WO2008035527A1 - ANTI-C1q MONOCLONAL ANTIBODY - Google Patents
ANTI-C1q MONOCLONAL ANTIBODY Download PDFInfo
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- WO2008035527A1 WO2008035527A1 PCT/JP2007/066200 JP2007066200W WO2008035527A1 WO 2008035527 A1 WO2008035527 A1 WO 2008035527A1 JP 2007066200 W JP2007066200 W JP 2007066200W WO 2008035527 A1 WO2008035527 A1 WO 2008035527A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
Definitions
- the present invention relates to an anti-Clq monoclonal antibody, a force, a method for determining the presence or amount of Clq using such an antibody, and a kit for the same.
- RA Rheumatoid arthritis
- Non-patent Document 1 It has been reported that the prognosis of whether or not the joint is destroyed can be made by the amount of Clq protein in the blood of RA patients (Non-patent Document 1). However, in the clinic, an anti-Clq antibody that can be used for the quantification of Clq protein in patient samples has not been established, and its development has been desired.
- Non-Patent Document 1 Ochi T et al., Arthritis Rheum. 1988 Jan; 31 (1): 37-43
- the problem to be solved by the present invention is to obtain an anti-Clq antibody that can be used for the quantification of Clq protein, a method for producing such an antibody, a specific and reproducible method for quantifying Clq protein, and a method therefor Is to develop a kit.
- the present invention provides:
- the antibody according to (1) which can specifically recognize a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7, (3)
- the antibody according to (1) or (2) which is produced by a hyperidoma having the patent biological deposit center accession number FERM BP-10649, AIST,
- a kit for determining the presence or amount of Clq protein in a sample comprising the antibody according to any one of (1) to (8) as an essential component;
- an anti-Clq monoclonal antibody that specifically recognizes Clq protein a method for producing the same, a hybridoma that produces an anti-Clq monoclonal antibody, and an anti-Clq monoclonal antibody that can be used for quantification of Clq protein are provided.
- Methods for determining the presence or amount of Clq protein in the sample used, as well as kits and the like are provided.
- Fig. 1 shows the results of ELISA using the culture supernatant of Hypridoma.
- FIG. 2 is a graph showing the results of sandwich ELISA using anti-Clq antibody # 8 as a solid phase antibody and anti-Clq antibody # 35 (square) as a sensitizing antibody.
- Figure 3 shows anti-Clq antibody # 33 as the solid phase antibody, anti-Clq antibody # 3 5 (square), anti-Clq antibody # 54 (circle), and anti-Clq antibody # 76 (diamond) as the sensitizing antibody. It is a graph which shows the result of the sandwich ELISA method using this.
- Figure 4 shows anti-Clq antibody # 35 as the solid phase antibody, anti-Clq antibody # 3 5 (square), anti-Clq antibody # 54 (circle), and anti-Clq antibody # 76 (diamond) as the sensitizing antibody. It is a graph which shows the result of the sandwich ELISA method using this.
- Figure 5 shows anti-Clq antibody # 40 as solid phase antibody and anti-Clq antibody # 5 as sensitizing antibody. 4 (circle) is a drape showing the results of sandwich ELISA using anti-Clq antibody # 76 (diamond).
- Figure 6 shows the results of sandwich ELISA using anti-Clq antibody # 54 as solid phase antibody, anti-Clq antibody # 3 5 (square) and anti-Clq antibody # 54 (circle) as sensitizing antibodies. It is a dull that shows.
- FIG. 7 is a graph showing the results of sandwich ELISA using anti-Clq antibody # 76 as a solid-phase antibody and anti-Clq antibody # 35 (square) as a sensitizing antibody.
- FIG. 8 shows the results of sandwich ELISA using anti-Clq antibody # 107 as a solid phase antibody, anti-Clq antibody # 33 (triangle) and anti-Clq antibody # 35 (square) as sensitizing antibodies. It is a graph.
- Figure 9 shows anti-Clq antibody # 112 as solid phase antibody, anti-Clq antibody # 35 (square), anti-Clq antibody # 54 (circle), and anti-Clq antibody # 76 (diamond) as sensitizing antibodies. It is a graph which shows the result of the used San-Germany ELISA method.
- FIG. 10 is a graph showing the results of sandwich ELISA using anti-Clq antibody # 119 as a solid phase antibody and anti-Clq antibody # 33 (triangle) as a sensitizing antibody.
- the present invention relates to an anti-Clq monoclonal antibody (hereinafter also referred to as “anti-Clq antibody”) capable of specifically recognizing Clq protein.
- the anti-Clq monoclonal antibody of the present invention increases the amount of antigen-antibody conjugate formed depending on the amount of protein that can bind specifically to Clq protein and the amount of protein, that is, Clq protein. It is excellent in that it can be used for quantitative determination.
- the antibody of the present invention can be obtained, for example, by immunizing a mammal such as a mouse with Clq protein, fusing a lymphocyte of the immunized animal with a myeloma cell line to produce a hyperidoma, and culturing this. That power S. Other known methods such as gene recombination and chemical synthesis may be used to produce such antibodies!
- the anti-Clq monoclonal antibody of the present invention is preferably (a) a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7, (b) SEQ ID NO: In the amino acid sequence of!
- a peptide having an amino acid sequence deleted, substituted or appended and (c) at least 50% or more of the amino acid sequence of SEQ ID NOs: 1 to 7, for example, 60, 70, 75, 80, It is possible to specifically recognize a peptide selected from the group consisting of peptides having an amino acid sequence having 85, 90, and 93% of the same age.
- the homology of amino acid sequences can be measured using, for example, FASTA, BLAST, DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.), GENETYX (manufactured by Genenetics Co., Ltd.).
- An anti-Clq monoclonal antibody may be obtained by immunizing an animal such as a mouse with the above peptide or a protein containing the peptide, and fusing a lymphocyte of the immunized mouse with a myeloma cell line to produce a hyperprideoma.
- the antibody of the present invention is a hybridoma of a mouse myeloma cell line and a mouse lymphocyte, such as Neubridoma KS— 0131 # 8, KS — 0131 # 33, KS — 0131 # 35, KS — 0131 # 40, KS— 0131 # 54, or KS— 0131 # 76.
- the Hypridoma was deposited at the Patent Organism Depositary Center (National Institute of Advanced Industrial Science and Technology) (Tsukuba Ito, 1-chome, 1-chome, 1-Chuo 6) and received on August 2, 2006.
- Such hypridoma can be prepared by using known culture conditions, for example, composition: RPMI 1640 (ohjin 16005005) 425 mL, L-Glut amine (SIGMA G7513) 5 mL, sodium pyruvate (SIGMA S8636) 5 mL, HAT supplement (GIBCO 21060-017) lOmL, penicillin-streptomycin (SIGMA P4 333) 2. 37 ⁇ 0.1 in medium of 5 mL, FBS 75 mL. C, cultured at 5 ⁇ 0.15% CO
- the anti-Clq monoclonal antibody can be produced by recovering and purifying the produced anti-Clq monoclonal antibody by a known means / method.
- the anti-Clq monoclonal antibodies produced by # 40, KS— 0131 # 54, and KS— 0131 # 76 are anti-Clq antibody # 8, anti-Clq antibody # 33, anti-tagged; ⁇ antibody # 35, anti-Clq antibody, respectively. Denote # 40, anti-Clq antibody # 54, and anti-Clq antibody # 76.
- the anti-Clq monoclonal antibodies of the present invention include fragments thereof, modified antibodies such as chimeric antibodies and humanized antibodies, and mutant antibodies. These fragments, modified antibodies or mutant antibodies also have the same specificity for Clq protein as the original antibody. These can be produced by means and methods known to those skilled in the art.
- the present invention provides a patent biological deposit center accession number FERM BP-10649, FERM BP-10650, FERM BP-10651, FERM BP 10652, FERM BP—
- the present invention relates to a hybridoma producing an anti-Clq monoclonal antibody having 10653 or FERM BP-10654.
- the hyperidoma of the present invention is a cell obtained by fusing a mouse lymphocyte immunized with Clq protein and a mouse myeloma cell line.
- the present invention relates to a method for producing an anti-Clq monoclonal antibody, wherein a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7 is used.
- a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7 and (b) one or more, preferably 1, in the amino acid sequence of SEQ ID NOs: 1 to 7 (Masame, Arrangement (, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 (A solid amino acid has been deleted, substituted or added)
- a peptide having an amino acid sequence or (c) having at least 50% or more homology with the amino acid IJ of SEQ ID NO:!-7, such as 60, 70, 75, 80, 85, 90, 93%
- a peptide having an amino acid sequence may be used.
- the production method of this aspect of the present invention may include a step of purifying an anti-Clq monoclonal antibody, etc., so as to immunize an animal such as a mouse with the peptide.
- Immunization with peptides and purification of anti-Clq monoclonal antibodies can be performed by methods known to those skilled in the art.
- the present invention relates to a method for producing an anti-Clq monoclonal antibody, characterized by culturing the hyperpridoma of the present invention.
- Hypridoma culture includes both in vitro and in vivo culture. In vitro culture is performed under known culture conditions, for example, composition: RPMI 1640 (ohjin 16005005) 425 mL, L-G1 utamine (SIGMA G7513) 5 mL, sodium pyruvate (SIGMA S8636) 5 mL, HA T supplement (GIBCO 21060-017 ) 10mL, penicillin 'streptomycin (SIGMA P4333) 2. 37 ⁇ 0.1 in medium of 5mL, FBS 75mL. C, 5 ⁇ 0. 15% CO
- Swelling power S In vivo culture is achieved, for example, by transplanting the hyperidoma of the present invention into the abdominal cavity of an animal such as a mouse.
- the production method of the present invention may further include a step of purifying the obtained antibody. Those skilled in the art can appropriately select and use the purification method.
- the present invention relates to a method for determining the presence or amount of Clq protein in a sample, characterized by using an anti-Clq monoclonal antibody of the present invention. Since the anti-Clq monoclonal antibody of the present invention has the property that the amount of the antigen-antibody conjugate increases depending on the amount of Clq protein, the presence of the Clq protein in the sample will reduce the force. Can be determined. Two or more anti-Clq monoclonal antibodies may be used in the determination method of the present invention. Using two or more anti-Clq monoclonal antibodies increases the sensitivity and specificity of determination and improves accuracy.
- the body 76 is used in the determination method of the present invention.
- the antibody used in the determination method of the present invention may be labeled. Various labels are known and can be appropriately selected and used by those skilled in the art.
