-homologous region, a 22 Gly-Xaa-Pro or Gly-Xaa-Xaa collagen repetition region, and a C-terminal globular domain.
  • amino acid sequence of adiponectin is as shown in
  • Adiponectin is a protein abundant in human serum that is controlled by insulin, and sequence homology between complement C1q and hibernating Siberian chipmunk
  • TNF tumor necrosis factor
  • a decrease in blood adiponectin concentration and single nucleotide polymorphism are known as one of the causes of obesity and Type II diabetes.
  • adiponectin expression is induced at a rate of more than 100 times normal, which indicates that adiponectin controls energy balance and can be used for detection of adipocytes contained in a sample.
  • Insulin-independent diabetes namely
  • Type II diabetes is manifested due to problems of signal transfer such as insulin resistance, and blood glucose influx and efflux into muscle or liver cells are disturbed to increase blood glucose thereby causing complications such as multi-organ failure, hypertension, arteriosclerosis, cataracts, etc.
  • signal transfer such as insulin resistance
  • blood glucose influx and efflux into muscle or liver cells are disturbed to increase blood glucose thereby causing complications such as multi-organ failure, hypertension, arteriosclerosis, cataracts, etc.
  • abnormalities in signal transfer and glucose metabolism affect physiology of adipocytes to increase fat, and thereby obesity is involved, and obesity in turn involves Type II diabetes, indicating that obesity and Type II diabetes have a pathological cause in common.
  • Hotta et al. compared blood adiponectin level in diabetic patients having coronary arteriosclerosis, diabetic patients without coronary artery disease, and normal persons, and reported that a decrease in blood adiponectin in diabetic patients correlates to vascular disease (Hotta, et al., Arteriosclerosis, Thrombosis & Vascular Biology 20(6): 1595-9, 2000). Yamauchi, et al.
  • adiponectin because of the clinical significance of adiponectin, it is important to measure adiponectin blood concentration for diagnosis and prognosis of obesity and Type II diabetes, and to develop a treating agent.
  • adiponectin blood concentration for diagnosis and prognosis of obesity and Type II diabetes, and to develop a treating agent.
  • immunological tolerance due to a high amino acid similarity between human and mouse adiponectin protein, it is very difficult to secure a monoclonal antibody having high specificity and sensitivity, and until now, a detection method that can be clinically used has not been developed.
  • Fig. 1 shows a structure of adiponectin.
  • Fig. 2 compares amino acid sequences of human and mouse.
  • Fig. 3 is an electrophoresis photograph showing Western Blot results of ADI773, ADI741 , and ADI943, which are monoclonal antibodies according to the present invention.
  • Fig. 4 shows the results of Western Blot of ADI773 and ADI741 , which are monoclonal antibodies that recognize a full length adiponectin, a globular domain of adiponectin, recombinant adiponectin without a globular domain, and human serum adiponectin.
  • Fig. 5 shows the results of Western Blot of ADI773 and ADI741 , which are monoclonal antibodies that recognize serum of various mammals including a human, a mouse, a rat, a cow, a horse, and a rabbit.
  • Fig. 6 is a graph showing binding sensitivity of ADI773, ADI741 , and ADI943, which are monoclonal antibodies according to the present invention, to human full length adiponectin, examined by ELISA.
  • Fig. 7 is a graph showing binding sensitivity of ADI773, ADI741 , and ADI943, which are monoclonal antibodies according to the present invention, to a globular domain of human adiponectin, examined by ELISA.
  • Fig. 8 is an electrophoresis photograph showing the results of Western Blot of adiponectin existing in plasma of a normal person (lanes 1 , 2, 3) and a Type II diabetic patient (lanes 4-13).
  • Fig. 9 is a graph showing the analysis results of adiponectin in plasma of a normal person, examined by ELISA
  • Fig. 10a to Fig. 10c shows reaction conditions of serum pre- treatment for ELISA using the monoclonal antibody according to the present invention.
  • Fig. 11 shows the results of Western Blot and ELISA of adiponectin in a sample of a Type II diabetic patient using ADI773 and ADI741. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention relates to a monoclonal antibody that recognizes adiponectin, its preparation method, and its use.
  • the monoclonal antibody according to the present invention includes an adiponectin antibody produced using human adiponectin or its fragment as an antigen, and which recognizes adiponectin or its fragment as an epitope.
  • a monoclonal antibody that recognizes adiponectin can be prepared a hybhdoma cell produced by fusing a mouse cell expressing human adiponectin and a mouse myeloma cell.
  • a human adiponectin antigen can be prepared by amplifying cDNA separated from a human adipocyte with a specific primer through PCR, connecting the amplified product to an expression vector with an Fc domain of immunoglobulin, introducing it into a mammalian cell, and expressing it.
  • the adiponectin antigen that can be used in the present invention includes a full length adiponectin and a fragment of adiponectin capable of producing a monoclonal antibody, and is preferably an amino acid 1-247 fragment of human adiponectin.
  • the present inventors in order to solve the difficulty of production of human adiponectin due to immune tolerance, prepared an antigen fused with immunoglobulin, which can be effectively introduced into a mouse dendritic cell to activate the dendritic cell, thereby inducing a strong immune response.
  • the expression vector that can be used to prepare an antigen includes all the expression vectors for mammals comprising a heavy chain Fc domain of human immunoglobulin, for example a constant region of lgG1.
  • the present invention in order to increase the production of monoclonal antibodies, fuses the Fc domain of an immunoglobulin heavy chain with an adiponectin gene and expresses it in a mammalian cell, for example in an HEK-293 cell, to prepare an fusion antigen.
  • the recombinant adiponectin of the present invention can retain similar structure of adiponectin in plasma, and has property to produce effective antibody.
  • the antigen used in the present invention to prepare a monoclonal antibody that recognizes adiponectin may have different properties from that expressed in E.coli by Arita et al (Arita, et al, Biochem. Biophy. Res. Commun, vol259, pp. 79-83, 1999).
  • the mammalian cell used to prepare an antigen of the present invention includes a mammalian cell expressing known foreign genes, for examples, a human embryonic kidney (HEK) cell 293, a 293T cell, an African green monkey kidney cell Cos 1 , a Cos 7 cell, a Chinese hamster ovary cell, CHO, etc.
  • the monoclonal antibody according to the present invention is prepared using human adiponectin-Fc fusion protein as an antigen, and it recognizes a full length adiponectin and serum adiponectin.
  • the monoclonal antibody includes a monoclonal antibody that recognizes only a globular domain as an epitope, and a monoclonal antibody that recognizes adiponectin excluding a globular domain, and preferably ADI773, ADI741 , and ADI943.
  • ADI773 and ADI741 recognize both plasma adiponectin and recombinant full length adiponectin, and they do not recognize an N-terminal variable region of adiponectin.
  • ADI773 recognizes a recombinant adiponectin protein excluding a globular domain
  • ADI741 recognizes a globular domain.
  • ADI741 recognizes adiponectin of mammals of various kinds such as a human, a mouse, a rat, a cow, a horse, a rabbit, a goat, etc., while ADI773 strongly binds to human adiponectin but slightly recognizes cow adiponectin.
  • ADI773 and ADI741 recognize different epitopes.
  • ADI741 is useful for quantifying many kinds of adiponectin, and it can be particularly used for screening of medicines for diagnosis or treatment of obesity or related diseases, for example Type II diabetes.
  • ADI773 is a monoclonal antibody raised from mice immunized with adiponectin-Fc and it can be identified by conducting Western Blot in human plasma. It does not react in rat or mouse adiponectin, and has an lgG1 /kappa isoform. The monoclonal antibodies do neither recognize a globular domain of human adiponectin nor the N-terminal variable region, hence its epitope could be collagenous domain.
  • ADI741 of the present invention recognizes only a globular domain of adiponectin. Since the nucleotide sequences of the globular head domains of humans and mice are almost identical, it may be difficult to produce an antibody that recognizes this region. Therefore, ADI741's recognition of such a globular domain means formation of an autoimmune antibody, as evidence of such, mouse adiponectin is much more strongly detected than human adiponectin. ADI741 can detect • a full length adiponectin in plasma, and it has an lgG1 /kappa isoform. The biding specificity of ADI943 of the present invention can be identified through ELISA, and it recognizes only a globular domain of human adiponectin.
  • the monoclonal antibodies that recognize human adiponectin of the present invention is prepared by substantially the same method as a common monoclonal antibody preparation method, except for the antigen used. Specifically, according to the present invention, a positive clone is identified among primarily prepared hybridoma cells, and hybridoma cells producing a monoclonal antibody are selected on the basis of recognition and properties of the antibody produced by each clone.
  • the hybridoma cell producing a monoclonal antibody that recognizes adiponectin of the present invention is a fusion cell of a myeloma cell and a splenocyte producing an antibody that recognizes the adiponectin, and it is preferably a hybridoma cell line producing the monoclonal antibodies ADI773, ADI741 , and ADI943.
  • hybridoma cells producing these adiponectins were deposited on June 29, 2002, with the Bioengineering Research Institute GeneBank, located in Eoeundong 52, Yoosung-ku, Daejun Kwang-yuk City, South Korea, under KCTC 10288BP for a hybridoma cell producing an antibody ADI773, under KCTC 10289BP for a hybridoma cell producing ADI741 , and under KCTC 10290BP for a hybridoma cell producing ADI943.
  • the present invention provides an analysis method and kit for quantification or detection of adiponectin in a sample using the monoclonal antibody that recognizes adiponectin in an immunoassay or in a kit for immunoassay.
  • All the immunoassays and kits for immunoassay known in the art, for example, ELISA and radioimmunoassay, etc. can be used in the present invention.
  • the adiponectin is preferably attached with detecting means such as an enzyme, a radio element, etc.
