WO2015152280A1 - 抗コンドロイチン硫酸e抗体 - Google Patents
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- WO2015152280A1 WO2015152280A1 PCT/JP2015/060226 JP2015060226W WO2015152280A1 WO 2015152280 A1 WO2015152280 A1 WO 2015152280A1 JP 2015060226 W JP2015060226 W JP 2015060226W WO 2015152280 A1 WO2015152280 A1 WO 2015152280A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/04—Immortalised cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
- G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
Definitions
- the present invention relates to a novel anti-chondroitin sulfate E antibody that reacts with chondroitin sulfate E but does not react with chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate D.
- CS is a kind of GAG, and is a linear polymer polysaccharide composed of a repeating structure of GlcA and GalNAc disaccharides.
- the number of sulfate groups bonded to the disaccharide unit, the difference in the bonding position, and the presence of uronic acid isomers diversify the CS structure.
- isomers of CS, CSA, CSB (also referred to as DS), CSC, CSD, CSE and the like exist.
- CSE is a sulfated polysaccharide having a repeating structure of a basic disaccharide unit [GlcA ⁇ 1-3GalNAc (4S, 6S)] in which positions 4 and 6 of GlcA and GalNAc are modified by sulfation.
- Non-patent Document 1 As an antibody against CSE, Ch-1 antibody and Ch-3 antibody (Patent Document 1), CSD, which have been reported to react with CSB having a repeating structure of CSE and a basic disaccharide unit [IdoA ⁇ 1-3GalNAc (4S)], CSD MO-225 antibody that mainly recognizes the D structure [GlcA (2S) ⁇ 1-3GalNAc (6S)], which is a basic disaccharide unit, but has also been reported to react with CSE (Non-patent Documents 1 and 2) 3), GD3G7 antibody (Non-patent Document 4) reported to react with CSE, shark skin-derived DS, and CSA is known.
- An object of the present invention is to provide an antibody that reacts with high specificity to CSE and can be used for detection or isolation of a specific sulfated polysaccharide.
- the present inventor tried to produce a novel anti-CSE antibody using a substance obtained by binding lipid and CSE as an antigen.
- the present inventor has found that the obtained antibody reacts with CSE and does not react with CSA, CSB and CSD, and has invented an anti-CSE antibody that reacts specifically with CSE.
- the present invention is as follows. (1) An antibody that reacts with chondroitin sulfate E but does not react with chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate D. (2) The antibody according to (1), which does not react with any of chondroitin sulfate C, fully desulfated chondroitin sulfate C, heparin, heparan sulfate, heparosan, keratan sulfate, hyaluronic acid and 6-O-desulfated chondroitin sulfate E. .
- the antibody according to (1) or (2) which does not react with sulfated chondroitin sulfate B.
- the antibody according to any one of (1) to (3) which is obtained by immunization using a substance formed by binding lipid and chondroitin sulfate E as an antigen.
- the antibody according to (4), wherein the lipid is dipalmitoyl-L- ( ⁇ -phosphatidyl) ethanolamine.
- the antibody according to any one of (1) to (5) which is a monoclonal antibody.
- a hybridoma that produces the monoclonal antibody according to (6) A hybridoma that produces the monoclonal antibody according to (6).
- the hybridoma according to (8) which is formed by cell fusion between an antibody-producing cell derived from an animal immunized with a substance formed by binding lipid and chondroitin sulfate E as an antigen and a tumor cell.
- the hybridoma according to (9), wherein the lipid is dipalmitoyl-L- ( ⁇ -phosphatidyl) ethanolamine.
- a method for detecting chondroitin sulfate E present in a sample comprising at least a step of bringing the antibody according to any one of (1) to (7) into contact with the sample.
- a detection kit for chondroitin sulfate E comprising at least the antibody according to any one of (1) to (7).
- the antibody of the present invention specifically reacts with CSE, it can be suitably used for detection of CSE. Moreover, the antibody of the present invention can be efficiently produced by using the hybridoma of the present invention. Further, the CSE can be detected with high accuracy by the CSE detection method of the present invention. Further, by using the CSE detection kit of the present invention, CSE can be detected more efficiently and simply by the CSE detection method of the present invention.
- FIG. 5 is a diagram (photograph) showing the staining ability of E-12C antibody on tissues treated with C-ABC.
- the present invention provides antibodies that react with CSE and do not react with CSA, CSB and CSD.
- CSE is a sulfated polysaccharide having a repeating structure of a basic disaccharide unit [GlcA ⁇ 1-3GalNAc (4S, 6S)] in which positions 4 and 6 of GlcA and GalNAc are modified by sulfation.
- the CSE to which the antibody of the present invention reacts may be natural or chemically synthesized as long as it is such a sulfated polysaccharide.
- the thing derived from a squid is mentioned, for example.