- HRP horseradish peroxidase
- ALP enzymes such as Glueose oxidase ⁇ ⁇ -galactosidase, FITC, Rhodamine, Cy3, y5, Texas Red, Alexa Fluors, BODIPYs, IRDyes, MFPs, Quantum Dot, Light labels such as AMCA, Allophycocyanin, BMP, Cy2, Cy3.5, Cy5.5, DTAF, DyLight 547, DyLight 647, FluoroNano gold, Phycoerythrin, Phycocyanin, R_PE, Saporin, TRITC, Biotin, Digoxigenin (DIG), Acridium Ester, chemicals such Flashlight, 60 mm MicroBead of which beads, magnetic beads, such as MagCellect Ferrofluid [R], the radiation-labeled, such as 1 1, gold particles, labels such as Agarose used.
- the determination method of the present invention can be performed efficiently by attaching a force and a
- the sample may be any sample that may contain Clq protein. Examples include serum, joint fluid, cartilage, bone, and each tissue.
- the sample is incubated with the anti-Clq monoclonal antibody of the present invention to form a conjugate of the anti-Clq antibody and the Clq protein in the sample, and then the conjugate. May be carried out by measuring.
- ELISA methods such as sandwich ELISA, immunoblot method, radioimmunoassay (RIA) method, immunoprecipitation method, method using protein array, method using flow cytometer, chemiluminescent enzyme immunoassay (CLEIA), bioluminescent enzyme
- the determination method of the present invention is performed using immunoassay (BLEIA), measurement using a developing device (immunoassay test piece) that can be infused by capillary action, immunochromatography, immunostaining, aggregation, etc. can do.
- the ELISA method is preferably used because it does not require special equipment or the like, and the presence or amount of Clq protein can be determined quickly and easily in an actual medical field.
- the determination method of the present invention is used for the prognosis diagnosis of RA. .
- the present invention provides a method for diagnosing the prognosis of RA in a patient, comprising determining the presence or amount of Clq protein in a sample using the anti-Clq monoclonal antibody of the present invention. It is about.
- the presence or amount of Clq protein may be determined as described above! /.
- the present invention relates to a method for treating RA in a patient, which comprises administering the anti-Clq monoclonal antibody of the present invention.
- the present invention relates to a kit for determining the presence or amount of Clq protein in a sample, comprising the anti-Clq monoclonal antibody of the present invention as an essential component.
- Two or more anti-Clq monoclonal antibodies may be included in the kit of the present invention.
- the kit of the present invention may contain, for example, a sample collection means, a label, a reaction container, a determination reagent and the like.
- an instruction manual is attached to the kit.
- anti-Clq monochrome The final antibody may be labeled.
- kits of the present invention for example, ELISA methods such as sandwich ELISA method, Imnobutt method, Radioimmunoassay (RIA) method, immunoprecipitation method, method using protein array, flow cytometer , Chemiluminescent enzyme immunoassay (CLEIA), bioluminescent enzyme immunoassay (BLEIA), measuring method using expandable device (immunoassay specimen) that can be infused by capillary action, immunochromatography Alternatively, an immunostaining method, an aggregation method, or the like may be used. Since the kit of the present invention can be used to quickly and easily determine the presence or amount of Clq protein, it is possible to prognose RA patients in clinical settings. In addition, pathological studies of RA can be performed using the kit of the present invention.
- ELISA methods such as sandwich ELISA method, Imnobutt method, Radioimmunoassay (RIA) method, immunoprecipitation method, method using protein array, flow cytometer , Che
- mice Four female BALB / C mice were given Clq purified protein (purity 99% or higher, Calbiochem 20 4876) 0.1 mg with adjuvant (Titermax, SIGMA) 2-3 times every 2 weeks subcutaneously on the footpad and back And immunized. Lymphocytes were collected from the mouse spleen and cultured in a medium containing the antigen 30, ig for 3 days. After culture, lymphocytes and mouse myeloma cell line P3U1 cells were fused using PEG1500 according to a conventional method.
- the cells were composed of: R PMI 1640 (ohjin 16005005) 425 mL, L-Glutamine (SIGMA G7513) 5 mL, sodium pyruvate (SIGMA S8636) 5 mL, HAT supplement (GIBCO 21060-017) lOmL, penicillin streptomycin (SIGMA P4333 2. Suspended in 5 mL, 75 mL FBS medium and dispensed into 96 well plates previously seeded with mouse thymocytes. After 8 days, the presence of antibody in the supernatant was screened by ELISA. The procedure for ELISA is as follows.
- a solution of 1 ag / ml Clq in PBS was prepared, added to an ELISA microplate (3912 Falcon), and allowed to bind by incubation at room temperature for 2 hours. Next, blocking was performed using 1% skim milk / PBS to prepare an antigen plate. The culture supernatant was added to the antigen plate for reaction, and after washing, ALP-labeled goat anti-IgG (Zymet) was bound. Add ALP substrate, allow enzyme reaction, and absorb at 492 nm The light intensity was measured and antibody positive wells were selected. As a control, a polyclonal antibody isolated from the serum of a similarly immunized rabbit was used. The results are shown in Figure 1.
- the amino acid sequences of human Clq subunits A chain, B chain, and C chain are shown in SEQ ID NOs: 17 to 19, and the nucleotide sequences are shown in SEQ ID NOs: 20 to 22, respectively.
- a sequence obtained by shifting the amino acid sequence of each subunit by 15 amino acids (residues) at intervals of 3 amino acids is a synthetic peptide (in order, peptide numbers;! -78, 97-; 117, 193-270) on a glass array.
- Each peptide was synthesized at a specific position on the array, and a peptide array containing a synthetic peptide covering the entire amino acid sequence of the C1 q subunit was prepared.
- the array was manufactured by JPT.
- # 8, # 33, # 40, # 54, and # 76 were purified using a Protein G column.
- the purified antibody was diluted 1,000 times in PBS (10 mM phosphate buffer pH 7.0, 0.1 M NaCl) 330,11 on the peptide array, sealed, and sealed at 4 ° C. Incubated for 12 hours. Thereafter, it was washed once with methanol and then with 0 water for 5 minutes 5 times. Centrifuge to dry the array slide, scan with a fluorescence scanner (Agilent DNA microarray scanner; Agilent), and use software (Feature Extraction software; Agilent) to determine the fluorescence intensity of each peptide spot on the array. Turned into. Several spots showing strong fluorescence intensity were detected.
- peptide spots that are 1,300 or more higher than the background level! / And show fluorescence intensity are shown in Table 1 (amino acid sequences of peptide numbers 149, 150, and 244 to 246 are shown in SEQ ID NOs: 3 to 7, respectively) ).
- the peptides 149 and 150 are sequences derived from the ClqB subunit (B chain), and the peptides 24 to 246 are sequences derived from the ClqC subunit (C chain). From these sequences, the amino acid sequence of Clq epitope was predicted to contain 9 residues or a part of CKVPGLYYF (SEQ ID NO: 2).
- CKVPGLYY (SEQ ID NO: 23), KVPGLYYF (SEQ ID NO: 24)), 7 amino acids (CKVPG LY (SEQ ID NO: 25), KVPGLYY (SEQ ID NO: 26), VPGLYYF (SEQ ID NO: 27)), 6 amino acids ( CKVPGL (SEQ ID NO: 28), KVPGLY (SEQ ID NO: 29), VPGLYY (SEQ ID NO: 30), PGLYYF (SEQ ID NO: 1)), 5 amino acids (CKVPG (SEQ ID NO: 31), KVPGL (SEQ ID NO: 32), VPGLY (SEQ ID NO: 32) 33), PGLYY (SEQ ID NO: 34), GLYYF ( (Column number 3 5)), 4 amino acids (CKVP (SEQ ID NO: 36), KVPG (SEQ ID NO: 37), VPGL (SEQ ID NO: 3 8), PGLY (SEQ ID NO: 39), GLYY (SEQ ID NO: 40),
- Glycellonore ( ⁇ ⁇ 2 ⁇ 2) was dissolved to prepare ZOO ⁇ g / ml and 50 g / ml human Clq protein solutions.
- the human Clq protein solution was spotted on a nitrocellulose membrane (Hybond C; manufactured by Amersham) having a size of 5 mm ⁇ 15 mm as shown in Table 3 and air-dried at room temperature for about 1 hour. Soak the nitrocellulose membrane in TBS, let it fit for about 5 minutes, and then use TBS (20 mM Tris-HC1 pH 7.5, 150 mM NaCl) containing 5% blocking agent (Amersham ECL blocking reagent; manufactured by GE Healthcare) at room temperature. Blocking was performed for 1 hour.
- the blocked nitrocellulose membrane was gently washed with TBS, immersed in a solution (201) containing each anti-Clq antibody and synthetic peptide, and allowed to react at room temperature for 1 hour.
- the nitrocellulose membrane was lightly washed once with TTBS (TBS added with Tween 20 at a final concentration of 0.05%), and then washed with a sufficient amount of TTBS for 10 minutes x 3 times with shaking. Binding detection was performed as follows.
- Nitrocellulose membrane with ALP coloring buffer (lOOmM NaCl, 5mM MgCl in Tri
- the plate was immersed in ALP color buffer 30 containing BCIP / NBT solution (Promega) and developed for 10 minutes at room temperature. When the stained image was obtained, The trocellulose membrane was immersed in a sufficient amount of distilled water to wash away the color developing buffer. After washing, the nitrocellulose membrane was air-dried and stained images were captured using an Epson digital scanner GT-8300UF.
- the nitrosenololose membrane was immersed in a mixed solution of equal amounts of A and B in Amersham ECL Advance Western Blotting Detection Kit (manufactured by GE healthcare) and allowed to react at room temperature for 1 minute. After that, the reaction solution was lightly shaken, the nitrocellulose membrane was sandwiched between plastic films, and the chemiluminescence on the membrane was detected by exposing the membrane to a bolloid film.
- a secondary antibody (avidinated ALP antibody) was added at 100 1 / well, and reacted at 4 ° C to room temperature;! ⁇ 2 hours. After washing 4 times with pure water, ALP substrate coloring solution was added to cause color development. Absorbance (OD) at 492 nm was measured with a microplate reader (reference wavelength: 660 nm). The results are shown in FIGS. A positive correlation was observed between Clq protein concentration and absorbance, and it was found that Clq protein in the sample could be quantified by sandwich ELISA using anti-Clq monoclonal antibody. Although not shown, it was quantifiable for Clq protein at concentrations of ⁇ 400 g / mL.
- an anti-Clq monoclonal antibody that can be used for quantification of Clq protein, a method for detecting and / or quantifying Clq protein using such antibody, and a kit therefor can be obtained. It is extremely useful in fields such as diagnostics and (RA pathological) research.
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Abstract
Description
明 細 書 Specification
抗 Clqモノクローナル抗体 Anti-Clq monoclonal antibody
技術分野 Technical field
[0001] 本発明は、抗 Clqモノクローナル抗体、力、かる抗体を用いた Clqの存在または量を 決定する方法、ならびにそのためのキットなどに関するものである。 [0001] The present invention relates to an anti-Clq monoclonal antibody, a force, a method for determining the presence or amount of Clq using such an antibody, and a kit for the same.