  • the sample can be blood or plasma or serum of mammals such as a human, a mouse, a rat, a cow, a horse, a rabbit, a goat, etc.
  • the human includes a normal person, an obese person, or a Type II diabetic patient.
  • a method for quantification of adiponectin comprising the steps of adding a sample to a microtiter plate coated with a monoclonal antibody according to the present invention, reacting an enzyme-antibody complex, treating a substrate of the enzyme, measuring absorbance, and comparing the absorbance with a standard curve to quantify adiponectin in a sample.
  • Biotin can be bound to the antibody of the enzyme-antibody complex.
  • the enzyme can be HRP (horse radish peroxidase), but is not limited thereto, and all the enzymes and substrates used in ELISA can be used in the present invention.
  • An ELISA assay can detect and quantify an antigen existing in a sample using an antibody-antigen reaction.
  • Two or three antibodies are required for applying an ELISA kit.
  • the Kit consists of 3 antibodies.
  • the construction and principle for preparing the ELISA kit are as follows.
  • an ADI741 monoclonal antibody (primary antibody) is coated on a 96-well plate, a sample comprising adiponectin is added to the well and reacted, a polyclonal antibody (secondary antibody) marked with an enzyme is reacted, a substrate capable of reacting with the marked enzyme is treated, and an enzyme-substrate reaction is then detected and compared with a standard curve to quantify adiponectin in the sample.
  • the enzyme and substrate biotin and streptavidin conjugated HRP can be used, but it is not limited thereto.
  • a sample plasma can be pre-treated, which comprises the steps of diluting a sample and/or boiling the sample, wherein a dilution rate is 10-20 fold and boiling time is 1-10 minutes, preferably 4-6 minutes.
  • the present invention also provides a method or kit for diagnosing obesity or Type II diabetes, by quantifying adiponectin in plasma using the monoclonal antibody according to the present invention. Comparing concentrations of adiponectins in plasma of a normal person and a Type II diabetic patient, it can be seen that the plasma of a normal person includes adiponectin in a higher concentration (Figs. 8 and 9). Therefore, according to the present invention, obesity and Type II diabetes could be diagnosed by quantifying adiponectin in a sample.
  • variations in a living body of a subject blood adiponectin distribution of a normal person and a diabetic patient, sensitivity and specificity of an adiponectin value that can distinguish a normal person from a diabetic patient, etc. can be examined.
  • Human adiponectin DNA was amplified by 35 times using human adipocyte cDNA (Clonetech, Inc.) as a template and using a primer pair having a Hind III restriction enzyme recognition site at 5' terminal and a
  • BamH I recognition site at 3' terminal wherein temperature and time were set as follows: PCR at 95 ° C for 2 minutes, denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, and chain extension at 72 ° C for 1 minute (MJ Research, minicycler PTC150 model).
  • Forward direction primer 5'-cccaagcttgggctctaagattgtcagt-3' (Sequence No. 1)
  • Reverse direction primer 5'-gaaggatccggggttggtatcatggta-3'
  • the amplified fragment was inserted in a pCEP4 mammal expression vector (Invitrogen, cat# VO44-50) comprising Fc (Fc domain of immunoglobulin) to prepare a recombinant vector, and an HEK 293-EBNA cell was transformed with the recombinant vector and then cultured in a pCEP4 mammal expression vector (Invitrogen, cat# VO44-50) comprising Fc (Fc domain of immunoglobulin) to prepare a recombinant vector, and an HEK 293-EBNA cell was transformed with the recombinant vector and then cultured in a pCEP4 mammal expression vector (Invitrogen, cat# VO44-50) comprising Fc (Fc domain of immunoglobulin) to prepare a recombinant vector, and an HEK 293-EBNA cell was transformed with the recombinant vector and then cultured in a pCEP4 mammal expression vector (Invitrogen
  • DMEM culture medium comprising the antibiotic G418.
  • the culture medium for the transformant was changed every 3 days, when the culture supernatant was collected with a pipet.
  • a protein A affinity column (Amersham Bioscience, HiTrap rProteinA FF) was homogenized with DPBS of 10 times the amount at a speed of 1 ml/min, and then passed through the collected cell culture at a speed of 1 ml/min with an AKTA purifier (Amersham Bioscience).
  • the collected cell culture was eluted with 100 mM glycine, pH 3.0 at a speed of 1 ml/min and neutralized with 2 M Tris, pH 8.0 to separate adiponectin-Fc fusion protein.
  • the separated adiponectin-Fc fusion protein was used as an antigen for preparing an antibody in the subsequent process.
  • Electrophoresis of the adiponectin-Fc fusion protein prepared in Example 1 was conducted on a gel containing PBS (phosphate-buffered saline) or through SDS-PAGE (polyacrylaminde gel electrophoresis), and the fragment of the gel was cut and an emulsion mixed with complete Freund's adjuvant of the same volume was prepared.
  • the emulsion was injected into abdominal cavities of 5 6-8 week old BALB/C mice. 20-50 ⁇ g of antigen was injected into each mouse such that the total volume reached 200-400 ⁇ L
  • the emulsion was injected twice at an interval of 2 weeks to induce antibody production, and an antigen dissolved in PBS was injected into the tail veins, 3 days before conducting cell fusion. In order to prevent contamination, all the mice were grown in the selected area. 2-2. Identification and selection of antibody production cell
  • E.coli BL21 strain was transformed with the cloned vector.
  • the transformed strain was cultured in an LB medium to which 1 Mm of IPTG was added at 20 ° C for 16 hours to induce desired protein expression.
  • the transformed strain was treated with lysozyme at room temperature for 30 minutes, ultrasonic-treated, and centrifuged at 15,000 rpm at 4 ° C for 30 minutes.
  • the supernatant was passed through a Nikel column.
  • the Nikel column was washed with a washing buffer solution (60mM imidazole, 500mM NaCI, 20Mm Tris-HCI), and then eluted with an eluting buffer solution (1 Mm imidazole, 500mM NaCI, 20Mm Tris-HCI).
  • the purified protein was introduced into Slide-A- Lyzer dialysis cassettes (PIERCE, Cat. 66370) to dialyze in 2L PBS for 16 hours.
  • the adiponectin expressed in E.coli was coated on a 96-well plate in an amount of 0.5 g/well and reacted overnight, and then washed with PBST (PBS buffer, 0.05% Tween 20) three times, and reaction was terminated with 2% BSA.
  • PBST PBS buffer, 0.05% Tween 20
  • the adiponectin was again washed with PBST three times, and treated with serum diluted to 1 :100-10,000 to react for 1 hour. Then, the adiponectin was washed with PBST three times and treated with anti-mouse IgG conjugate HRP to react for 1 hour.
  • the adiponectin was again washed with PBST three times, treated with 100 ⁇ l of OPD (Sigma), and color reaction was conducted for 20 minutes. 50 ⁇ l of reaction termination solution (0.5 M sulfuric acid) was added thereto, and an O.D. value was obtained at 492 nm.
  • the mouse was killed to separate antibody production cells (splenocyte), which were fused with myeloma cells SP2, according to a modified method originated from Cesar Milstein and Georges Kohler's method (Method in enzymology, vol. 73, p3. Academic Press, New York). Specifically, an SP2 cell of the mouse was maintained in a culture plate until an optimum growth period began using a 10% FBS DMEM medium. One day before conducting cell fusion, the SP2 cell was diluted to 2-3 x 10 5 cell/ml, and 50 ml was taken the next day, which was cent fuged at 400g for 3 minutes. After washing with a serum-free medium twice, the concentration was controlled to 1-2 x 10 7 cell/ml.
  • the mouse was killed by cervical dislocation to obtain its spleen, which was introduced into a mesh container to separate individual cells.
  • the medium used was serum-free.
  • the completely separated antibody production cell was centrifuged at 400g for 5 minutes, washed with a serum-free medium twice, and suspended in 10 ml of medium.
  • the number of lymphocytes was calculated with a haemocytomer, and 10 8 lymphocytes were mixed with 2 x 10 7 SP2 cells (5: 1 ) to centrifuge at 400g for 3 minutes. 1 ml of PEG (Sigma) solution pre-treated at 37 ° C was slowly dripped into the supernatant over 1 minute to mix them.
  • the produced mixed solution was diluted with DMEM over 2ml/2 minutes and 5ml/min, centrifuged at 400g for 3 minutes, and then cells were suspended in 200ml of HAT selection medium. 200 ⁇ l of the suspension was sprayed into a 96-well plate coated with feeder cells (macrophage separated from abdominal cavity of the mouse using PBS) to culture in a 5% CO medium at 37 °C for 7-14 days. 2-4. Selection and separation of hybridoma producing each antibody
  • the culture supernatant was taken in 2-3 above, and positive clones for adiponectin were selected by ELISA of 2-2 above and then identified by Western Blot. Therein, among a number of hybridoma, those having high antibody activities for full length adiponectin were selected, among which those having high antibody activities for globular domain were selected to obtain 4 kinds of hybridoma cells.
  • the identified positive clones were diluted, the fusion cells were sprayed into a 96-well plate at 0.5 cell/well to culture for 7 days, and they were examined again by ELISA. According to antibody properties and recognition sites, the cells were divided into ADI773, ADI741 , and ADI943. The finally-examined fusion cells were transferred to a 24-well plate, and finally transferred to a T25 flask to separate and culture.
  • the hybridoma cells producing antibody recognizing adiponectin were deposited on June 29, 2002, with the Bioengineering Research Institute GeneBank, located in Eoeundong 52, Yoosung-ku, Taejun Kwang- yuk City, South Korea, under KCTC 10288BP for those producing ADI773, under KCTC 10289BP for those producing ADI741 , and under KCTC 10290BP for those producing ADI943.
  • Example 3 Production and separation of antibody
  • mice abdominal culture liquids were prepared, from which antibodies were cloned. Preparation and separation of abdominal culture liquid were conducted by the same method, except that hybridoma cells differed for each antibody.