- Examples of chemically synthesized compounds include those chemically synthesized from CSA (hereinafter also referred to as sulfated CSA) by the method described in Example 6 (1) described later.
- the abundance ratio of the basic disaccharide unit in such CSE is preferably 15% or more, more preferably 30% or more, further preferably 40% or more, and 50% or more. Is very preferable, 60% or more is particularly preferable, and 70% or more is most preferable.
- the abundance ratio of basic disaccharide units can be determined by the method described in the examples described later.
- the “reaction” of an antibody means an immunological reaction or an antigen-antibody reaction and can be evaluated by binding to an antigen.
- the binding between the antibody and the antigen can be examined by conventional methods such as ELISA, RIA, plaque, agglutination, flow cytometry, tissue staining, Western blot, surface plasmon resonance, and the like.
- Examples of the ELISA method include the method described in Example 3 (2) described later. For example, when an ELISA method is performed using an antibody, if the reaction signal is enhanced in proportion to an increase in the concentration of the antigen, it can be said that the antibody reacts with the antigen.
- the reactivity of the antibody of the present invention to the antigen is, for example, relative reactivity based on the reactivity of the antibody of the present invention to CSE measured by the method described in Example 3 (2) described later. be able to.
- “does not react” means that the reactivity of an antigen and an antibody is comparable to that of a negative control or a blank.
- the difference between the reactivity of antigen and antibody and the negative control or blank is less than 10%, preferably 5% Less than, more preferably less than 4%, even more preferably less than 3%, very particularly preferably less than 2%, particularly preferably less than 1%, most preferably less than 0.5%, it is judged as "not reacting" be able to.
- the antibody of the present invention is not particularly limited as long as it reacts with CSE and does not react with CSA, CSB and CSD, but also reacts with CSC, deS-CSC, Hep, HS, HPN, KS, HA, and de6S-CSE. It is preferable that the antibody does not react with sulfated CSB.
- the sulfated CSB refers to that obtained by the method described in Example 6 (1) described later.
- the class of the antibody of the present invention is not particularly limited, but is preferably IgM, more preferably IgM or ⁇ chain.
- the type of the antibody of the present invention is not particularly limited, and may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferable.
- the monoclonal antibody may be a part of an antibody, that is, an antibody fragment.
- the antibody fragment include Fab, F (ab ′) 2 , Fab ′, Fv and the like.
- Such an antibody fragment can be obtained, for example, by digesting a monoclonal antibody with peptidase or the like.
- the Fab fragment can be obtained by treating the monoclonal antibody of the present invention with papain
- the F (ab ′) 2 fragment can be obtained by treating the monoclonal antibody of the present invention with pepsin.
- the method for obtaining the antibody of the present invention will be described in the following items. However, the method for obtaining the antibody of the present invention is not limited to the following. 1. 1. Preparation of antigen 2. Immunization method Acquisition of polyclonal antibody 4. Acquisition of monoclonal antibodies 5. Production of hybridoma Hybridoma selection and cloning
- the antibody of the present invention can be obtained, for example, by immunizing an animal that can produce an antibody using a substance formed by binding lipid and CSE as an antigen in order to enhance the antigenicity of CSE.
- CSE is not particularly limited, but those derived from squid are preferred.
- the lipid is not particularly limited as long as it binds to CSE, but phospholipid is preferable, and PE is more preferable.
- the bond between the lipid and CSE is not particularly limited, but is preferably a chemical bond, more preferably a covalent bond, and even more preferably an aminoalkyl bond.
- GAG and PE can be combined with CSE and PE by the method of lactonizing the reducing end described in JP-A-4-80201, the method of limited oxidation at the reducing end described in JP-A-4-80202, And a preparation method through an intermediate of a GAG-tetrabutylammonium (GAG-But 4 N + ) salt described in Kaikai 2003-335801.
- GAG-But 4 N + GAG-tetrabutylammonium
- the animal to be immunized is not particularly limited as long as it can produce an antibody that reacts with CSE, but is preferably an animal such as a pig, cow, rabbit, sheep, chicken, rat, mouse, and the like. Is preferred.
- the adjuvant for immunization is not particularly required, but when it is used, those that are expected to have an adjuvant effect such as alum, gram positive bacteria, gram negative bacteria, nucleic acids and the like are preferable, and Salmonella cell membrane is more preferable.
- Immunization can be performed, for example, by intravenously injecting an antigen and an adjuvant into an animal.
- the injection method is not limited to this, and may be subcutaneous injection or intraperitoneal injection. Usually, administration is carried out in several parts.
- the polyclonal antibody can be obtained by collecting blood from the immunized animal. Whether or not a polyclonal antibody is present in the collected blood can be evaluated using an antibody reaction as an index. Examples of the antibody reaction measurement method include ELISA method, RIA method, plaque method, agglutination reaction method, flow cytometry method, tissue staining method, Western blot method and the like.