背景技術 Background art
[0002] 関節リウマチ (RA)患者は、将来重篤な関節破壊に陥る群と、そうでない群とに二 分される。 RA患者がいずれの群に属するかで必要な治療方法は異なる力 かかる 予後を有効に診断する方法はなレ、。 [0002] Rheumatoid arthritis (RA) patients are divided into two groups: those who will suffer severe joint destruction in the future and those who will not. Necessary treatment methods differ depending on which group RA patients belong to. There is no way to effectively diagnose the prognosis.
[0003] RA患者の血中 Clqタンパク質の量によって、関節破壊に陥るか否かの予後診断 が可能であることが報告されている（非特許文献 1)。し力もながら、臨床において、患 者試料中の Clqタンパク質の定量に用いることができる抗 Clq抗体は確立されてお らず、その開発が望まれていた。 [0003] It has been reported that the prognosis of whether or not the joint is destroyed can be made by the amount of Clq protein in the blood of RA patients (Non-patent Document 1). However, in the clinic, an anti-Clq antibody that can be used for the quantification of Clq protein in patient samples has not been established, and its development has been desired.
非特許文献 1 : Ochi T et al.， Arthritis Rheum. 1988 Jan; 31(1): 37-43 Non-Patent Document 1: Ochi T et al., Arthritis Rheum. 1988 Jan; 31 (1): 37-43
発明の開示 Disclosure of the invention
発明が解決しょうとする課題 Problems to be solved by the invention
[0004] 本発明の解決課題は、 Clqタンパク質の定量に用いることができる抗 Clq抗体を得 ること、かかる抗体の製造方法、特異的かつ再現性のある Clqタンパク質の定量方 法、ならびにそのためのキットを開発することである。 [0004] The problem to be solved by the present invention is to obtain an anti-Clq antibody that can be used for the quantification of Clq protein, a method for producing such an antibody, a specific and reproducible method for quantifying Clq protein, and a method therefor Is to develop a kit.
課題を解決するための手段 Means for solving the problem
[0005] 本発明者らは、上記事情に鑑み鋭意研究を重ねた結果、 Clqタンパク質量に依存 して抗原と抗体の結合体量が増大する抗 Clqモノクローナル抗体を産生するハイブ リドーマの作製に成功し、本発明を完成するに至った。 [0005] As a result of intensive studies in view of the above circumstances, the present inventors have succeeded in producing a hybridoma that produces an anti-Clq monoclonal antibody that increases the amount of antigen-antibody conjugate depending on the amount of Clq protein. Thus, the present invention has been completed.
[0006] すなわち、本発明は、 That is, the present invention provides:
(1) Clqタンパク質を特異的に認識することができる、抗 Clqモノクローナル抗体、 (1) an anti-Clq monoclonal antibody capable of specifically recognizing Clq protein,
(2)配列番号 1〜 7のアミノ酸配列を有するペプチドを特異的に認識することができる 、（1)記載の抗体、 (3)独立行政法人産業技術総合研究所 特許生物寄託センター受託番号 FERM BP— 10649を有するハイプリドーマにより産生される、（1)または（2)記載の抗体、(2) The antibody according to (1), which can specifically recognize a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7, (3) The antibody according to (1) or (2), which is produced by a hyperidoma having the patent biological deposit center accession number FERM BP-10649, AIST,
(4)独立行政法人産業技術総合研究所 特許生物寄託センター受託番号 FERM BP— 10650を有するハイプリドーマにより産生される、（1)または（2)記載の抗体、(4) The antibody according to (1) or (2), which is produced by a hyperidoma having the patent biological deposit center accession number FERM BP-10650, AIST,
(5)独立行政法人産業技術総合研究所 特許生物寄託センター受託番号 FERM BP— 10651を有するハイプリドーマにより産生される、（1)または（2)記載の抗体、(5) The antibody according to (1) or (2), which is produced by a hyperidoma having the patent biological deposit center accession number FERM BP-10651, the National Institute of Advanced Industrial Science and Technology,
(6)独立行政法人産業技術総合研究所 特許生物寄託センター受託番号 FERM BP— 10652を有するハイプリドーマにより産生される、（1)または（2)記載の抗体、(6) The antibody according to (1) or (2), which is produced by a hyperidoma having the patent biological deposit center accession number FERM BP-10652, AIST,
(7)独立行政法人産業技術総合研究所 特許生物寄託センター受託番号 FERM BP— 10653を有するハイプリドーマにより産生される、（1)または（2)記載の抗体、(7) The antibody according to (1) or (2), which is produced by a hyperidoma having the patent biological deposit center accession number FERM BP-10653, the National Institute of Advanced Industrial Science and Technology,
(8)独立行政法人産業技術総合研究所 特許生物寄託センター受託番号 FERM BP - 10654を有するハイプリドーマにより産生される、（ 1 )または（2)記載の抗体、(8) The antibody according to (1) or (2), which is produced by a hyperidoma having the patent biological deposit center accession number FERM BP-10654
(9)独立行政法人産業技術総合研究所 特許生物寄託センター受託番号 FERM BP— 10649、 FERM BP— 10650、 FERM BP— 10651、 FERM BP— 1065 2、 FERM BP— 10653、または FERM BP— 10654を有する、抗 Clqモノクロ一 ナル抗体を産生するハイブリドーマ、 (9) National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center Accession No. FERM BP—10649, FERM BP—10650, FERM BP—10651, FERM BP—1065 2, FERM BP—10653, or FERM BP—10654 A hybridoma producing an anti-Clq monoclonal antibody,
(10)配列番号 1〜7のアミノ酸配列を有するペプチドを用いることを特徴とする、抗 C lqモノクローナル抗体の製造方法、 (10) A method for producing an anti-Clq monoclonal antibody, comprising using a peptide having an amino acid sequence of SEQ ID NOs: 1 to 7,
(11) (9)記載のハイプリドーマを培養することを特徴とする、抗 Clqモノクローナル抗 体の製造方法、 (11) A method for producing an anti-Clq monoclonal antibody, comprising culturing the hyperpridoma according to (9),
(12) (1)〜（8)の!/、ずれか記載の抗体を用いることを特徴とする、試料中の Clqタン パク質の存在または量を決定する方法、 (12) A method for determining the presence or amount of Clq protein in a sample, characterized by using the antibody according to (1) to (8)!
(13) ELISA法によるものである、（12)記載の方法、 (13) The method according to (12), which is based on an ELISA method,
(14) (1)〜（8)のいずれか記載の抗体を 2種以上用いるものである、（12)または（1 3)記載の方法、 (14) The method according to (12) or (13), wherein two or more antibodies according to any one of (1) to (8) are used.
(15) (4)記載の抗体と（7)記載の抗体、（4)記載の抗体と（8)記載の抗体、（6)記載 の抗体と（7)記載の抗体、または（6)記載の抗体と（8)記載の抗体を用いるものであ る、（14)記載の方法、 (16)関節リウマチの予後診断のための、（12)から（15)のいずれか記載の方法、(15) The antibody according to (4) and the antibody according to (7), the antibody according to (4) and the antibody according to (8), the antibody according to (6) and the antibody according to (7), or the description according to (6) Using the antibody of (8) and the antibody of (8), (16) The method according to any one of (12) to (15), for prognosis of rheumatoid arthritis,
(17) (1)〜（8)のいずれか記載の抗体を必須構成成分として含む、試料中の Clqタ ンパク質の存在または量を決定するためのキット、 (17) A kit for determining the presence or amount of Clq protein in a sample, comprising the antibody according to any one of (1) to (8) as an essential component;
(18) ELISA法によるものである、（17)記載のキット、 (18) The kit according to (17), which is based on an ELISA method,
(19) (1)〜（8)のいずれか記載の抗体を 2種以上含むものである、（17)または（18) 記載のキット、 (19) The kit according to (17) or (18), comprising two or more antibodies according to any one of (1) to (8),
(20) (4)記載の抗体と（7)記載の抗体、（4)記載の抗体と（8)記載の抗体、（6)記載 の抗体と（7)記載の抗体、または（6)記載の抗体と（8)記載の抗体を含むものである 、（19)記載のキット、 (20) The antibody according to (4) and the antibody according to (7), the antibody according to (4) and the antibody according to (8), the antibody according to (6) and the antibody according to (7), or the description according to (6) A kit according to (19), comprising the antibody according to (8) and the antibody according to (8),
(21)関節リウマチの予後診断のための、（17)〜（20)の!/、ずれか記載のキット、 を提供するものである。 (21) A kit according to (17) to (20)! / Or any of the above, for prognosis of rheumatoid arthritis.
発明の効果 The invention's effect
[0007] 本発明により、 Clqタンパク質の定量に用いることができる、 Clqタンパク質を特異 的に認識する抗 Clqモノクローナル抗体およびその製造方法、抗 Clqモノクローナ ル抗体を産生するハイブリドーマ、抗 Clqモノクローナル抗体を用いた試料中の Clq タンパク質の存在または量を決定する方法、ならびにそのためのキットなどが提供さ れる。 [0007] According to the present invention, an anti-Clq monoclonal antibody that specifically recognizes Clq protein, a method for producing the same, a hybridoma that produces an anti-Clq monoclonal antibody, and an anti-Clq monoclonal antibody that can be used for quantification of Clq protein are provided. Methods for determining the presence or amount of Clq protein in the sample used, as well as kits and the like are provided.
図面の簡単な説明 Brief Description of Drawings
[0008] [図 1]図 1は、ハイプリドーマの培養上清を用いた ELIS Aの結果を示す。 [0008] Fig. 1 shows the results of ELISA using the culture supernatant of Hypridoma.
[図 2]図 2は、固相抗体として抗 Clq抗体 # 8を、感作抗体として抗 Clq抗体 # 35 (四角）を用いたサンドイッチ ELISA法の結果を示すグラフである。 FIG. 2 is a graph showing the results of sandwich ELISA using anti-Clq antibody # 8 as a solid phase antibody and anti-Clq antibody # 35 (square) as a sensitizing antibody.
[図 3]図 3は、固相抗体として抗 Clq抗体 # 33を、感作抗体として抗 Clq抗体 # 3 5 (四角）、抗 Clq抗体 # 54 (丸）、抗 Clq抗体 # 76 (菱形）を用いたサンドイッチ ELISA法の結果を示すグラフである。 [Figure 3] Figure 3 shows anti-Clq antibody # 33 as the solid phase antibody, anti-Clq antibody # 3 5 (square), anti-Clq antibody # 54 (circle), and anti-Clq antibody # 76 (diamond) as the sensitizing antibody. It is a graph which shows the result of the sandwich ELISA method using this.