  • the obtained ascites were purified through a protein A affinity column (Amersham Bioscience, HuTrap rProtein A FF). Specifically, the protein A column was homogenized with DPBS at 10 times the volume at a speed of 1 ml/min, and then passed through the ascites at a speed of 1 ml/min with an AKRA purifier (Amersham Bioscience). The ascites were eluted with 100mM glycine, pH 3.0 at a speed of 1 ml/min, and then neutralized with 1/10 the amount of 2M Tris, pH 8.0 to purify and separate each antibody.
  • Example 3 For each antibody of ADI73, ADI741 , and ADI943 separated in Example 3, ELISA and Western Blot were conducted by the same method as in Example 2, and immuno-precipitation was additionally conducted.
  • adiponectin and human adiponectin recombinant globular domain 5 ng
  • binding specificity of each antibody was examined, and the results are shown in Fig. 3.
  • 20ml of human blood was taken, and centrifuged at 1600 rpm for 10 minutes to separate supernatant, i.e., plasma.
  • Plasma protein was quantified, and each 40 g of protein was electrophoresed on 12% PAGE (first three lanes excluding a marker), and the serum protein was transferred to a nitrocellulose membrane and left overnight. The membrane was blocked with 5% skim milk, and washed with PBST twice.
  • the membrane was treated with ADI773, ADI741 , and ADI943, and then cultured for 1 hour, washed with PBST, treated with mouse anti-lgG-HRP diluted to 1 :5000, and cultured for 40 minutes.
  • 1 ml of ECL reagent (Pierce Inc.) was uniformly sprayed into the membrane, and let stand for 1 min. Then, the ECL reagent was completely removed, and color reaction to adiponectin was identified by an ECL detector (Kodak). Additionally, the globular domain of a recombinant protein was electrophoresed under the same conditions, and reaction specificity was identified by the same method (Fig. 3 lanes 4-6). As shown in Fig. 3, ADI773 can recognize only full length human adiponectin, and as a result of Western Blot, it is shown that it recognizes human full length adiponectin in plasma and serum.
  • ADI 1943 could not be identified by Western Blot, and it can be identified by ELISA. As a result, it can recognize only the globular domain of human adiponectin. Therefore, it can also be used in the ELISA kit.
  • ADI 741 recognizes only the globular domain of human adiponectin. It is very difficult to prepare an antibody that recognizes only the globular domain due to sequence homology of the globular domain, and the antibody is likely to be an autoimmune antibody. It was identified that the antibody can detect full length human adiponectin, but it binds to mouse adiponectin very strongly.
  • adiponectin protein (ALEXIS Biochemicals) without a globular domain, a globular domain (prepared by the method of Example 2-2), and full length adiponectin (prepared by the method of Example 2-2) were performed by electrophoresis.
  • Western Blot was conducted using ADI741 and ADI773 monoclonal antibodies. The results are shown in Fig. 4.
  • the serum protein and full length adiponectin were recognized by
  • ADI773 and ADI741 recognized only the globular domain of adiponectin, and ADI773 recognized a recombinant adiponectin protein without a globular domain. Therefore, it can be seen that ADI741 and ADI773 are monoclonal antibodies that recognize different epitopes.
  • N-terminal variable regions of human, mouse, and rat adiponectin were synthesized with peptide, and a control peptide (15aa-peptide) was prepared.
  • peptide e was diluted in a carbonate buffer solution (pH 9.6), coated on a 96-well plate in an amount of 0.5 g/well, and then reacted overnight. After washing with PBST (PBS buffer, 0.05% Tween 20) three times, reaction was stopped with 1 % BSA. After again washing with PBST three times, the reactant was treated with ADI773 and ADI 741 monoclonal antibodies diluted with 1 % BSA-PBST buffer solution to 500-fold, and then reacted for 1 hour. After washing with PBST three times, the reactants were treated with anti-mouse IgG conjugate HRP and reacted for 1 hour.
  • PBST PBS buffer, 0.05% Tween 20
  • ADI773 and ADI 741 did not recognize the adiponectin N-terminal variable region. Therefore, it can be seen that ADI773 recognizes the collagenous domain and ADI 741 recognizes the globular domain.
  • Fig. 6 is a graph showing binding specificity to full length adiponectin according to concentrations of ADI 773, ADI 741 , and ADI 949. As shown in Fig. 6, ADI 773 and ADI 741 bind to recombinant full length adiponectin.
  • Example 5-1 Example 5-1 , and the results are shown in Fig. 7.
  • Fig. 7 is a graph showing binding specificity to the adiponectin globular domain of ADI773, ADI741 , and ADI 949 according to antibody concentrations. As shown in Fig. 7, compared to ADI773, AD741 more specifically binds to the adiponectin globular domain hence can be detected, which indicates that the ADI773 epitope is located in a variable region or collagen-like domain of adiponectin.
  • Example 6 Sandwich ELISA of adiponectin in clinical blood
  • ADI773 antibody was used in the same way as the recombinant adiponectin in the above.
  • a marked antibody a biotin-bound polyclonal antibody was used, and color reaction was conducted using peroxidase
  • HRP (originated from Western mustard, Sigma, hereinafter referred to HRP)- bound streptavidin.
  • adiponectin 50 ⁇ g of purified adiponectin was mixed with Freund's complete adjuvant in a ratio of 1 :1 to make a suspension, and then injected hypodermically into the back of a rabbit in an amount of 5-7 ⁇ L 50j «g of adiponectin was mixed with Freund's incomplete adjuvant in a ratio of 1 :1 , and injected by the same method as above twice with 2 weeks therebetween to amplify antibody production. After 6 weeks, the rabbit was killed and blood was taken from its heart. The blood was centrifuged to separate serum, which was passed through a protein-A column to purify the polyclonal antibody.
  • adiponectin purified in 1mg/1ml of protein-A-column and 4.5 ⁇ l of biotin-7-NHS solution dissolved in DMSO were mixed to react at 20 ° C for 2 hours.
  • the mixed solution of polyclonal antibody and biotin-7-NHS was introduced into a Sephadex G25 column saturated with 3.5 ml of PBS solution, 3.5 ml of PBS was added to fractionate every 0.5 ml, and it was eluted to receive the fractions in an Eppendorf tube.
  • the received polyclonal antibody and biotin were read using an O.D. measuring instrument at 280 nm to measure the eluted amount.
  • the pre-treated serum was finally diluted to 1 :100-5000 in PBS (serum dilution solution) comprising 1 % BSA, 0.01 % NaN3, and 0.05% tween20, 100 ⁇ of the diluent was introduced into a 96- well plate, and reacted in an incubator of 37 °C for 1 hour.
  • ADI773 was coated on the 96-well plate in an amount of 0.5 g/well, and reacted overnight.
  • the plate was then treated with 100 ⁇ of serum sample and reacted for 1 hour. In order to measure enzyme activity, the plate was washed with
  • ELISA indicates that ADI773 can recognize adiponectin in blood.
  • Example 7 Sample pre-treatment for ELISA
  • a dilution amount suitable for pre- treatment and boiling time was optimized.
  • the experiment was designed so that ELISA could be applied to ADI741. Because adiponectin in human plasma consists of multiple bodies, it should be changed to a monomer for recognition of epitopes.
  • serum was diluted in a pre-treatment solution (30mM Tris-HCI (pH 6.8) + 2% SDS + 1 Mm EDTA) and boiled.
  • 50, 20, 10, 5, 2, and 1 ⁇ l of human serum were respectively taken and diluted in 50 (1 :2 dilution), 80 (1 :5 dilution), 90 (1 :10 dilution), 95 (1 :20 dilution), 98 (1:50 dilution), and 99 ⁇ l (1 :100 dilution) of pre-treatment buffer solution, and then boiled for 5 minutes.
  • 100/i ⁇ of a sample diluted to 1 :500 was reacted with coated ADI741 and washed three times.
  • the biotinylated human adiponectin polyclonal antibody prepared in Example 6-1 was added and reacted for 1 hour.
  • Fig. 10a results at 10-20 fold are optimum, so the following experiment was conducted with a 10-fold dilution.
  • plasma was diluted 10-fold in a sample buffer, reacted at 100°C in a heating block for 0.5, 1 , 3, 5, and 10 minutes to boil, and finally diluted to
  • Fig. 10b shows the results.
  • Fig. 10c In order to examine the optimum conditions, human serum was diluted to 2, 5, 10, and 20-fold, boiled for 5 minutes, and then electrophoresed on a native gel to conduct Western Blot thereby identifying monomer production. The results are shown in Fig. 10c. As shown in Fig. 10c, since the lane 5 is a sample without pre-treatment, most adiponectin cannot move and thus it is hung in a well of the gel, and the lane 1 shows little monomer production at a 1 :2 dilution, at which it is highly likely that serum protein and adiponectin are coagulated to be lost during centrifugation.
  • Example 8 Detection of adiponectin in a sample of a Type II diabetic patient using ADI773, ADI741
  • adiponectin 1 ⁇ l of plasma of each of 3 normal persons and 6 Type II diabetic patients was electrophoresed, adiponectin was detected using ADI773 and ADI741 antibodies, and the results are shown in Fig. 11 (A). It can be seen that the adiponectin level in the Type II diabetic patients was decreased, and that ADI 773 / ADI 741 can be used in Western Blot to qualitatively measure adiponectin concentration.
  • the present invention relates to a monoclonal antibody that recognizes adiponectin, a preparation thereof, and a method and kit for quantification of adiponectin using the same.
  • the monoclonal antibody that recognizes adiponectin according to the present invention can be used to measure blood adiponectin concentration, and thus can be used in a kit for diagnosis of obesity and diabetes.