- the polyclonal antibody may be collected blood itself, may be serum obtained by centrifugation, or may be purified. Purification of the antibody can be achieved by appropriately combining biochemical techniques such as salting out, ion exchange, gel filtration, affinity chromatography, electrophoresis and the like.
- a method for obtaining a monoclonal antibody includes a method of culturing the obtained hybridoma in vitro or in vivo, and can be selected according to the purpose. In the case of in vitro, it can be obtained by collecting the cell culture medium when the hybridoma is cultured, and in the case of in vivo, it can be obtained from the ascites of the mouse transplanted with the hybridoma.
- a cell culture solution or ascites may be used as it is, or a product obtained by centrifuging to remove cells or the like may be used, or a purified product may be used. Purification of the antibody can be achieved by appropriately combining biochemical techniques such as salting out, ion exchange, gel filtration, affinity chromatography, electrophoresis and the like.
- Monoclonal antibodies that react with CSE include monoclonal antibodies produced by hybridomas named E-12C and E-18H (hereinafter referred to as E-12C antibody and E-18H antibody).
- E-12C antibody and E-18H antibody react with CSE, and other GAGs (CSA, CSB, CSC, CSD, deS-CSC, Hep, HS, HPN, KS, HA, de6S-CSE, sulfated CSB ) Does not react.
- Hybridomas are produced by cell fusion of antibody-producing cells and tumor cells obtained by removing lymph nodes or spleens from immunized animals.
- the antibody-producing cells are preferably lymphocytes.
- Tumor cells are preferably myeloma cells, preferably derived from mammals, and more preferably derived from mice.
- the selection of the hybridoma can be performed, for example, using the growth rate of the hybridoma and the antibody reaction as an index.
- the antibody reaction measurement method include ELISA method, RIA method, plaque method, aggregation reaction method, flow cytometry method, tissue staining method, Western blot method, surface plasmon resonance method and the like.
- a hybridoma producing an antibody that reacts with CSE by the above method is selected, and the obtained hybridoma is cloned.
- Examples of cloning methods include limiting dilution. In the limiting dilution method, a 96-well plate is seeded so that the number of cells per well is 1 / well (5.0 cells / ml) or 0.5 / well (2.5 cells / ml). preferable.
- the cloning operation is preferably repeated twice or more, and preferably a single clone.
- hybridomas may include hybridomas named E-12C and E-18H.
- the CSE detection method of the present invention is not particularly limited as long as it includes a step of bringing the antibody of the present invention into contact with a sample.
- the above-mentioned “sample” is not particularly limited as long as it contains or has a possibility of containing CSE.
- sources of the sample include urine, blood, plasma, serum, joint fluid, spinal cord.
- body fluids such as fluid, secretions, cells such as animal cells or plant cells, tissues, organs, cultures of cells or microorganisms, and the like.
- a specific method for detecting CSE present in a sample is not particularly limited, but ELISA method, RIA method, plaque method, agglutination reaction method, flow cytometry method, tissue staining method, Examples include western blotting and surface plasmon resonance.
- the detection in the CSE detection method of the present invention may be quantitative detection or qualitative detection.
- the CSE concentration present in the sample is prepared by, for example, preparing a calibration curve for the relationship between the CSE concentration and the detection result using a CSE standard solution having a known concentration in advance, and the sample having an unknown CSE concentration. This can be done by comparing the detection result for and the calibration curve.
- the CSE detection kit of the present invention is not particularly limited as long as it contains at least the antibody of the present invention. According to the CSE detection kit of the present invention, the CSE detection method of the present invention can be performed more simply.
- the “kit including at least the antibody of the present invention” includes, for example, a kit including the antibody of the present invention itself (for example, the antibody of the present invention dissolved in a solution) as a constituent component, and a solid phase on which the antibody of the present invention is fixed. Examples include kits containing the components, kits containing the antibodies of the present invention labeled with enzymes, and the like as components.
- the components of the CSE detection kit of the present invention may contain, for example, a secondary antibody, a reaction buffer, a washing solution, a reaction substrate, a CSE standard solution, and the like.
- the method for producing the antibody of the present invention is not particularly limited as long as it includes at least a step of immunizing a substance formed by binding lipid and chondroitin sulfate E as an antigen.
- the specific method for producing the antibody is described in 1. above. ⁇ 6. And the methods described in the examples described later can be used.
- CSE having a weight average molecular weight of 14.6 kDa, a sulfur content of 10.0%, and an E structure content of 70% or more was obtained (analysis method of CSE is as follows).
- the CSE was designated as antigen CSE.
- the average molecular weight was determined using a CS molecular weight standard as a control.
- the molecular weight of standard CS was measured by a light scattering method.
- the calculated weight average molecular weight (Mw) and number average molecular weight (Mn) of CSE were 14.6 kDa and 7.7 kDa, respectively, and the molecular weight dispersity (Mw / Mn) was 1.8. there were.