[図 4]図 4は、固相抗体として抗 Clq抗体 # 35を、感作抗体として抗 Clq抗体 # 3 5 (四角）、抗 Clq抗体 # 54 (丸）、抗 Clq抗体 # 76 (菱形）を用いたサンドイッチ ELISA法の結果を示すグラフである。 [Figure 4] Figure 4 shows anti-Clq antibody # 35 as the solid phase antibody, anti-Clq antibody # 3 5 (square), anti-Clq antibody # 54 (circle), and anti-Clq antibody # 76 (diamond) as the sensitizing antibody. It is a graph which shows the result of the sandwich ELISA method using this.
[図 5]図 5は、固相抗体として抗 Clq抗体 # 40を、感作抗体として抗 Clq抗体 # 5 4 (丸）、抗 Clq抗体 # 76 (菱形）を用いたサンドイッチ ELISA法の結果を示すダラ フである。 [Figure 5] Figure 5 shows anti-Clq antibody # 40 as solid phase antibody and anti-Clq antibody # 5 as sensitizing antibody. 4 (circle) is a drape showing the results of sandwich ELISA using anti-Clq antibody # 76 (diamond).
[図 6]図 6は、固相抗体として抗 Clq抗体 # 54を、感作抗体として抗 Clq抗体 # 3 5 (四角）、抗 Clq抗体 # 54 (丸）を用いたサンドイッチ ELIS A法の結果を示すダラ フである。 [Figure 6] Figure 6 shows the results of sandwich ELISA using anti-Clq antibody # 54 as solid phase antibody, anti-Clq antibody # 3 5 (square) and anti-Clq antibody # 54 (circle) as sensitizing antibodies. It is a dull that shows.
[図 7]図 7は、固相抗体として抗 Clq抗体 # 76を、感作抗体として抗 Clq抗体 # 3 5 (四角）を用いたサンドイッチ ELISA法の結果を示すグラフである。 FIG. 7 is a graph showing the results of sandwich ELISA using anti-Clq antibody # 76 as a solid-phase antibody and anti-Clq antibody # 35 (square) as a sensitizing antibody.
[図 8]図 8は、固相抗体として抗 Clq抗体 # 107を、感作抗体として抗 Clq抗体 # 33 (三角）、抗 Clq抗体 # 35 (四角）を用いたサンドイッチ ELISA法の結果を示す グラフである。 [FIG. 8] FIG. 8 shows the results of sandwich ELISA using anti-Clq antibody # 107 as a solid phase antibody, anti-Clq antibody # 33 (triangle) and anti-Clq antibody # 35 (square) as sensitizing antibodies. It is a graph.
[図 9]図 9は、固相抗体として抗 Clq抗体 # 112を、感作抗体として抗 Clq抗体 # 35 (四角）、抗 Clq抗体 # 54 (丸）、抗 Clq抗体 # 76 (菱形）を用いたサンドイツ チ ELISA法の結果を示すグラフである。 [Figure 9] Figure 9 shows anti-Clq antibody # 112 as solid phase antibody, anti-Clq antibody # 35 (square), anti-Clq antibody # 54 (circle), and anti-Clq antibody # 76 (diamond) as sensitizing antibodies. It is a graph which shows the result of the used San-Germany ELISA method.
[図 10]図 10は、固相抗体として抗 Clq抗体 # 119を、感作抗体として抗 Clq抗体 # 33 (三角）を用いたサンドイッチ ELISA法の結果を示すグラフである。 FIG. 10 is a graph showing the results of sandwich ELISA using anti-Clq antibody # 119 as a solid phase antibody and anti-Clq antibody # 33 (triangle) as a sensitizing antibody.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
[0009] 本発明は、 1の態様において、 Clqタンパク質を特異的に認識することができる、抗 Clqモノクローナル抗体（以下、「抗 Clq抗体」ともいう）に関するものである。本発明 の抗 Clqモノクローナル抗体は、 Clqタンパク質と特異的に結合できるだけでなぐ 力、かるタンパク質量に依存して、形成される抗原と抗体の結合体量が増大する点、す なわち、 Clqタンパク質の定量に用いることができる点で非常に優れている。本発明 の抗体は、例えば、マウスのような哺乳動物を Clqタンパク質で免疫し、免疫動物の リンパ球と骨髄腫細胞株を融合させてハイプリドーマを作製し、これを培養することに より、得ること力 Sできる。他の既知の方法、例えば、遺伝子組換え法、化学合成法など を用いて力、かる抗体を製造してもよ!/、。 In one embodiment, the present invention relates to an anti-Clq monoclonal antibody (hereinafter also referred to as “anti-Clq antibody”) capable of specifically recognizing Clq protein. The anti-Clq monoclonal antibody of the present invention increases the amount of antigen-antibody conjugate formed depending on the amount of protein that can bind specifically to Clq protein and the amount of protein, that is, Clq protein. It is excellent in that it can be used for quantitative determination. The antibody of the present invention can be obtained, for example, by immunizing a mammal such as a mouse with Clq protein, fusing a lymphocyte of the immunized animal with a myeloma cell line to produce a hyperidoma, and culturing this. That power S. Other known methods such as gene recombination and chemical synthesis may be used to produce such antibodies!
[0010] Clqタンパク質のェピトープのアミノ酸配列およびヌクレオチド配列をそれぞれ配列 番号 1〜7および配列番号 8〜； 16に示す。従って、本発明の抗 Clqモノクローナル抗 体は、好ましくは、（a)配列番号 1〜7のアミノ酸配列を有するペプチド、（b)配列番号 ；!〜 7のアミノ酸配列において、 1個以上、好ましくは、 1または数個、例えば、 2、 3、 4 、 5、 6、 7、 8、 9、 10、 11、 12、 13、または 14固のアミノ酸カ欠失、置換若しくは付カロ されたアミノ酸配列を有するペプチド、および (c)配列番号 1〜7のアミノ酸配列と少 なくとも 50%以上、 列えば、、 60、 70、 75、 80、 85、 90、 93%の申目同十生を有するァミノ 酸配列を有するペプチドからなる群より選択されるペプチドを特異的に認識すること ができるものである。アミノ酸配列の相同性は、例えば、 FASTA、 BLAST, DNASI S (日立ソフトウェアエンジニアリング (株)製）、 GENETYX ( (株）ジエネテイクス製）を 用いて測定することができる。上記ペプチドまたはそれを含むタンパク質などでマウス などの動物を免疫し、かかる免疫マウスのリンパ球と骨髄腫細胞株を融合させてハイ プリドーマを作製し、抗 Clqモノクローナル抗体を得てもよい。 [0010] The amino acid and nucleotide sequences of the Clq protein epitopes are shown in SEQ ID NOs: 1-7 and SEQ ID NOs: 8-16, respectively. Accordingly, the anti-Clq monoclonal antibody of the present invention is preferably (a) a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7, (b) SEQ ID NO: In the amino acid sequence of! -7, one or more, preferably one or several, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 A peptide having an amino acid sequence deleted, substituted or appended, and (c) at least 50% or more of the amino acid sequence of SEQ ID NOs: 1 to 7, for example, 60, 70, 75, 80, It is possible to specifically recognize a peptide selected from the group consisting of peptides having an amino acid sequence having 85, 90, and 93% of the same age. The homology of amino acid sequences can be measured using, for example, FASTA, BLAST, DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.), GENETYX (manufactured by Genenetics Co., Ltd.). An anti-Clq monoclonal antibody may be obtained by immunizing an animal such as a mouse with the above peptide or a protein containing the peptide, and fusing a lymphocyte of the immunized mouse with a myeloma cell line to produce a hyperprideoma.
本発明の抗体の 1の具体例は、マウス骨髄腫細胞株とマウスリンパ球との融合細胞 であるノヽイブリドーマ KS— 0131 # 8、KS— 0131 # 33、KS— 0131 # 35、K S - 0131 # 40、 KS— 0131 # 54、または KS— 0131 # 76により産生される抗 Clqモノクローナル抗体である。当該ハイプリドーマは、独立行政法人産業技術総合 研究所 特許生物寄託センター（茨城県つくば巿東 1丁目 1番地 1中央第 6)に寄託 され、平成 18年 8月 2日付で受領され、それぞれ受託番号 FERM BP— 10649、 F ERM BP- 10650, FERM BP— 10651、 FERM BP— 10652、 FERM BP 10653、および FERM BP— 10654を付与された。かかるハイプリドーマを、公 知の培養条件、例えば、組成： RPMI 1640 ( ohjin 16005005) 425mL、 L-Glut amine (SIGMA G7513) 5mL、ピルビン酸ナトリウム（SIGMA S8636) 5mL、 HAT サプリメント（GIBCO 21060-017) lOmL、ペニシリン ·ストレプトマイシン（SIGMA P4 333) 2. 5mL, FBS 75mLの培地中、 37 ± 0. 1。C、 5 ± 0. 15% COにて培養 One specific example of the antibody of the present invention is a hybridoma of a mouse myeloma cell line and a mouse lymphocyte, such as Neubridoma KS— 0131 # 8, KS — 0131 # 33, KS — 0131 # 35, KS — 0131 # 40, KS— 0131 # 54, or KS— 0131 # 76. The Hypridoma was deposited at the Patent Organism Depositary Center (National Institute of Advanced Industrial Science and Technology) (Tsukuba Ito, 1-chome, 1-chome, 1-Chuo 6) and received on August 2, 2006. FERM BP—10649, F ERM BP-10650, FERM BP—10651, FERM BP—10652, FERM BP 10653, and FERM BP—10654. Such hypridoma can be prepared by using known culture conditions, for example, composition: RPMI 1640 (ohjin 16005005) 425 mL, L-Glut amine (SIGMA G7513) 5 mL, sodium pyruvate (SIGMA S8636) 5 mL, HAT supplement (GIBCO 21060-017) lOmL, penicillin-streptomycin (SIGMA P4 333) 2. 37 ± 0.1 in medium of 5 mL, FBS 75 mL. C, cultured at 5 ± 0.15% CO
2 し、産生された抗 Clqモノクローナル抗体を公知の手段 ·方法にて回収 ·精製するこ とにより、抗 C lqモノクローナル抗体を製造することができる。本明細書において、ハ イブリドーマ KS— 0131 # 8、KS— 0131 # 33、 KS— 0131 # 35、 KS— 0131 The anti-Clq monoclonal antibody can be produced by recovering and purifying the produced anti-Clq monoclonal antibody by a known means / method. In this specification, hybridoma KS— 0131 # 8, KS— 0131 # 33, KS— 0131 # 35, KS— 0131
# 40、 KS— 0131 # 54、および KS— 0131 # 76により産生される抗 Clqモノ クローナル抗体をそれぞれ、抗 Clq抗体 # 8、抗 Clq抗体 # 33、抗じ；^抗体 # 35、抗 Clq抗体 # 40、抗 Clq抗体 # 54、および抗 Clq抗体 # 76と表す。 [0012] 本発明の抗 Clqモノクローナル抗体は、そのフラグメント、およびそのキメラ抗体お よびヒト化抗体などの改変抗体ならびに変異抗体を含む。これらのフラグメント、改変 抗体または変異抗体もまた、元の抗体と同様の Clqタンパク質に対する特異性を有 するものである。これらは、当業者に既知の手段 ·方法により製造され得る。 The anti-Clq monoclonal antibodies produced by # 40, KS— 0131 # 54, and KS— 0131 # 76 are anti-Clq antibody # 8, anti-Clq antibody # 33, anti-tagged; ^ antibody # 35, anti-Clq antibody, respectively. Denote # 40, anti-Clq antibody # 54, and anti-Clq antibody # 76. [0012] The anti-Clq monoclonal antibodies of the present invention include fragments thereof, modified antibodies such as chimeric antibodies and humanized antibodies, and mutant antibodies. These fragments, modified antibodies or mutant antibodies also have the same specificity for Clq protein as the original antibody. These can be produced by means and methods known to those skilled in the art.