Abstract

The present invention relates to a monoclonal antibody that recognizes adiponectin, and a preparation method and use of the same. The present invention provides a measuring method for concentration of the adiponectin in a sample, especially blood with the monoclonal antibody, thereby rendering diagnosis of adiponectin-associated diseases such as Type II diabetes and obesity.

Description

MONOCLONAL ANTIBODY AGAINST ADIPONECTIN, PREPARATION
METHOD AND USE THEREOF
BACKGROUND OF THE INVENTION (a) Field of the Invention
The present invention relates to a monoclonal antibody that recognizes adiponectin, and a preparation method and use thereof, and more particularly to a monoclonal antibody that recognizes adiponectin, quantification of adiponectin in a sample, and a use of the monoclonal antibody for diagnosis of diabetes and obesity. (b) Description of the Related Art
A deficiency in energy homeostasis causes an excess or lack of fat tissue. Fat tissue plays an important role in energy storage, fatty acid metabolism, sugar homeostasis, etc. According to studies on humans and animals, it is reported that an excess of fat tissue in an obese condition and a lack of fat tissue in a lipodystrophic condition are related to insulin-resistant diabetes, also called Type II diabetes and insulin-independent diabetes.
According to recent endocrinological studies, adipocytes secrete various cytokines called adipokines, as well as functioning for energy storage and, signal transfer between adipocytes which controls insulin signal transfer to maintain blood sugar homeostasis. As such adipokines, TNF-α, adipsin, adiponectin, leptin, etc. have been reported.
Adiponectin, which is an adipocyte-specific secretion protein initially called Acrp30, is now called AdipoQ, apM1 , GBP28, adiponectin, etc.
Adiponectin is one of the adipokines that is attracting the most attention, and it is a polypeptide consisting of 247 amino acids expressed by an adipocyte.
The structure of adiponectin, as shown in Fig. 1 , consists of an N-terminal signal sequence, a 27 amino acid non-homologous region, a 22 Gly-Xaa-Pro or Gly-Xaa-Xaa collagen repetition region, and a C-terminal globular domain. In addition, the amino acid sequence of adiponectin is as shown in
Fig. 2, and those of mice and humans slightly differ. Adiponectin is a protein abundant in human serum that is controlled by insulin, and sequence homology between complement C1q and hibernating Siberian chipmunk
(Hib27), and the C-terminal globular head domain, is very high. The structure of the adiponectin globular domain shows significant homology with tumor necrosis factor (TNF).
According to the results of various studies in the fields of genetics, physiology, kinetics, etc., a decrease in blood adiponectin concentration and single nucleotide polymorphism (SNP) are known as one of the causes of obesity and Type II diabetes.
When an adipocyte is differentiated, adiponectin expression is induced at a rate of more than 100 times normal, which indicates that adiponectin controls energy balance and can be used for detection of adipocytes contained in a sample. Insulin-independent diabetes, namely
Type II diabetes, is manifested due to problems of signal transfer such as insulin resistance, and blood glucose influx and efflux into muscle or liver cells are disturbed to increase blood glucose thereby causing complications such as multi-organ failure, hypertension, arteriosclerosis, cataracts, etc. Particularly, it has been reported that abnormalities in signal transfer and glucose metabolism affect physiology of adipocytes to increase fat, and thereby obesity is involved, and obesity in turn involves Type II diabetes, indicating that obesity and Type II diabetes have a pathological cause in common.
Many studies have proven that adiponectin is a critical factor for early diagnosis and treatment of obesity and diabetes. Hotta, et al. compared blood adiponectin level in diabetic patients having coronary arteriosclerosis, diabetic patients without coronary artery disease, and normal persons, and reported that a decrease in blood adiponectin in diabetic patients correlates to vascular disease (Hotta, et al., Arteriosclerosis, Thrombosis & Vascular Biology 20(6): 1595-9, 2000). Yamauchi, et al. reported that in a mouse model, there is a high correlation between insulin resistance and adiponectin decrease, and when adiponectin and reptin are administrated to a fat-deficient mouse in a physiological amount, insulin resistance is recovered (Nature Medicine 7(8): 941-6, 2001). Yang et al. reported that blood adiponectin levels increase in obese patients undergoing gastric partition surgery, and obesity inhibits blood adiponectin level increases (J Clinical Endocrinology & Metabolism 86(8): 3815-9, 2001). Sardas et al. reported that in diabetic patients, oxidative stress increases to increase DNA damage to a level greater than in normal persons (Mutation Research 490(2): 123-9, 2001).
Accordingly, because of the clinical significance of adiponectin, it is important to measure adiponectin blood concentration for diagnosis and prognosis of obesity and Type II diabetes, and to develop a treating agent. However, because of immunological tolerance due to a high amino acid similarity between human and mouse adiponectin protein, it is very difficult to secure a monoclonal antibody having high specificity and sensitivity, and until now, a detection method that can be clinically used has not been developed.
SUMMARY OF THE INVENTION It is an object of the present invention to provide a monoclonal antibody that recognizes adiponectin, its preparation method, and its use. It is another object of the present invention to provide a hybridoma cell that produces a monoclonal antibody that recognizes adiponectin.
It is another object of the present invention to provide a kit for quantifying adiponectin in a sample, comprising an immunoassay kit using a monoclonal antibody that recognizes adiponectin. It is another object of the present invention to provide a method for quantification of adiponectin in a sample using a monoclonal antibody that recognizes human adiponectin.
It is another object of the present invention to provide a method or kit for diagnosing obesity or Type II diabetes using a monoclonal antibody that recognizes adiponectin.
It is another object of the present invention to provide a method and kit for screening a treating agent for obesity or Type II diabetes using a monoclonal antibody that recognizes adiponectin.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a structure of adiponectin. Fig. 2 compares amino acid sequences of human and mouse. Fig. 3 is an electrophoresis photograph showing Western Blot results of ADI773, ADI741 , and ADI943, which are monoclonal antibodies according to the present invention.
Fig. 4 shows the results of Western Blot of ADI773 and ADI741 , which are monoclonal antibodies that recognize a full length adiponectin, a globular domain of adiponectin, recombinant adiponectin without a globular domain, and human serum adiponectin.
Fig. 5 shows the results of Western Blot of ADI773 and ADI741 , which are monoclonal antibodies that recognize serum of various mammals including a human, a mouse, a rat, a cow, a horse, and a rabbit.
Fig. 6 is a graph showing binding sensitivity of ADI773, ADI741 , and ADI943, which are monoclonal antibodies according to the present invention, to human full length adiponectin, examined by ELISA.
Fig. 7 is a graph showing binding sensitivity of ADI773, ADI741 , and ADI943, which are monoclonal antibodies according to the present invention, to a globular domain of human adiponectin, examined by ELISA.
Fig. 8 is an electrophoresis photograph showing the results of Western Blot of adiponectin existing in plasma of a normal person (lanes 1 , 2, 3) and a Type II diabetic patient (lanes 4-13). Fig. 9 is a graph showing the analysis results of adiponectin in plasma of a normal person, examined by ELISA
Fig. 10a to Fig. 10c shows reaction conditions of serum pre- treatment for ELISA using the monoclonal antibody according to the present invention. Fig. 11 shows the results of Western Blot and ELISA of adiponectin in a sample of a Type II diabetic patient using ADI773 and ADI741. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to a monoclonal antibody that recognizes adiponectin, its preparation method, and its use. The monoclonal antibody according to the present invention includes an adiponectin antibody produced using human adiponectin or its fragment as an antigen, and which recognizes adiponectin or its fragment as an epitope. In the present invention, a monoclonal antibody that recognizes adiponectin can be prepared a hybhdoma cell produced by fusing a mouse cell expressing human adiponectin and a mouse myeloma cell.
In one embodiment of the present invention, a human adiponectin antigen can be prepared by amplifying cDNA separated from a human adipocyte with a specific primer through PCR, connecting the amplified product to an expression vector with an Fc domain of immunoglobulin, introducing it into a mammalian cell, and expressing it. The adiponectin antigen that can be used in the present invention includes a full length adiponectin and a fragment of adiponectin capable of producing a monoclonal antibody, and is preferably an amino acid 1-247 fragment of human adiponectin.
The present inventors, in order to solve the difficulty of production of human adiponectin due to immune tolerance, prepared an antigen fused with immunoglobulin, which can be effectively introduced into a mouse dendritic cell to activate the dendritic cell, thereby inducing a strong immune response. The expression vector that can be used to prepare an antigen includes all the expression vectors for mammals comprising a heavy chain Fc domain of human immunoglobulin, for example a constant region of lgG1.
The present invention, in order to increase the production of monoclonal antibodies, fuses the Fc domain of an immunoglobulin heavy chain with an adiponectin gene and expresses it in a mammalian cell, for example in an HEK-293 cell, to prepare an fusion antigen. Thus, the recombinant adiponectin of the present invention can retain similar structure of adiponectin in plasma, and has property to produce effective antibody. The antigen used in the present invention to prepare a monoclonal antibody that recognizes adiponectin may have different properties from that expressed in E.coli by Arita et al (Arita, et al, Biochem. Biophy. Res. Commun, vol259, pp. 79-83, 1999). The mammalian cell used to prepare an antigen of the present invention includes a mammalian cell expressing known foreign genes, for examples, a human embryonic kidney (HEK) cell 293, a 293T cell, an African green monkey kidney cell Cos 1 , a Cos 7 cell, a Chinese hamster ovary cell, CHO, etc. The monoclonal antibody according to the present invention is prepared using human adiponectin-Fc fusion protein as an antigen, and it recognizes a full length adiponectin and serum adiponectin. The monoclonal antibody includes a monoclonal antibody that recognizes only a globular domain as an epitope, and a monoclonal antibody that recognizes adiponectin excluding a globular domain, and preferably ADI773, ADI741 , and ADI943.
ADI773 and ADI741 recognize both plasma adiponectin and recombinant full length adiponectin, and they do not recognize an N-terminal variable region of adiponectin. ADI773 recognizes a recombinant adiponectin protein excluding a globular domain, and ADI741 recognizes a globular domain. Additionally, ADI741 recognizes adiponectin of mammals of various kinds such as a human, a mouse, a rat, a cow, a horse, a rabbit, a goat, etc., while ADI773 strongly binds to human adiponectin but slightly recognizes cow adiponectin. Therefore, ADI773 and ADI741 recognize different epitopes. ADI741 is useful for quantifying many kinds of adiponectin, and it can be particularly used for screening of medicines for diagnosis or treatment of obesity or related diseases, for example Type II diabetes. ADI773 is a monoclonal antibody raised from mice immunized with adiponectin-Fc and it can be identified by conducting Western Blot in human plasma. It does not react in rat or mouse adiponectin, and has an lgG1 /kappa isoform. The monoclonal antibodies do neither recognize a globular domain of human adiponectin nor the N-terminal variable region, hence its epitope could be collagenous domain.
ADI741 of the present invention recognizes only a globular domain of adiponectin. Since the nucleotide sequences of the globular head domains of humans and mice are almost identical, it may be difficult to produce an antibody that recognizes this region. Therefore, ADI741's recognition of such a globular domain means formation of an autoimmune antibody, as evidence of such, mouse adiponectin is much more strongly detected than human adiponectin. ADI741 can detect • a full length adiponectin in plasma, and it has an lgG1 /kappa isoform. The biding specificity of ADI943 of the present invention can be identified through ELISA, and it recognizes only a globular domain of human adiponectin.
The properties of ADI773, ADI741 , and ADI943 according to the present invention are summarized in Table 1. [Table 1]
Figure imgf000012_0001
The monoclonal antibodies that recognize human adiponectin of the present invention is prepared by substantially the same method as a common monoclonal antibody preparation method, except for the antigen used. Specifically, according to the present invention, a positive clone is identified among primarily prepared hybridoma cells, and hybridoma cells producing a monoclonal antibody are selected on the basis of recognition and properties of the antibody produced by each clone. The hybridoma cell producing a monoclonal antibody that recognizes adiponectin of the present invention is a fusion cell of a myeloma cell and a splenocyte producing an antibody that recognizes the adiponectin, and it is preferably a hybridoma cell line producing the monoclonal antibodies ADI773, ADI741 , and ADI943. The hybridoma cells producing these adiponectins were deposited on June 29, 2002, with the Bioengineering Research Institute GeneBank, located in Eoeundong 52, Yoosung-ku, Daejun Kwang-yuk City, South Korea, under KCTC 10288BP for a hybridoma cell producing an antibody ADI773, under KCTC 10289BP for a hybridoma cell producing ADI741 , and under KCTC 10290BP for a hybridoma cell producing ADI943.
In addition, the present invention provides an analysis method and kit for quantification or detection of adiponectin in a sample using the monoclonal antibody that recognizes adiponectin in an immunoassay or in a kit for immunoassay. All the immunoassays and kits for immunoassay known in the art, for example, ELISA and radioimmunoassay, etc. can be used in the present invention. The adiponectin is preferably attached with detecting means such as an enzyme, a radio element, etc. The sample can be blood or plasma or serum of mammals such as a human, a mouse, a rat, a cow, a horse, a rabbit, a goat, etc. The human includes a normal person, an obese person, or a Type II diabetic patient.
In a preferred embodiment of the present invention, a method is provided for quantification of adiponectin comprising the steps of adding a sample to a microtiter plate coated with a monoclonal antibody according to the present invention, reacting an enzyme-antibody complex, treating a substrate of the enzyme, measuring absorbance, and comparing the absorbance with a standard curve to quantify adiponectin in a sample. Biotin can be bound to the antibody of the enzyme-antibody complex. And, the enzyme can be HRP (horse radish peroxidase), but is not limited thereto, and all the enzymes and substrates used in ELISA can be used in the present invention.
An ELISA assay can detect and quantify an antigen existing in a sample using an antibody-antigen reaction. Two or three antibodies are required for applying an ELISA kit. For example, when monoclonal antibody against adiponectin is used for an ELISA kit, the Kit consists of 3 antibodies. The construction and principle for preparing the ELISA kit are as follows. First, an ADI741 monoclonal antibody (primary antibody) is coated on a 96-well plate, a sample comprising adiponectin is added to the well and reacted, a polyclonal antibody (secondary antibody) marked with an enzyme is reacted, a substrate capable of reacting with the marked enzyme is treated, and an enzyme-substrate reaction is then detected and compared with a standard curve to quantify adiponectin in the sample. In one example of the present invention, as the enzyme and substrate, biotin and streptavidin conjugated HRP can be used, but it is not limited thereto.
Preferably, before conducting an ELISA of a sample, a sample plasma can be pre-treated, which comprises the steps of diluting a sample and/or boiling the sample, wherein a dilution rate is 10-20 fold and boiling time is 1-10 minutes, preferably 4-6 minutes.
The present invention also provides a method or kit for diagnosing obesity or Type II diabetes, by quantifying adiponectin in plasma using the monoclonal antibody according to the present invention. Comparing concentrations of adiponectins in plasma of a normal person and a Type II diabetic patient, it can be seen that the plasma of a normal person includes adiponectin in a higher concentration (Figs. 8 and 9). Therefore, according to the present invention, obesity and Type II diabetes could be diagnosed by quantifying adiponectin in a sample. And, according to the present invention, variations in a living body of a subject, blood adiponectin distribution of a normal person and a diabetic patient, sensitivity and specificity of an adiponectin value that can distinguish a normal person from a diabetic patient, etc. can be examined.
The present invention will be explained with reference to the following Examples. However, these are only to illustrate the present invention and the present invention is not limited to them.
EXAMPLES Example 1 : Preparation of antigen
Human adiponectin DNA was amplified by 35 times using human adipocyte cDNA (Clonetech, Inc.) as a template and using a primer pair having a Hind III restriction enzyme recognition site at 5' terminal and a
BamH I recognition site at 3' terminal, wherein temperature and time were set as follows: PCR at 95 °C for 2 minutes, denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and chain extension at 72 °C for 1 minute (MJ Research, minicycler PTC150 model).
Forward direction primer: 5'-cccaagcttgggctctaaagattgtcagt-3' (Sequence No. 1) Reverse direction primer: 5'-gaaggatccggggttggtatcatggta-3'
(Sequence No. 2)
The amplified fragment was inserted in a pCEP4 mammal expression vector (Invitrogen, cat# VO44-50) comprising Fc (Fc domain of immunoglobulin) to prepare a recombinant vector, and an HEK 293-EBNA cell was transformed with the recombinant vector and then cultured in a
DMEM culture medium comprising the antibiotic G418.
The culture medium for the transformant was changed every 3 days, when the culture supernatant was collected with a pipet. A protein A affinity column (Amersham Bioscience, HiTrap rProteinA FF) was homogenized with DPBS of 10 times the amount at a speed of 1 ml/min, and then passed through the collected cell culture at a speed of 1 ml/min with an AKTA purifier (Amersham Bioscience). The collected cell culture was eluted with 100 mM glycine, pH 3.0 at a speed of 1 ml/min and neutralized with 2 M Tris, pH 8.0 to separate adiponectin-Fc fusion protein. The separated adiponectin-Fc fusion protein was used as an antigen for preparing an antibody in the subsequent process.
All the antibodies for producing ADI773, ADI741 , and ADI943 were prepared from the same antigen, which was produced by the same method according to this example.
Example 2: Preparation of hybridoma cell
2-1. Antibody production cell
Electrophoresis of the adiponectin-Fc fusion protein prepared in Example 1 was conducted on a gel containing PBS (phosphate-buffered saline) or through SDS-PAGE (polyacrylaminde gel electrophoresis), and the fragment of the gel was cut and an emulsion mixed with complete Freund's adjuvant of the same volume was prepared. The emulsion was injected into abdominal cavities of 5 6-8 week old BALB/C mice. 20-50 μg of antigen was injected into each mouse such that the total volume reached 200-400 μL The emulsion was injected twice at an interval of 2 weeks to induce antibody production, and an antigen dissolved in PBS was injected into the tail veins, 3 days before conducting cell fusion. In order to prevent contamination, all the mice were grown in the selected area. 2-2. Identification and selection of antibody production cell
Blood samples were taken from the eyes of mice immunized according to the above method, introduced into 1.5 ml centrifuge tubes to separate blood serum (centrifugation at 1800 rpm for 10 minutes), and stored at -20 °C until antibody production was tested. Antibody production was identified by ELISA using human full length adiponectin protein and the human adiponectin globular domain, and then fusion of the antibody production cells began.
First, in order to prepare adiponectin expressed in E.coli, full length (30 kDa) and globular domain (22kDa) of human adiponectin were respectively cloned in a bacteria protein expression vector (pET-21a) so that 6 histidine tags were connected at the C-terminal. E.coli BL21 strain was transformed with the cloned vector. The transformed strain was cultured in an LB medium to which 1 Mm of IPTG was added at 20 °C for 16 hours to induce desired protein expression. The transformed strain was treated with lysozyme at room temperature for 30 minutes, ultrasonic-treated, and centrifuged at 15,000 rpm at 4 °C for 30 minutes. The supernatant was passed through a Nikel column. The Nikel column was washed with a washing buffer solution (60mM imidazole, 500mM NaCI, 20Mm Tris-HCI), and then eluted with an eluting buffer solution (1 Mm imidazole, 500mM NaCI, 20Mm Tris-HCI). The purified protein was introduced into Slide-A- Lyzer dialysis cassettes (PIERCE, Cat. 66370) to dialyze in 2L PBS for 16 hours. The adiponectin expressed in E.coli was coated on a 96-well plate in an amount of 0.5 g/well and reacted overnight, and then washed with PBST (PBS buffer, 0.05% Tween 20) three times, and reaction was terminated with 2% BSA. The adiponectin was again washed with PBST three times, and treated with serum diluted to 1 :100-10,000 to react for 1 hour. Then, the adiponectin was washed with PBST three times and treated with anti-mouse IgG conjugate HRP to react for 1 hour. The adiponectin was again washed with PBST three times, treated with 100 μl of OPD (Sigma), and color reaction was conducted for 20 minutes. 50 μl of reaction termination solution (0.5 M sulfuric acid) was added thereto, and an O.D. value was obtained at 492 nm.