[0013] 本発明は、別の態様において、独立行政法人産業技術総合研究所 特許生物寄 託センター受託番号 FERM BP— 10649、 FERM BP— 10650、 FERM BP— 10651、 FERM BP— 10652、 FERM BP— 10653、または FERM BP— 106 54を有する、抗 Clqモノクローナル抗体を産生するハイブリドーマに関するものであ る。本発明のハイプリドーマは、 Clqタンパク質で免疫されたマウス由来のリンパ球と マウス骨髄腫細胞株を融合させて得られた細胞である。 [0013] In another aspect, the present invention provides a patent biological deposit center accession number FERM BP-10649, FERM BP-10650, FERM BP-10651, FERM BP 10652, FERM BP— The present invention relates to a hybridoma producing an anti-Clq monoclonal antibody having 10653 or FERM BP-10654. The hyperidoma of the present invention is a cell obtained by fusing a mouse lymphocyte immunized with Clq protein and a mouse myeloma cell line.
[0014] 本発明は、別の態様において、配列番号 1〜7のアミノ酸配列を有するペプチドを 用いることを特徴とする、抗 Clqモノクローナル抗体の製造方法に関するものである 。本発明のこの態様の製造方法において、（a)配列番号 1〜7のアミノ酸配列を有す るペプチド、（b)配列番号 1〜7のアミノ酸配列において、 1個以上、好ましくは、 1ま た (ま数固、 列え ( 、 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、また (ま 14固のアミノ 酸が欠失、置換若しくは付加されたアミノ酸配列を有するペプチド、または（c)配列 番号；!〜 7のアミノ酸酉己歹 IJと少なくとも 50%以上、 列えば、 60、 70、 75、 80、 85、 90 、 93%の相同性を有するアミノ酸配列を有するペプチドを用いてもよい。 [0014] In another aspect, the present invention relates to a method for producing an anti-Clq monoclonal antibody, wherein a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7 is used. In the production method of this aspect of the present invention, (a) a peptide having the amino acid sequence of SEQ ID NOs: 1 to 7, and (b) one or more, preferably 1, in the amino acid sequence of SEQ ID NOs: 1 to 7 (Masame, Arrangement (, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 (A solid amino acid has been deleted, substituted or added) A peptide having an amino acid sequence, or (c) having at least 50% or more homology with the amino acid IJ of SEQ ID NO:!-7, such as 60, 70, 75, 80, 85, 90, 93% A peptide having an amino acid sequence may be used.
[0015] 本発明のこの態様の製造方法は、上記ペプチドでマウスなどの動物を免疫するェ 程、抗 Clqモノクローナル抗体を精製する工程などを含んでいてもよい。ペプチドで の免疫、および抗 Clqモノクローナル抗体の精製は、当業者に既知の方法により行 われ得る。 [0015] The production method of this aspect of the present invention may include a step of purifying an anti-Clq monoclonal antibody, etc., so as to immunize an animal such as a mouse with the peptide. Immunization with peptides and purification of anti-Clq monoclonal antibodies can be performed by methods known to those skilled in the art.
[0016] 本発明は、さらに別の態様において、本発明のハイプリドーマを培養することを特 徴とする、抗 Clqモノクローナル抗体の製造方法に関するものでる。ハイプリドーマの 培養は、インビトロ培養、およびインビボ培養の両方を含む。インビトロ培養は、公知 の培養条件にて、例えば、組成： RPMI 1640 ( ohjin 16005005) 425mL、 L-G1 utamine (SIGMA G7513) 5mL、ピルビン酸ナトリウム（SIGMA S8636) 5mL、 HA Tサプリメント（GIBCO 21060-017) 10mL、ペニシリン 'ストレプトマイシン（SIGMA P4333) 2. 5mL、FBS 75mLの培地中、 37 ± 0. 1。C、 5 ± 0. 15% COにて行 [0016] In still another aspect, the present invention relates to a method for producing an anti-Clq monoclonal antibody, characterized by culturing the hyperpridoma of the present invention. Hypridoma culture includes both in vitro and in vivo culture. In vitro culture is performed under known culture conditions, for example, composition: RPMI 1640 (ohjin 16005005) 425 mL, L-G1 utamine (SIGMA G7513) 5 mL, sodium pyruvate (SIGMA S8636) 5 mL, HA T supplement (GIBCO 21060-017 ) 10mL, penicillin 'streptomycin (SIGMA P4333) 2. 37 ± 0.1 in medium of 5mL, FBS 75mL. C, 5 ± 0. 15% CO
2 うこと力 Sできる。インビボ培養は、例えば、マウスのごとき動物の腹腔に本発明のハイ プリドーマを移植することで、達成される。本発明の製造方法は、得られた抗体を精 製する工程をさらに含んでいてもよい。精製方法を当業者は適宜選択して用いること ができる。 2 Swelling power S In vivo culture is achieved, for example, by transplanting the hyperidoma of the present invention into the abdominal cavity of an animal such as a mouse. The production method of the present invention may further include a step of purifying the obtained antibody. Those skilled in the art can appropriately select and use the purification method.
[0017] 本発明は、さらなる態様において、本発明の抗 Clqモノクローナル抗体を用いるこ とを特徴とする、試料中の Clqタンパク質の存在または量を決定する方法に関するも のである。本発明の抗 Clqモノクローナル抗体は、 Clqタンパク質の量に依存して、 抗原と抗体の結合体量が増大するという特性を有するので、試料中の Clqタンパク 質の存在ば力、りでなぐ量を決定することができる。本発明の決定方法において用い られる抗 C lqモノクローナル抗体は 2種以上であってもよい。 2種以上の抗 Clqモノク ローナル抗体を用いることで、決定感度および特異性が増大し、精度が向上する。 好ましくは、抗 Clq抗体 # 33と抗 Clq抗体 # 54、抗 Clq抗体 # 33と抗 Clq抗 体 # 76、抗 Clq抗体 # 40と抗 Clq抗体 # 54、抗 C lq抗体 # 40と抗 Clq抗 体 76とが本発明の決定方法にお!/、て用いられる。本発明の決定方法にぉレ、て用い られる抗体は、標識されたものであってもよい。様々な標識が公知であり、当業者は 適宜選択して用いることができる。例えば、 horseradish peroxidase (HRP)、 ALP、 Glue ose oxidase^ β - galactosidaseなどの酵素、 FITC、 Rhodamine、 Cy3、し y5、 Texas Red 、 Alexa Fluor類、 BODIPY類、 IRDye類、 MFP類、 Quantum Dot類、 AMCA、 Allophyc ocyanin, BMP, Cy2、 Cy3.5、 Cy5.5、 DTAF、 DyLight 547、 DyLight 647、 FluoroNano gold, Phycoerythrin, Phycocyanin, R_PE、 Saporin, TRITCなどの 光標識、 Biotin、 Digoxigenin (DIG)、 Acridium Ester, Flashlightなどの化学物質、 60mm Microbeadな どのビーズ、 MagCellect Ferrofluid [登録商標]などの磁気ビーズ、 1 1などの放射標 識、金粒子、 Agaroseなどの標識が用いられる。力、かる標識を付すことで、本発明の 決定方法を効率よく行うことができる。 [0017] In a further aspect, the present invention relates to a method for determining the presence or amount of Clq protein in a sample, characterized by using an anti-Clq monoclonal antibody of the present invention. Since the anti-Clq monoclonal antibody of the present invention has the property that the amount of the antigen-antibody conjugate increases depending on the amount of Clq protein, the presence of the Clq protein in the sample will reduce the force. Can be determined. Two or more anti-Clq monoclonal antibodies may be used in the determination method of the present invention. Using two or more anti-Clq monoclonal antibodies increases the sensitivity and specificity of determination and improves accuracy. Preferably, anti-Clq antibody # 33 and anti-Clq antibody # 54, anti-Clq antibody # 33 and anti-Clq antibody # 76, anti-Clq antibody # 40 and anti-Clq antibody # 54, anti-Clq antibody # 40 and anti-Clq anti The body 76 is used in the determination method of the present invention. The antibody used in the determination method of the present invention may be labeled. Various labels are known and can be appropriately selected and used by those skilled in the art. For example, horseradish peroxidase (HRP), ALP, enzymes such as Glueose oxidase ^ β-galactosidase, FITC, Rhodamine, Cy3, y5, Texas Red, Alexa Fluors, BODIPYs, IRDyes, MFPs, Quantum Dot, Light labels such as AMCA, Allophycocyanin, BMP, Cy2, Cy3.5, Cy5.5, DTAF, DyLight 547, DyLight 647, FluoroNano gold, Phycoerythrin, Phycocyanin, R_PE, Saporin, TRITC, Biotin, Digoxigenin (DIG), Acridium Ester, chemicals such Flashlight, 60 mm MicroBead of which beads, magnetic beads, such as MagCellect Ferrofluid [R], the radiation-labeled, such as 1 1, gold particles, labels such as Agarose used. The determination method of the present invention can be performed efficiently by attaching a force and a mark.