2-3 Preparation of hybridoma cell
After identifying antibody production, the mouse was killed to separate antibody production cells (splenocyte), which were fused with myeloma cells SP2, according to a modified method originated from Cesar Milstein and Georges Kohler's method (Method in enzymology, vol. 73, p3. Academic Press, New York). Specifically, an SP2 cell of the mouse was maintained in a culture plate until an optimum growth period began using a 10% FBS DMEM medium. One day before conducting cell fusion, the SP2 cell was diluted to 2-3 x 105 cell/ml, and 50 ml was taken the next day, which was cent fuged at 400g for 3 minutes. After washing with a serum-free medium twice, the concentration was controlled to 1-2 x 107 cell/ml. The mouse was killed by cervical dislocation to obtain its spleen, which was introduced into a mesh container to separate individual cells. The medium used was serum-free. The completely separated antibody production cell was centrifuged at 400g for 5 minutes, washed with a serum-free medium twice, and suspended in 10 ml of medium. The number of lymphocytes was calculated with a haemocytomer, and 108 lymphocytes were mixed with 2 x 107 SP2 cells (5: 1 ) to centrifuge at 400g for 3 minutes. 1 ml of PEG (Sigma) solution pre-treated at 37 °C was slowly dripped into the supernatant over 1 minute to mix them. The produced mixed solution was diluted with DMEM over 2ml/2 minutes and 5ml/min, centrifuged at 400g for 3 minutes, and then cells were suspended in 200ml of HAT selection medium. 200 μl of the suspension was sprayed into a 96-well plate coated with feeder cells (macrophage separated from abdominal cavity of the mouse using PBS) to culture in a 5% CO medium at 37 °C for 7-14 days. 2-4. Selection and separation of hybridoma producing each antibody
The culture supernatant was taken in 2-3 above, and positive clones for adiponectin were selected by ELISA of 2-2 above and then identified by Western Blot. Therein, among a number of hybridoma, those having high antibody activities for full length adiponectin were selected, among which those having high antibody activities for globular domain were selected to obtain 4 kinds of hybridoma cells.
The identified positive clones were diluted, the fusion cells were sprayed into a 96-well plate at 0.5 cell/well to culture for 7 days, and they were examined again by ELISA. According to antibody properties and recognition sites, the cells were divided into ADI773, ADI741 , and ADI943. The finally-examined fusion cells were transferred to a 24-well plate, and finally transferred to a T25 flask to separate and culture.
The hybridoma cells producing antibody recognizing adiponectin were deposited on June 29, 2002, with the Bioengineering Research Institute GeneBank, located in Eoeundong 52, Yoosung-ku, Taejun Kwang- yuk City, South Korea, under KCTC 10288BP for those producing ADI773, under KCTC 10289BP for those producing ADI741 , and under KCTC 10290BP for those producing ADI943. Example 3: Production and separation of antibody
Using the positive clones for each antibody obtained in Example 2, mouse abdominal culture liquids were prepared, from which antibodies were cloned. Preparation and separation of abdominal culture liquid were conducted by the same method, except that hybridoma cells differed for each antibody.
In order to obtain abdominal culture liquids (ascites) of the three kinds of antibodies classified in Example 2, 1 x 106 positive clone cells respectively producing three kinds of monoclonal antibodies identified by ELISA and Western Blot were injected into the abdominal cavity of BALB/C mice previously treated with prestin. After 2 to 3 weeks, ascites were extracted with 3ml of PBS. The three kinds of monoclonal antibodies, ADI773, ADI743, and ADI943 were prepared by the same method, except that in the final examination step antibodies were divided into four kinds. In order to prepare each antibody, ascites were independently obtained, and from the ascites comprising each antibody, antibody separation was conducted by the following procedure.
The obtained ascites were purified through a protein A affinity column (Amersham Bioscience, HuTrap rProtein A FF). Specifically, the protein A column was homogenized with DPBS at 10 times the volume at a speed of 1 ml/min, and then passed through the ascites at a speed of 1 ml/min with an AKRA purifier (Amersham Bioscience). The ascites were eluted with 100mM glycine, pH 3.0 at a speed of 1 ml/min, and then neutralized with 1/10 the amount of 2M Tris, pH 8.0 to purify and separate each antibody.
Example 4: ELISA for classification of antibody isoforms
In order to classify isoforms of the ADI773, ADI741 , and ADI943 antibodies prepared in Example 3, an ELISA kit purchased from Pierce Inc. was used. Specifically, 100 ng of adiponectin was coated on a 96-well plate, and blocked with 1mg/ml BSA. Four kinds of antibodies were diluted to 100 pg~ 1 βg, and OPD color reaction was conducted using an HRP-conjugated antibody. Example 5: Specificity of binding domain of monoclonal antibody
For each antibody of ADI73, ADI741 , and ADI943 separated in Example 3, ELISA and Western Blot were conducted by the same method as in Example 2, and immuno-precipitation was additionally conducted.
5-1. Antibody binding specificity for human full length adiponectin and globular domain
For human plasma (40 tg) adiponectin and human adiponectin recombinant globular domain (5 ng), binding specificity of each antibody was examined, and the results are shown in Fig. 3. Specifically, 20ml of human blood was taken, and centrifuged at 1600 rpm for 10 minutes to separate supernatant, i.e., plasma. Plasma protein was quantified, and each 40 g of protein was electrophoresed on 12% PAGE (first three lanes excluding a marker), and the serum protein was transferred to a nitrocellulose membrane and left overnight. The membrane was blocked with 5% skim milk, and washed with PBST twice. The membrane was treated with ADI773, ADI741 , and ADI943, and then cultured for 1 hour, washed with PBST, treated with mouse anti-lgG-HRP diluted to 1 :5000, and cultured for 40 minutes. 1 ml of ECL reagent (Pierce Inc.) was uniformly sprayed into the membrane, and let stand for 1 min. Then, the ECL reagent was completely removed, and color reaction to adiponectin was identified by an ECL detector (Kodak). Additionally, the globular domain of a recombinant protein was electrophoresed under the same conditions, and reaction specificity was identified by the same method (Fig. 3 lanes 4-6). As shown in Fig. 3, ADI773 can recognize only full length human adiponectin, and as a result of Western Blot, it is shown that it recognizes human full length adiponectin in plasma and serum.
ADI 1943 could not be identified by Western Blot, and it can be identified by ELISA. As a result, it can recognize only the globular domain of human adiponectin. Therefore, it can also be used in the ELISA kit.
ADI 741 recognizes only the globular domain of human adiponectin. It is very difficult to prepare an antibody that recognizes only the globular domain due to sequence homology of the globular domain, and the antibody is likely to be an autoimmune antibody. It was identified that the antibody can detect full length human adiponectin, but it binds to mouse adiponectin very strongly.
5-2 Recognition of ADI741 and ADI773
1 βl of human serum, a recombinant adiponectin protein (ALEXIS Biochemicals) without a globular domain, a globular domain (prepared by the method of Example 2-2), and full length adiponectin (prepared by the method of Example 2-2) were performed by electrophoresis. Western Blot was conducted using ADI741 and ADI773 monoclonal antibodies. The results are shown in Fig. 4.
The serum protein and full length adiponectin were recognized by
ADI773 and ADI741. However, ADI741 recognized only the globular domain of adiponectin, and ADI773 recognized a recombinant adiponectin protein without a globular domain. Therefore, it can be seen that ADI741 and ADI773 are monoclonal antibodies that recognize different epitopes.
5-3 Recognition of ADI741 for adiponectin globular domain of various animals.
1 βl of each of human, rat, mouse, cow, horse, and rabbit serum were electrophoresed, then Western Blot was conducted using ADI773 and ADI741. The results are shown in Fig. 5.
ADI773 strongly recognized human adiponectin and weakly recognized cow adiponectin, but ADI741 recognized all kinds of adiponectin, indicating that globular domains of these animals have significant amounts of stored amino acid. 5-4 Recognition of N-terminal
In order to confirm the adiponectin recognition region of ADI773 and ADI741 , N-terminal variable regions of human, mouse, and rat adiponectin were synthesized with peptide, and a control peptide (15aa-peptide) was prepared.
Human: DQETTTQGPGVLLPLPKGAC (Sequence No. 3) RAT: EGITATEGPGALVPPPKETC (Sequence No. 4) Mouse: DVTTTEELAPALVPPPKGTC (Sequence No. 5) 15aa: CLEENHLQDEGVCSL (Sequence No. 6)
In order to conduct ELISA, peptide e was diluted in a carbonate buffer solution (pH 9.6), coated on a 96-well plate in an amount of 0.5 g/well, and then reacted overnight. After washing with PBST (PBS buffer, 0.05% Tween 20) three times, reaction was stopped with 1 % BSA. After again washing with PBST three times, the reactant was treated with ADI773 and ADI 741 monoclonal antibodies diluted with 1 % BSA-PBST buffer solution to 500-fold, and then reacted for 1 hour. After washing with PBST three times, the reactants were treated with anti-mouse IgG conjugate HRP and reacted for 1 hour. After washing with PBST three times, the reactant was treated with hydrogen peroxide and 100μ of OPD (Sigma), and then color reaction was conducted for 20 minutes. After adding 15 C of reaction terminating solution (8N sulfuric acid), O.D. value was obtained at 492 nm (Table 2).