[0018] 試料は、 Clqタンパク質が含まれる可能性があればいずれのものであってもよぐ 例えば、血清、関節液、軟骨や骨及び各組織などが挙げられる。本発明の決定方法 を用いることで、力、かる試料中の Clqタンパク質の存在または量を迅速かつ容易に 決定すること力 Sでさる。 [0018] The sample may be any sample that may contain Clq protein. Examples include serum, joint fluid, cartilage, bone, and each tissue. By using the determination method of the present invention, the force, the presence or amount of Clq protein in the sample can be quickly and easily determined. Determining power S
[0019] 本発明の決定方法は、例えば、上記試料を本発明の抗 Clqモノクローナル抗体と インキュベーションし、該抗 Clq抗体と試料中の Clqタンパク質との結合体を形成さ せ、次に該結合体を測定することにより、行われてもよい。例えば、サンドイッチ ELISA 法などの ELISA法、ィムノブロット法、 Radioimmunoassay (RIA)法、免疫沈降法、プロ ティンアレイを用いる方法、フローサイトメーターを用いる方法、化学発光酵素免疫 測定法 (CLEIA)、生物発光酵素免疫測定法 (BLEIA)、毛細管現象によって輸液可 能な展開器材 (免疫測定用試験片）を用いる測定法、イミュノクロマト法、免疫染色法 、凝集法などを用いて本発明の決定方法を実施することができる。 ELISA法は、特 殊な設備等を必要とせず、実際の医療現場において Clqタンパク質の存在または量 の決定を迅速かつ容易に行うことが可能であるので、好ましく用いられる。 [0019] In the determination method of the present invention, for example, the sample is incubated with the anti-Clq monoclonal antibody of the present invention to form a conjugate of the anti-Clq antibody and the Clq protein in the sample, and then the conjugate. May be carried out by measuring. For example, ELISA methods such as sandwich ELISA, immunoblot method, radioimmunoassay (RIA) method, immunoprecipitation method, method using protein array, method using flow cytometer, chemiluminescent enzyme immunoassay (CLEIA), bioluminescent enzyme The determination method of the present invention is performed using immunoassay (BLEIA), measurement using a developing device (immunoassay test piece) that can be infused by capillary action, immunochromatography, immunostaining, aggregation, etc. can do. The ELISA method is preferably used because it does not require special equipment or the like, and the presence or amount of Clq protein can be determined quickly and easily in an actual medical field.
[0020] 本発明の抗 Clqモノクローナル抗体を用いて、 Clqタンパク質の存在または量を迅 速かつ容易に決定することができるので、本発明の決定方法を RAの予後診断に用 いることあでさる。 [0020] Since the presence or amount of Clq protein can be determined quickly and easily using the anti-Clq monoclonal antibody of the present invention, the determination method of the present invention is used for the prognosis diagnosis of RA. .
[0021] 本発明は、なおさらなる態様において、本発明の抗 Clqモノクローナル抗体を用い て試料中の Clqタンパク質の存在または量を決定することを特徴とする、患者におけ る RAの予後の診断方法に関するものである。 Clqタンパク質の存在または量は上述 の方法にて決定されてもよ!/、。 [0021] In a still further aspect, the present invention provides a method for diagnosing the prognosis of RA in a patient, comprising determining the presence or amount of Clq protein in a sample using the anti-Clq monoclonal antibody of the present invention. It is about. The presence or amount of Clq protein may be determined as described above! /.
[0022] 本発明は、もう 1つの態様において、本発明の抗 Clqモノクローナル抗体を投与す ることを特徴とする、患者における RAの治療方法に関するものである。 [0022] In another aspect, the present invention relates to a method for treating RA in a patient, which comprises administering the anti-Clq monoclonal antibody of the present invention.
[0023] 本発明は、さらなる態様において、本発明の抗 Clqモノクローナル抗体を必須構成 成分として含む、試料中の Clqタンパク質の存在または量を決定するためのキットに 関するものである。本発明のキットに含まれる抗 Clqモノクローナル抗体は 2種以上 であってもよい。 2種以上の抗 Clqモノクローナル抗体を含むことで、本発明のキット を用いて行われる Clqタンパク質の存在または量の決定の感度、特異性、精度を向 上させること力 Sできる。本発明のキットは、本発明の抗 Clqモノクローナル抗体の他に 、例えば、試料採取手段、標識、反応容器、決定用試薬等を含んでいてもよい。一 般的に、キットには取扱説明書を添付する。本発明のキットにおいて、抗 Clqモノクロ ーナル抗体は標識されたものであってもょレ、。 [0023] In a further aspect, the present invention relates to a kit for determining the presence or amount of Clq protein in a sample, comprising the anti-Clq monoclonal antibody of the present invention as an essential component. Two or more anti-Clq monoclonal antibodies may be included in the kit of the present invention. By including two or more types of anti-Clq monoclonal antibodies, it is possible to improve the sensitivity, specificity and accuracy of determination of the presence or amount of Clq protein performed using the kit of the present invention. In addition to the anti-Clq monoclonal antibody of the present invention, the kit of the present invention may contain, for example, a sample collection means, a label, a reaction container, a determination reagent and the like. In general, an instruction manual is attached to the kit. In the kit of the present invention, anti-Clq monochrome The final antibody may be labeled.
[0024] 本発明のキットにお!/、て、例えば、サンドイッチ ELISA法などの ELISA法、ィムノブ口 ット法、 Radioimmunoassay (RIA)法、免疫沈降法、プロテインアレイを用いる方法、フ ローサイトメーターを用いる方法、化学発光酵素免疫測定法 (CLEIA)、生物発光酵 素免疫測定法 (BLEIA)、毛細管現象によって輸液可能な展開器材 (免疫測定用試 験片）を用いる測定法、イミュノクロマト法、免疫染色法、凝集法などを用いてもよい。 本発明のキットを用いて、 Clqタンパク質の存在または量を迅速かつ容易に決定す ること力 Sできるので、臨床現場において RA患者の予後診断が可能になる。また、本 発明のキットを用いて RAの病理学的研究等を行うこともできる。 [0024] In the kit of the present invention, for example, ELISA methods such as sandwich ELISA method, Imnobutt method, Radioimmunoassay (RIA) method, immunoprecipitation method, method using protein array, flow cytometer , Chemiluminescent enzyme immunoassay (CLEIA), bioluminescent enzyme immunoassay (BLEIA), measuring method using expandable device (immunoassay specimen) that can be infused by capillary action, immunochromatography Alternatively, an immunostaining method, an aggregation method, or the like may be used. Since the kit of the present invention can be used to quickly and easily determine the presence or amount of Clq protein, it is possible to prognose RA patients in clinical settings. In addition, pathological studies of RA can be performed using the kit of the present invention.
[0025] 以下に実施例を示して本発明を詳細かつ具体的に説明するが、実施例は本発明 を限定するものではない。 Hereinafter, the present invention will be described in detail and specifically with reference to Examples, but the Examples are not intended to limit the present invention.
実施例 1 Example 1
[0026] 抗 Clqモノクローナル抗体産生ハイブリドーマの作製 [0026] Preparation of anti-Clq monoclonal antibody-producing hybridoma
雌の BALB/Cマウス 4匹に Clq精製タンパク質（純度 99%以上、 Calbiochem 20 4876) 0. lmgをアジュバント（タイターマックス、 SIGMA)と共に 2週間おきに 2〜3回、 フットパットおよび背中皮下に投与し、免疫した。マウスの脾臓からリンパ球を回収し、 抗原 30 ,i gを含む培地にて 3日間培養した。培養後、リンパ球とマウス骨髄腫細胞株 P3U1細胞を、 PEG1500を用いて常法に従い融合させた。融合後、細胞を組成: R PMI 1640 ( ohjin 16005005) 425mL、 L-Glutamine (SIGMA G7513) 5mL、 ピルビン酸ナトリウム（SIGMA S8636) 5mL、 HATサプリメント（GIBCO 21060-017) lOmL、ペニシリン ·ストレプトマイシン（SIGMA P4333) 2. 5mL、 FBS 75mLの 培地に懸濁し、あらかじめマウス胸腺細胞を播種した 96ゥエルプレートに分注した。 8 日後、上清中の抗体の有無を ELISA法によりスクリーニングした。 ELISA法の手順 は以下の通りである。 1 a g/ml Clqの PBS中溶液を調製し、 ELISA用マイクロプ レート（3912 ファルコン）に添加し、室温にて 2時間インキュベーションすることで結 合させた。次に、 1 % スキムミルク/ PBSを用いてブロッキングを行い、抗原プレート とした。抗原プレートに培養上清を添加して反応させ、洗浄後、 ALP標識ャギ抗 IgG (ザィメット）を結合させた。 ALP基質を添加して、酵素反応させ、 492nmにおける吸 光度を測定し、抗体陽性ゥエルを選択した。対照として、同様に免疫したゥサギの血 清から分離したポリクローナル抗体を用いた。結果を図 1に示す。抗原を結合させて いない ELISA用マイクロプレートを用い、非特異的反応を除去し、抗 Clqモノクロ一 ナノレ抗体産生ノヽィフ、、リドーマ KS— 0131 # 8、KS— 0131 # 25、KS— 0131 # 33、KS— 0131 # 35、KS— 0131 # 40、KS— 0131 # 54、KS— 0131 # 76、KS— 0131 # 100、KS— 0131 # 102、KS— 0131 # 107、KS— 013 1 # 112、および KS— 0131 # 119などを得た。 Four female BALB / C mice were given Clq purified protein (purity 99% or higher, Calbiochem 20 4876) 0.1 mg with adjuvant (Titermax, SIGMA) 2-3 times every 2 weeks subcutaneously on the footpad and back And immunized. Lymphocytes were collected from the mouse spleen and cultured in a medium containing the antigen 30, ig for 3 days. After culture, lymphocytes and mouse myeloma cell line P3U1 cells were fused using PEG1500 according to a conventional method. After fusion, the cells were composed of: R PMI 1640 (ohjin 16005005) 425 mL, L-Glutamine (SIGMA G7513) 5 mL, sodium pyruvate (SIGMA S8636) 5 mL, HAT supplement (GIBCO 21060-017) lOmL, penicillin streptomycin (SIGMA P4333 2. Suspended in 5 mL, 75 mL FBS medium and dispensed into 96 well plates previously seeded with mouse thymocytes. After 8 days, the presence of antibody in the supernatant was screened by ELISA. The procedure for ELISA is as follows. A solution of 1 ag / ml Clq in PBS was prepared, added to an ELISA microplate (3912 Falcon), and allowed to bind by incubation at room temperature for 2 hours. Next, blocking was performed using 1% skim milk / PBS to prepare an antigen plate. The culture supernatant was added to the antigen plate for reaction, and after washing, ALP-labeled goat anti-IgG (Zymet) was bound. Add ALP substrate, allow enzyme reaction, and absorb at 492 nm The light intensity was measured and antibody positive wells were selected. As a control, a polyclonal antibody isolated from the serum of a similarly immunized rabbit was used. The results are shown in Figure 1. Using an ELISA microplate without antigen binding, nonspecific reactions were removed, and anti-Clq monoclonal monoclonal antibody production noise, Ridooma KS— 0131 # 8, KS— 0131 # 25, KS— 0131 # 33, KS— 0131 # 35, KS— 0131 # 40, KS— 0131 # 54, KS— 0131 # 76, KS— 0131 # 100, KS— 0131 # 102, KS— 0131 # 107, KS— 013 1 # 112 , And KS-0131 # 119 etc.
実施例 2 Example 2
[0027] Clqェピトープの同定 [0027] Identification of Clq epitopes
ヒト Clqのサブユニット A鎖、 B鎖、 C鎖のアミノ酸配列を配列番号 17〜19に、ヌク レオチド配列を配列番号 20〜22に示す。それぞれのアミノ酸配列をもとにして、各サ ブユニットのアミノ酸配列を 15アミノ酸 (残基）ずつ 3アミノ酸の間隔でずらした配列を 合成ペプチド（順に、ペプチド番号；!〜 78、 97〜； 117、 193〜270番）としてガラスァ レイ上に合成した。それぞれのペプチドの合成はアレイ上の特定の位置で行い、 C1 qのサブユニットの全アミノ酸配列を網羅する合成ペプチド含むペプチドアレイを作 製した。尚、アレイの作製は JPT社に委託して行った。 The amino acid sequences of human Clq subunits A chain, B chain, and C chain are shown in SEQ ID NOs: 17 to 19, and the nucleotide sequences are shown in SEQ ID NOs: 20 to 22, respectively. Based on each amino acid sequence, a sequence obtained by shifting the amino acid sequence of each subunit by 15 amino acids (residues) at intervals of 3 amino acids is a synthetic peptide (in order, peptide numbers;! -78, 97-; 117, 193-270) on a glass array. Each peptide was synthesized at a specific position on the array, and a peptide array containing a synthetic peptide covering the entire amino acid sequence of the C1 q subunit was prepared. The array was manufactured by JPT.
[0028] 得られた抗 Clqモノクローナル抗体産生ハイブリドーマから産生された抗 Clq抗体 [0028] Anti-Clq antibody produced from the resulting anti-Clq monoclonal antibody-producing hybridoma
# 8、 # 33、 # 40、 # 54、および # 76を Protein Gカラムを用いて精製した。精製し た抗体の PBS (10mM リン酸緩衝液 pH7. 0、0. 1M NaCl)での 1 , 000倍希釈 液 330 ,1 1をペプチドアレイ上に塗布し、シールをした後、 4°Cで 12時間インキュべ ートした。その後メタノール 1回、 ー0水で5分間 5回洗浄した。遠心してアレイ スライドを乾燥させ、蛍光スキャナー（Agilent DNAマイクロアレイスキャナ；アジレント 社製）で、スキャンし、ソフトウェア（Feature Extractionソフトウェア；アジレント社製）を 用いてアレイ上のそれぞれのペプチドスポットの蛍光強度を数 化した。強い蛍光強 度を示すいくつかのスポットを検出した。このうち、バックグラウンドレベルより 13, 00 0以上高!/、蛍光強度を示すペプチドスポットを表 1に示す（ペプチド番号 149、 150、 244〜246番のアミノ酸配列をそれぞれ配列番号 3〜7に示す）。 # 8, # 33, # 40, # 54, and # 76 were purified using a Protein G column. The purified antibody was diluted 1,000 times in PBS (10 mM phosphate buffer pH 7.0, 0.1 M NaCl) 330,11 on the peptide array, sealed, and sealed at 4 ° C. Incubated for 12 hours. Thereafter, it was washed once with methanol and then with 0 water for 5 minutes 5 times. Centrifuge to dry the array slide, scan with a fluorescence scanner (Agilent DNA microarray scanner; Agilent), and use software (Feature Extraction software; Agilent) to determine the fluorescence intensity of each peptide spot on the array. Turned into. Several spots showing strong fluorescence intensity were detected. Among these, peptide spots that are 1,300 or more higher than the background level! / And show fluorescence intensity are shown in Table 1 (amino acid sequences of peptide numbers 149, 150, and 244 to 246 are shown in SEQ ID NOs: 3 to 7, respectively) ).
[0029] [表 1] ぺプチド 配列 蛍光強度 [0029] [Table 1] Peptide array Fluorescence intensity
番号 番号 # 8 # 33 #40 # 54 # 76 Number Number # 8 # 33 # 40 # 54 # 76
149 3 54， 01 7 1 3, 633 33, 732 26, 890 46， 230149 3 54, 01 7 1 3, 633 33, 732 26, 890 46, 230
1 50 4 44， 583 1 5, 765 43, 735 26, 50 1 64, 1 1 31 50 4 44, 583 1 5, 765 43, 735 26, 50 1 64, 1 1 3
244 5 56， 144 48, 01 2 57, 993 63, 408 65， 31 2244 5 56, 144 48, 01 2 57, 993 63, 408 65, 31 2
245 6 6 5， 29 7 65, 31 1 65, 3 12 65, 31 2 65， 31 2245 6 6 5, 29 7 65, 31 1 65, 3 12 65, 31 2 65, 31 2
246 7 6 5， 29 7 46, 299 63， 698 43, 644 65， 31 2246 7 6 5, 29 7 46, 299 63, 698 43, 644 65, 31 2
* バックグラウンドの蛍光強度は 2. 6 * Background fluorescence intensity is 2.6
[0030] 149番および 150番のペプチドは ClqBサブユニット（B鎖）由来の配列であり、 24 4番〜 246番のペプチドは ClqCサブユニット（C鎖）由来の配列である。これらの配 列から、 Clqェピトープのアミノ酸配列は CKVPGLYYF (配列番号 2)の 9残基ある いはその一部を含むと予測した。 [0030] The peptides 149 and 150 are sequences derived from the ClqB subunit (B chain), and the peptides 24 to 246 are sequences derived from the ClqC subunit (C chain). From these sequences, the amino acid sequence of Clq epitope was predicted to contain 9 residues or a part of CKVPGLYYF (SEQ ID NO: 2).
[0031] 「149番、 150番、 244〜246番のペプチドに共通するのは 9個のアミノ酸からなる 酉己列 CKVPGLYYF (配列番号 2)であることから、かかる配列自体、あるいはかかる 配列内に抗 Clq抗体のェピトープが存在すると考えられる。このアミノ酸配列に由来 するより短いペプチドを使用することによって、ェピトープとして必要な配列をさらに 特定すること力できる。そのような配列を有するペプチドとしては、例えば、 8アミノ酸（ CKVPGLYY (配列番号 23)、 KVPGLYYF (配列番号 24) )、 7アミノ酸 (CKVPG LY (配列番号 25)、 KVPGLYY (配列番号 26)、 VPGLYYF (配列番号 27) )、 6ァ ミノ酸（CKVPGL (配列番号 28)、 KVPGLY (配列番号 29)、 VPGLYY (配列番号 30)、 PGLYYF (配列番号 1))、 5アミノ酸（CKVPG (配列番号 31)、 KVPGL (配列 番号 32)、 VPGLY (配列番号 33)、 PGLYY (配列番号 34)、 GLYYF (配列番号 3 5) )、 4アミノ酸 (CKVP (配列番号 36)、 KVPG (配列番号 37)、 VPGL (配列番号 3 8)、 PGLY (配列番号 39)、 GLYY (配列番号 40)、 LYYF (配列番号 41))からなる ペプチド、またはそれ以下のアミノ酸からなるペプチドが挙げられる。 [0031] “Common to peptides No. 149, No. 150, No. 244 to No. 246 is a self-sequence CKVPGLYYF (SEQ ID No. 2) consisting of 9 amino acids. It is thought that there exists an anti-Clq antibody epitope, and by using a shorter peptide derived from this amino acid sequence, it is possible to further identify the sequence required as an epitope. , 8 amino acids (CKVPGLYY (SEQ ID NO: 23), KVPGLYYF (SEQ ID NO: 24)), 7 amino acids (CKVPG LY (SEQ ID NO: 25), KVPGLYY (SEQ ID NO: 26), VPGLYYF (SEQ ID NO: 27)), 6 amino acids ( CKVPGL (SEQ ID NO: 28), KVPGLY (SEQ ID NO: 29), VPGLYY (SEQ ID NO: 30), PGLYYF (SEQ ID NO: 1)), 5 amino acids (CKVPG (SEQ ID NO: 31), KVPGL (SEQ ID NO: 32), VPGLY (SEQ ID NO: 32) 33), PGLYY (SEQ ID NO: 34), GLYYF ( (Column number 3 5)), 4 amino acids (CKVP (SEQ ID NO: 36), KVPG (SEQ ID NO: 37), VPGL (SEQ ID NO: 3 8), PGLY (SEQ ID NO: 39), GLYY (SEQ ID NO: 40), LYYF (SEQ ID NO: 41)) or a peptide consisting of less amino acids.
[0032] CKVPGLYYF (配列番号 2)の 9残基のペプチドが Clqェピトープであるかどう力、 を決定するため、さらに以下の実験を行った。配列番号 2のアミノ酸配列を有するぺ プチド（R2)、およびかかるペプチドより短!/、アミノ酸配歹 IJPGLYYF (配列番号 1)を 有するペプチド（R1)を GL Biochem (Shanghai)社に委託して作製した。これらの ペプチドを表 2に示す。それぞれのペプチドを 10mg/mlとなるようジメチルスルホキ シドに溶解し、保存した。 [0032] In order to determine whether the 9-residue peptide of CKVPGLYYF (SEQ ID NO: 2) is a Clq epitope, the following experiment was further conducted. A peptide having the amino acid sequence of SEQ ID NO: 2. A peptide (R1) having a peptide (R2) and a shorter amino acid sequence IJPGLYYF (SEQ ID NO: 1) than that of the peptide was prepared by consigning to GL Biochem (Shanghai). These peptides are shown in Table 2. Each peptide was dissolved in dimethyl sulfoxide to a concentration of 10 mg / ml and stored.