[Table 2]
Figure imgf000026_0001
As shown in the above Table, ADI773 and ADI 741 did not recognize the adiponectin N-terminal variable region. Therefore, it can be seen that ADI773 recognizes the collagenous domain and ADI 741 recognizes the globular domain.
5-5 Binding specificity to recombinant full length adiponectin according to antibody concentration.
An experiment was conducted using human full length adiponectin expressed in E.coli, by the same method as ELISA of Example 5-1 , and the results are shown in Fig. 6. Fig. 6 is a graph showing binding specificity to full length adiponectin according to concentrations of ADI 773, ADI 741 , and ADI 949. As shown in Fig. 6, ADI 773 and ADI 741 bind to recombinant full length adiponectin.
Binding specificity to recombinant globular domain of adiponectin according to antibody concentration The experiment was conducted by the same method as ELISA of
Example 5-1 , and the results are shown in Fig. 7.
Fig. 7 is a graph showing binding specificity to the adiponectin globular domain of ADI773, ADI741 , and ADI 949 according to antibody concentrations. As shown in Fig. 7, compared to ADI773, AD741 more specifically binds to the adiponectin globular domain hence can be detected, which indicates that the ADI773 epitope is located in a variable region or collagen-like domain of adiponectin.
Example 6: Sandwich ELISA of adiponectin in clinical blood
ADI773 antibody was used in the same way as the recombinant adiponectin in the above. As a marked antibody, a biotin-bound polyclonal antibody was used, and color reaction was conducted using peroxidase
(originated from Western mustard, Sigma, hereinafter referred to HRP)- bound streptavidin.
6-1 Preparation of biotin-marked antibody In order to prepare a polyclonal antibody, full length adiponectin was inserted into a pET-21a vector (Novagen) by genetic recombination, and then transferred to E.coli. IPTG was added thereto to induce expression, and then adiponectin was expressed at 20 °C . The expressed adiponectin was separated and purified using a His-taq column. 50 μg of purified adiponectin was mixed with Freund's complete adjuvant in a ratio of 1 :1 to make a suspension, and then injected hypodermically into the back of a rabbit in an amount of 5-7 μL 50j«g of adiponectin was mixed with Freund's incomplete adjuvant in a ratio of 1 :1 , and injected by the same method as above twice with 2 weeks therebetween to amplify antibody production. After 6 weeks, the rabbit was killed and blood was taken from its heart. The blood was centrifuged to separate serum, which was passed through a protein-A column to purify the polyclonal antibody. In order to bind a biotin mark to the polyclonal antibody, adiponectin purified in 1mg/1ml of protein-A-column and 4.5μl of biotin-7-NHS solution dissolved in DMSO were mixed to react at 20 °C for 2 hours. The mixed solution of polyclonal antibody and biotin-7-NHS was introduced into a Sephadex G25 column saturated with 3.5 ml of PBS solution, 3.5 ml of PBS was added to fractionate every 0.5 ml, and it was eluted to receive the fractions in an Eppendorf tube. The received polyclonal antibody and biotin were read using an O.D. measuring instrument at 280 nm to measure the eluted amount.
6-2 Assay In order to conduct ELISA (step 2), an ADI773 monoclonal antibody
(1 mg/ml) was diluted in PBS to 5 g/ml to spray on a 96-well QOμl at one time (finally 0.5 g/well), and reacted overnight. In order to prepare a human serum sample, the following two steps were conducted. First, for p re-treatment, the serum was diluted 10-fold in PBS buffer solution (pre- treatment solution) comprising 2% SDS, 1% BSA, and 0.01% NaN3, and then boiled for 5 minutes. The pre-treated serum was finally diluted to 1 :100-5000 in PBS (serum dilution solution) comprising 1 % BSA, 0.01 % NaN3, and 0.05% tween20, 100 β of the diluent was introduced into a 96- well plate, and reacted in an incubator of 37 °C for 1 hour. ADI773 was coated on the 96-well plate in an amount of 0.5 g/well, and reacted overnight. The plate was then treated with 100 β of serum sample and reacted for 1 hour. In order to measure enzyme activity, the plate was washed with
PBST (PBS buffer, 0.05% Tween 20) three times, and the reaction was stopped with 2% BSA. After again washing with PBST three times, the reactant was treated with a biotin-marked polyclonal antibody diluted to 1 :5000 and reacted for 1 hour. After washing with PBST three times, the reactant was treated with streptavidin conjugate HRP and reacted for 1 hour. After washing with PBST three times, the reactant was treated with 100 ϊ OPD (Sigma) and color reaction was conducted for 20 minutes. After adding 50 μl of reaction-stopping solution (0.5M sulfuric acid), the enzyme activity value was obtained at 492 nm. 6-3 Standard curve
In order to quantify adiponectin in blood through O.D. obtained from ELISA, a standard curve relating concentrations and O.D. was obtained by sequentially diluting recombinant adiponectin and conducting ELISA to obtain a linear equation for the standard curve. Therefore, an O.D. value obtained from adiponectin in a sample can be substituted in the equation to quantify adiponectin in the sample.
As shown in Fig. 9, ELISA indicates that ADI773 can recognize adiponectin in blood.
Example 7: Sample pre-treatment for ELISA
In order to optimize ELISA results, a dilution amount suitable for pre- treatment and boiling time was optimized. The experiment was designed so that ELISA could be applied to ADI741. Because adiponectin in human plasma consists of multiple bodies, it should be changed to a monomer for recognition of epitopes. For this, serum was diluted in a pre-treatment solution (30mM Tris-HCI (pH 6.8) + 2% SDS + 1 Mm EDTA) and boiled.
50, 20, 10, 5, 2, and 1 βl of human serum were respectively taken and diluted in 50 (1 :2 dilution), 80 (1 :5 dilution), 90 (1 :10 dilution), 95 (1 :20 dilution), 98 (1:50 dilution), and 99 μl (1 :100 dilution) of pre-treatment buffer solution, and then boiled for 5 minutes. In addition, 100/iϋ of a sample diluted to 1 :500 was reacted with coated ADI741 and washed three times. The biotinylated human adiponectin polyclonal antibody prepared in Example 6-1 was added and reacted for 1 hour. After washing three times, streptavidin-HRP and an OPD substrate were introduced and reacted to determine O.D. at 492 nm, and the results are shown in Fig. 10a. As shown in Fig. 10a, results at 10-20 fold are optimum, so the following experiment was conducted with a 10-fold dilution. In order to determine boiling time suitable .for pre-treatment of plasma, plasma was diluted 10-fold in a sample buffer, reacted at 100°C in a heating block for 0.5, 1 , 3, 5, and 10 minutes to boil, and finally diluted to
500-fold, then 100 (! thereof was used for ELISA. Fig. 10b shows the results.
In order to examine the optimum conditions, human serum was diluted to 2, 5, 10, and 20-fold, boiled for 5 minutes, and then electrophoresed on a native gel to conduct Western Blot thereby identifying monomer production. The results are shown in Fig. 10c. As shown in Fig. 10c, since the lane 5 is a sample without pre-treatment, most adiponectin cannot move and thus it is hung in a well of the gel, and the lane 1 shows little monomer production at a 1 :2 dilution, at which it is highly likely that serum protein and adiponectin are coagulated to be lost during centrifugation.
• Example 8: Detection of adiponectin in a sample of a Type II diabetic patient using ADI773, ADI741
8-1 Western Blot using ADI 773 or ADI 741
1 βl of plasma of each of 3 normal persons and 6 Type II diabetic patients was electrophoresed, adiponectin was detected using ADI773 and ADI741 antibodies, and the results are shown in Fig. 11 (A). It can be seen that the adiponectin level in the Type II diabetic patients was decreased, and that ADI 773 / ADI 741 can be used in Western Blot to qualitatively measure adiponectin concentration.
8-2 Sandwich ELISA using ADI 741 as a capture antibody 10 βl of plasma of each of 3 normal persons and 6 Type II diabetic patients was added to a microtiter plate coated with ADI 741 , ELISA was conducted, then adiponectin concentration per 1 ml of plasma was measured, and the results are shown in Fig. 11 (B). It can be seen that Sandwich ELISA using ADI 741 enables measuring blood adiponectin concentration and that the results correspond to the qualitative results of 8-1.
The present invention relates to a monoclonal antibody that recognizes adiponectin, a preparation thereof, and a method and kit for quantification of adiponectin using the same. The monoclonal antibody that recognizes adiponectin according to the present invention can be used to measure blood adiponectin concentration, and thus can be used in a kit for diagnosis of obesity and diabetes.
BUDAPEST TTOΪΛTY OM TOE )M"j'l:.lVW/,T!ON. , RE ittlJlTlON 01" TUB DIΪ CώlT OF MOOOrfCΛ ISMS TOD f ME PLUMOSE OP
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Address'. Korea Research Institute of Bioscience and 35oter.'.J!'mo)og;y VD. *> (ETO B) u ^ j
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IDEN πLICA Jαw OF THE MICKQC GANRM
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II. SCIENTIFIC DESCRIPTION ANDβR PROPOSED TAJΪONO IC DESIGNATION
Tilt! iTjie.roorganis.ri'i identifier] un er I above; was accompanied by:
[ x. ] a scientific description
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Hi. RECEIPT AND ACCEPTANCE
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IV. RECEIPT OF REQUEST FOR COJl VEJftSJlOH
The. m roorβaπi rø identified under' ϊ above WUIΪ; rtctiviad by tlύr ϊitenιai:ion&) Depositary Authority on und j.ι request to convert th oriβ'imύ dtpcαii. to a deposit under the Budapest Treaty wins received y it on
V . INTERNATIONAL DEPOSITARY ΛϋTBDWTΪ
Name: Korea n Colteβiinn for Type Cυliϋi'et Signature^,) o:f person (a) having tbt power |;o represent the International Depositary Authority of ifbύriKed officiϋiKε):
Address: Korea Research Institute of Bioscience an Biotecbno.lo o (KRIBB) ' β, Ourrdong, Yυsαnir'ir.u, Taejon 3U&-S33, BAJE, yunrj Sook, Director Republic of Korea Date: jtme 292002
Form )-'M OCTC Form 57)

Claims

WHAT IS CLAIMED IS:
1. A monoclonal antibody which is produced using adiponectin or its fragment linked to immunoglobulin Fc as a fusion antigen, and which binds to full length adiponectin.