[表 2]
[0034] ヒト Clqタンパク質（Carbiochem社製）を 10mM HEPES、 300mM NaCl, 40% [0034] Human Clq protein (Carbiochem) 10 mM HEPES, 300 mM NaCl, 40%
グリセローノレ（ρΗ7· 2) ίこ溶角早し、 ZOO ^ g/mlおよび 50 g/ml ヒト Clqタンノ ク質溶液を調製した。ヒト Clqタンパク質溶液を 5mm X 15mmの大きさのニトロセル ロース膜（Hybond C ;アマシャム社製）に表 3の通りスポットし、室温にて約 1時間風 乾した。ニトロセルロース膜を TBSに浸し、 5分ほどなじませてから 5%ブロッキング剤 (Amersham ECL blocking reagent； GE Healthcare社製）を含む TBS (20mM Tris -HC1 pH7. 5、 150mM NaCl)を用いて室温にて 1時間ブロッキングを行った。 ブロッキングを行ったニトロセルロース膜を TBSで軽く洗浄し、各抗 Clq抗体および 合成ペプチドを含む溶液（20 1)に浸して室温にて 1時間反応させた。 TTBS (TBS に最終濃度 0. 05%で Tween 20を加えたもの）にてニトロセルロース膜を一度軽 く洗浄した後、十分量の TTBSで 10分 X 3回振盪しながら洗浄した。結合の検出を 以下の通り行った。 Glycellonore ( ρ · 2 · 2) was dissolved to prepare ZOO ^ g / ml and 50 g / ml human Clq protein solutions. The human Clq protein solution was spotted on a nitrocellulose membrane (Hybond C; manufactured by Amersham) having a size of 5 mm × 15 mm as shown in Table 3 and air-dried at room temperature for about 1 hour. Soak the nitrocellulose membrane in TBS, let it fit for about 5 minutes, and then use TBS (20 mM Tris-HC1 pH 7.5, 150 mM NaCl) containing 5% blocking agent (Amersham ECL blocking reagent; manufactured by GE Healthcare) at room temperature. Blocking was performed for 1 hour. The blocked nitrocellulose membrane was gently washed with TBS, immersed in a solution (201) containing each anti-Clq antibody and synthetic peptide, and allowed to react at room temperature for 1 hour. The nitrocellulose membrane was lightly washed once with TTBS (TBS added with Tween 20 at a final concentration of 0.05%), and then washed with a sufficient amount of TTBS for 10 minutes x 3 times with shaking. Binding detection was performed as follows.
[0035] [表 3] [0035] [Table 3]
ALP標識抗 Clq抗体を用いた場合の検出方法 Detection method using ALP-labeled anti-Clq antibody
ニトロセルロース膜を ALP発色バッファー（lOOmM NaCl、 5mM MgCl入り Tri Nitrocellulose membrane with ALP coloring buffer (lOOmM NaCl, 5mM MgCl in Tri
2 s -HCl, pH9. 5)で軽く洗浄後、 BCIP/NBT溶液（プロメガ社製）を含む ALP発 色バッファー 30 に浸し、室温で 10分間発色させた。染色像が得られたところで二 トロセルロース膜を十分量の蒸留水に浸し、発色バッファーを洗い流した。洗浄後、 ニトロセルロース膜を風乾してエプソン社製デジタルスキャナ GT— 8300UFを用い て染色像の取り込みを行った。 After lightly washing with 2 s-HCl, pH 9.5), the plate was immersed in ALP color buffer 30 containing BCIP / NBT solution (Promega) and developed for 10 minutes at room temperature. When the stained image was obtained, The trocellulose membrane was immersed in a sufficient amount of distilled water to wash away the color developing buffer. After washing, the nitrocellulose membrane was air-dried and stained images were captured using an Epson digital scanner GT-8300UF.
[0037] HRP標識抗 C lq抗体を用いた場合の検出方法 [0037] Detection method using HRP-labeled anti-Clq antibody
ニトロセノレロース膜を Amersham ECL Advance Western Blotting Detectionキット (G E healthcare社製）中の A液 · Β液等量混合液に浸し、室温にて 1分間反応させた。そ の後、反応液を軽く振り払い、ニトロセルロース膜をプラスチックフィルムで挟み、ボラ ロイドフィルムに感光させることで膜上の化学発光の検出を行った。 The nitrosenololose membrane was immersed in a mixed solution of equal amounts of A and B in Amersham ECL Advance Western Blotting Detection Kit (manufactured by GE healthcare) and allowed to react at room temperature for 1 minute. After that, the reaction solution was lightly shaken, the nitrocellulose membrane was sandwiched between plastic films, and the chemiluminescence on the membrane was detected by exposing the membrane to a bolloid film.
[0038] ALP標識抗 C lq抗体（1/1000希釈）と C lqの結合が、ペプチドにより阻害される 力、どうかを検討した。ペプチド R1および R2を添加した系は、ペプチドを添加していな い対照と比べて、色濃度が低ぐ C lqへの結合が大幅に阻害されていることがわかつ た。 9残基のアミノ酸からなるペプチド R2だけでなぐ 6残基のアミノ酸からなるぺプチ ド R1でも結合が阻害されていることから、 C lqのェピトープは、 9残基のアミノ酸、また は 6残基のアミノ酸からなることが確認できた。 [0038] The ability of the peptide to inhibit the binding between ALP-labeled anti-C lq antibody (1/1000 dilution) and C lq was examined. It was found that the system to which peptides R1 and R2 were added was significantly inhibited from binding to C1q, which had a lower color density, compared to the control to which no peptide was added. The peptide R1 consisting of 6 amino acids, which is composed of only the peptide R2 consisting of 9 residues of amino acids, also inhibits the binding, so the epitope of C lq has 9 amino acids or 6 residues It was confirmed to be composed of the amino acids.
[0039] 次に、 HRP標識抗 C lq抗体（1/20, 000希釈）と C lqの結合の阻害に必要なぺ プチドの濃度を検討した。 125、 62. 5、 31. 3、および 15 · 6 μ g/ml ペプチド Rl を抗 C lq抗体と混合し、 C lqとの結合を発色強度で評価した。 15. 6 g/mlの濃度 のペプチドを用いた場合に、抗 C lq抗体と C lqの結合が見られた力 S、それ以上の濃 度のペプチドを用いた場合には、抗 C l q抗体と C lqの結合が阻害され、発色強度が 下がっていることを確認した。この結果から、抗 C lq抗体と C lqとの結合は抗体濃度 にも依存する力 15. 6-31. 3 g/mlの濃度範囲の R1ペプチドで阻害されること カゎカゝつた。 [0039] Next, the concentration of the peptide necessary for inhibiting the binding of HRP-labeled anti-Clq antibody (diluted 1 / 200,000) and Clq was examined. 125, 62.5, 31.3, and 15 · 6 μg / ml peptide Rl was mixed with anti-C lq antibody, and the binding to C lq was evaluated by color intensity. 15. When using a peptide at a concentration of 6 g / ml, the force S was found to be binding between the anti-C lq antibody and C lq, and when using a peptide with a higher concentration, the anti-C lq antibody It was confirmed that the binding between C and qq was inhibited and the color intensity decreased. From this result, it was found that the binding between the anti-Clq antibody and Clq was inhibited by the R1 peptide in a concentration range of 15.6.3-31.3 g / ml depending on the antibody concentration.
実施例 3 Example 3
[0040] サンドイッチ ELISAによる C lqタンパク質の定量 [0040] Quantification of C lq protein by sandwich ELISA
5 μ g/mL 抗 C lqモノクローナル抗体（固相抗体）溶液 100 μ 1を ELISA用マイク 口プレートに結合させた。 1 % スキムミルク/ PBS 100 ^ 1を添カロし、室温にて 30〜 60分間インキュベーションした。次に、 10 g/mL C lqタンパク質抗原 2倍連続希 釈溶液 100 1を添加して、 37°Cで 1〜2時間反応させた。純水で 2回洗浄後、 5 μ g/mL Clqモノクローナル抗体（感作抗体）溶液 100 1を添加し、 4°Cにてー晚反 応させた。次に、 2次抗体（アビジン化 ALP抗体）を 100 1/ゥエルにて添加し、 4°C 〜室温で;!〜 2時間反応させた。純水で 4回洗浄後、 ALP基質発色液を添加して発 色させた。マイクロプレートリーダにて 492nmにおける吸光度（OD)を測定した（参 照波長： 660nm)。結果を図 2〜； 10に示す。 Clqタンパク質濃度と吸光度との間に 正の相関関係が認められ、抗 Clqモノクローナル抗体を用いたサンドイッチ ELISA 法により、試料中の Clqタンパク質を定量できることが分かった。図示してはいないが 、濃度〜 400 g/mLの Clqタンパク質について定量可能であった。 100 μl of a 5 μg / mL anti-Clq monoclonal antibody (solid phase antibody) solution was bound to the ELISA mouthplate. 1% skim milk / PBS 100 ^ 1 was added and incubated at room temperature for 30-60 minutes. Next, 10 g / mL Clq protein antigen 2-fold continuous dilution solution 1001 was added and reacted at 37 ° C for 1-2 hours. 5 μm after washing twice with pure water 100 1 of a g / mL Clq monoclonal antibody (sensitized antibody) solution was added and reacted at 4 ° C. Next, a secondary antibody (avidinated ALP antibody) was added at 100 1 / well, and reacted at 4 ° C to room temperature;! ~ 2 hours. After washing 4 times with pure water, ALP substrate coloring solution was added to cause color development. Absorbance (OD) at 492 nm was measured with a microplate reader (reference wavelength: 660 nm). The results are shown in FIGS. A positive correlation was observed between Clq protein concentration and absorbance, and it was found that Clq protein in the sample could be quantified by sandwich ELISA using anti-Clq monoclonal antibody. Although not shown, it was quantifiable for Clq protein at concentrations of ~ 400 g / mL.
産業上の利用可能性 Industrial applicability
本発明により、 Clqタンパク質の定量に用いることができる、抗 Clqモノクローナル 抗体、力、かる抗体を用いた Clqタンパク質の検出および/または定量方法、および そのためのキットなどが得られるので、 RA患者の予後診断、（RAの病理学的)研究 等の分野において極めて有用である。 According to the present invention, an anti-Clq monoclonal antibody that can be used for quantification of Clq protein, a method for detecting and / or quantifying Clq protein using such antibody, and a kit therefor can be obtained. It is extremely useful in fields such as diagnostics and (RA pathological) research.
Claims
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| KR20180100944A (en) * | 2017-03-03 | 2018-09-12 | 서울대학교병원 | Peptide derived from adiponectin and anti-aging or anti-wrinkle composition for skin comprising the same |
| CN112638358A (en) * | 2018-09-03 | 2021-04-09 | (株)郑振镐效果 | Hair growth promoting composition comprising adiponectin-derived peptide |
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| CN102361979A (en) * | 2009-01-23 | 2012-02-22 | 株式会社Mmt | Peptides with Immunoglobulin Binding Ability |
| EP2383336A4 (en) * | 2009-01-23 | 2013-01-23 | Mmt Co Ltd | Peptide capable of binding to immunoglobulin |
| CN102942616A (en) * | 2009-01-23 | 2013-02-27 | 株式会社Mmt | Peptide capable of binding to immunoglobulin |
| WO2012043397A1 (en) * | 2010-10-01 | 2012-04-05 | 株式会社エム・エム・ティー | Peptide capable of binding to immunoglobulin |
| WO2012067267A1 (en) * | 2010-11-18 | 2012-05-24 | Takahiro Ochi | TESTING METHOD USING ANTI-C1q MONOCLONAL ANTIBODY |
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