2. The monoclonal antibody according to claim 1 , wherein the immunoglobulin Fc is human lgG1 Fc.
3. The monoclonal antibody according to claim 1 , wherein the monoclonal antibody does not recognize a variable region of the N-terminal of human adiponectin.
4. The monoclonal antibody according to claim 3, wherein the monoclonal antibody recognizes a globular domain of human adiponectin as an epitope.
5. The monoclonal antibody according to claim 4, wherein the monoclonal antibody recognizes plasma adiponectin of a mammal.
6. The monoclonal antibody according to claim 5, wherein the mammal is human, a mouse, a rat, a cow, a horse, a rabbit, or a goat.
7. The monoclonal antibody according to claim 5, wherein the monoclonal antibody is produced by hybridoma deposited under deposition number of KCTC 10289BP.
8. The monoclonal antibody according to claim 3, wherein the monoclonal antibody recognizes a collagenous domain of human adiponectin as an epitope.
9. The monoclonal antibody according to claim 8, wherein the monoclonal antibody is produced by hybridoma deposited under the deposition number of KCTC 10288BP.
10. The monoclonal antibody according to claim 2, wherein the monoclonal antibody is produced by hybridoma deposited under the deposition number of KCTC 10290BP.
1 1. A hybridoma cell which produces the monoclonal antibody according to claim 1 that is produced using adiponectin or it fragment fused with immunoglobulin Fc as an antigen, and which binds to full length adiponectin.
12. The hybridoma cell according to claim 11 , wherein the hybridoma cell is one deposited under the deposition number of KCTC 10288BP, KCTC 10289BP, or KCTC 10290BP.
13. A kit for detection or quantification of adiponectin in a sample, which comprises the monoclonal antibody according to any one of claims 1 to 10.
14. The kit for detection or quantification of adiponectin according to claim 13, wherein the monoclonal antibody is attached to a detection means.
15. The kit for detection or quantification of adiponectin according to claim 13, wherein the sample is blood of a Type II diabetic patient or an obese person.
16. A method for quantifying adiponectin in a sample, which comprises the steps of: a) introducing a sample into a microtiter plate to which the monoclonal antibody recognizing adiponectin according to any one of claims 1 to 10 is attached, to react an enzyme-antibody complex; b) treating the sample with a substrate of the enzyme to measure absorbance thereof; and c) comparing the measured absorbance with a standard curve to quantify adiponectin existing in the sample.
17. The method for quantifying according to claim 16, which further comprises the pre-treatment step of diluting a sample and boiling it, before the step a).
18. A kit for diagnosis of obesity or Type II diabetes, which quantifies adiponectin in plasma using the monoclonal antibody that recognizes adiponectin according to any one of claims 1 to 10.
19. The kit for diagnosis according to claim 18, wherein the monoclonal antibody is attached with a detection means.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1744160A2 (en) * 2004-03-31 2007-01-17 Kazuhisa Maeda Method of invasiveness control and use of adiponectin
WO2007120311A3 (en) * 2005-12-07 2008-01-31 Siemens Medical Solutions Diag Detection of soluble adiponectin receptor peptides and use in diagnostics and therapeutics
WO2008084003A1 (en) * 2007-01-08 2008-07-17 F. Hoffmann-La Roche Ag Adiponectin antibodies and methods to measure adiponectin
US8017573B2 (en) 2007-11-30 2011-09-13 Siemens Healthcare Diagnostics Inc. Adiponectin receptor fragments and methods of use
US8865648B2 (en) 2009-04-23 2014-10-21 Siemens Healthcare Diagnostics Inc. Monomeric and dimeric forms of adiponectin receptor fragments and methods of use
CN112574306A (en) * 2020-12-17 2021-03-30 武汉华美生物工程有限公司 Adiponectin monoclonal antibody, antibody pair, preparation method and application thereof
WO2021222189A1 (en) * 2017-08-30 2021-11-04 First Fruits Business Ministry, Llc Method for increasing serum adiponectin for treatment and reduction of the risk of cardiovascular, vascular, and related diseases
CN116396384A (en) * 2023-01-17 2023-07-07 武汉爱博泰克生物科技有限公司 Rabbit monoclonal antibody aiming at mouse adiponectin, application of rabbit monoclonal antibody and double-antibody sandwich method ELISA (enzyme-linked immunosorbent assay) kit

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1002865A1 (en) * 1998-10-30 2000-05-24 Sanofi-Synthelabo Adipose specific protein
WO2002061076A1 (en) * 2001-02-01 2002-08-08 Mochida Pharmaceutical Co., Ltd. Adiponectin-associated protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1002865A1 (en) * 1998-10-30 2000-05-24 Sanofi-Synthelabo Adipose specific protein
WO2002061076A1 (en) * 2001-02-01 2002-08-08 Mochida Pharmaceutical Co., Ltd. Adiponectin-associated protein

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ARITA Y. ET AL.: "Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 257, 1999, pages 79 - 83, XP000867719, DOI: doi:10.1006/bbrc.1999.0255 *
HOTTA K. ET AL.: "Plasma concentration of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients", ARTERIOSCLER. THROMB. VASC. BIOL., vol. 20, 2000, pages 1595 - 1599, XP002952714 *
MAEDA K. ET AL.: "cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1)", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 221, 1996, pages 286 - 289, XP002922055, DOI: doi:10.1006/bbrc.1996.0587 *

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US9341634B2 (en) 2004-03-31 2016-05-17 Kazuhisa Maeda Method for controlling post-operative sepsis infection by administering adiponectin
EP1744160A4 (en) * 2004-03-31 2008-05-21 Kazuhisa Maeda Method of invasiveness control and use of adiponectin
EP1744160A2 (en) * 2004-03-31 2007-01-17 Kazuhisa Maeda Method of invasiveness control and use of adiponectin
WO2007120311A3 (en) * 2005-12-07 2008-01-31 Siemens Medical Solutions Diag Detection of soluble adiponectin receptor peptides and use in diagnostics and therapeutics
US8093017B2 (en) 2005-12-07 2012-01-10 Siemens Heathcare Diagnostics Inc. Detection of soluble adiponectin receptor peptides and use in diagnostics and therapeutics
NO345378B1 (en) * 2005-12-07 2021-01-11 Siemens Healthcare Diagnostics Inc Detection of soluble adisponectin receptor peptides and use in diagnostics and therapy
US8632990B2 (en) 2005-12-07 2014-01-21 Siemens Healthcare Diagnostics Inc. Detection of soluble adiponectin receptor peptides and use in diagnosis and therapeutics
US8846346B2 (en) 2005-12-07 2014-09-30 Siemens Healthcare Diagnostics Inc. Detection of soluble adiponectin receptor peptides and use in diagnosis and therapeutics
WO2008084003A1 (en) * 2007-01-08 2008-07-17 F. Hoffmann-La Roche Ag Adiponectin antibodies and methods to measure adiponectin
JP2010515668A (en) * 2007-01-08 2010-05-13 エフ．ホフマン−ラ ロシュ アーゲー Adiponectin antibody and method for measuring adiponectin
US8026345B2 (en) 2007-01-08 2011-09-27 Hoffmann-La Roche Inc. Characterization and identification of unique human adiponectin isoforms and antibodies
US8017573B2 (en) 2007-11-30 2011-09-13 Siemens Healthcare Diagnostics Inc. Adiponectin receptor fragments and methods of use
US8377889B2 (en) 2007-11-30 2013-02-19 Siemens Healthcare Diagnostics Inc. Adiponectin receptor fragments and methods of use
US8865648B2 (en) 2009-04-23 2014-10-21 Siemens Healthcare Diagnostics Inc. Monomeric and dimeric forms of adiponectin receptor fragments and methods of use
WO2021222189A1 (en) * 2017-08-30 2021-11-04 First Fruits Business Ministry, Llc Method for increasing serum adiponectin for treatment and reduction of the risk of cardiovascular, vascular, and related diseases
US11712427B2 (en) 2017-08-30 2023-08-01 First Fruits Business Ministry, Llc Method for increasing serum adiponectin for treatment and reduction of the risk of cardiovascular, vascular, and related diseases
CN112574306A (en) * 2020-12-17 2021-03-30 武汉华美生物工程有限公司 Adiponectin monoclonal antibody, antibody pair, preparation method and application thereof
CN112574306B (en) * 2020-12-17 2023-05-05 武汉华美生物工程有限公司 Adiponectin monoclonal antibody, antibody pair, preparation method and application thereof
CN116396384A (en) * 2023-01-17 2023-07-07 武汉爱博泰克生物科技有限公司 Rabbit monoclonal antibody aiming at mouse adiponectin, application of rabbit monoclonal antibody and double-antibody sandwich method ELISA (enzyme-linked immunosorbent assay) kit
CN116396384B (en) * 2023-01-17 2023-11-17 武汉爱博泰克生物科技有限公司 Rabbit monoclonal antibody aiming at mouse adiponectin, application of rabbit monoclonal antibody and double-antibody sandwich method ELISA (enzyme-linked immunosorbent assay) kit

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