- the disaccharide composition was analyzed using HPLC (Waters). Using a reverse phase column (SenshuhPak DOCOSIL SP100, DC-A151-SP, 1 mm ID ⁇ 15 cm, Senshu Science), the fluorescence wavelength at an excitation wavelength of 346 nm and a measurement wavelength of 410 nm was measured.
- the eluents are: A) DW (gradient), B) 0.2M NaCl (gradient), C) 10 mM mM Tetra-n-butylammonium hydrogen sulfate, 12% (always), D) 50% Acetonitrile, 17% (always) Was allowed to flow at a flow rate of 1.1 ml / min (Alliance 2695), and 0.5% 2-cyanoacetamide and 0.25M sodium hydroxide were allowed to flow at 0.7 ml / min (600E). The column temperature was developed at 55 ° C.
- Analysis of disaccharide composition is the amount of unsaturated disaccharide that can be identified obtained by disaccharide analysis by enzyme digestion ( ⁇ Di-0S, ⁇ Di-4S, ⁇ Di-6S, ⁇ Di-2S, ⁇ Di ⁇ (4,6) S, ⁇ Di ⁇ (2,4) S, ⁇ Di ⁇ (2,6) S, ⁇ Di ⁇ (2,4,6) S is the total of mol%), and the ratio of disaccharides having a specific structure This value reflects the sulfation of the sulfated polysaccharide before enzymatic digestion.
- the powder was analyzed according to the above (1) molecular weight analysis and (2) disaccharide composition analysis.
- the weight average molecular weight (Mw) and number average molecular weight (Mn) were 12.7 kDa and 6.5 kDa, respectively, and the molecular weight dispersity (Mw / Mn) was 1.9.
- the disaccharide composition is ⁇ Di-0S 18.0%, ⁇ Di-4S 82.0%, ⁇ Di-6S 0%, ⁇ Di-2S 0%, ⁇ Di- (4,6) S 0%, ⁇ Di- (2,4 ) S 0%, ⁇ Di- (2,6) S 0%, ⁇ Di- (2,4,6) S 0%, and it was confirmed that the 6-position sulfate group was selectively desulfated.
- deS-CSC Completely Desulfated CSC
- the CSC powder was analyzed according to the above (1) molecular weight analysis and (2) disaccharide composition analysis.
- the disaccharide composition of the CSC before desulfation is as follows: ⁇ Di-0S 1.9%, ⁇ Di-4S 17.8%, ⁇ Di-6S 68.2%, ⁇ Di-2S 0%, ⁇ Di- (4,6) S 1 .3%, ⁇ Di- (2,4) S 0%, ⁇ Di- (2,6) S 10.8%, ⁇ Di- (2,4,6) S 0%.
- the disaccharide composition was ⁇ Di-0S 91.2%, ⁇ Di-4S 0%, ⁇ Di-6S 8.7%, ⁇ Di-2S 0%, ⁇ Di- (4,6) S 0%, ⁇ Di- (2,4 ) S 0%, ⁇ Di- (2,6) S 0%, ⁇ Di- (2,4,6) S 0%, confirming desulfation.
- the freeze-dried powder was dissolved in 450 ⁇ l of PBS, 50 ⁇ l of biotin solution (Biotin 10 mg / 222 ⁇ l in DMF) was added, and reacted at room temperature for 30 minutes. After the reaction, the dialysis membrane (MWCO 12000-14000) was dialyzed overnight, the solution was lyophilized, and biotin-labeled GAG (CSE, CSA, CSB, CSC, CSD, deS-CSC, Hep, HS, HPN, KS) , HA, de6S-CSE).
- biotin-labeled GAG CSE, CSA, CSB, CSC, CSD, deS-CSC, Hep, HS, HPN, KS
- CSE-But 4 N + salt CSE tetrabutylammonium salt
- 1.0 g of CSE prepared in Reference Example 1 was dissolved in 50 ml of distilled water, and Dowex 50W-X8 H + form (217514-500 g, SIGMA) column (2.5 cm ⁇ ⁇ 6.5 cm) was applied.
- CSE which became sodium salt free was collected as an acidic fraction of the eluted solution.
- Tetrabutylammonium (10% in Water) T0955, Tokyo Chemical Industry Co., Ltd.
- the solution was lyophilized at room temperature for 3 days to recover 1.85 g of CSE-But 4 N + salt as a dry powder.
- the column solution was taken out, washed with 200 ⁇ m of 0.2 ⁇ M NaCl, and then eluted with 50 ⁇ m of distilled water and 200 ⁇ m of a 30% (v / v) methanol-distilled water mixture (methanol elution fraction).
- the methanol-eluted fraction was concentrated to dryness under reduced pressure and redissolved with distilled water.
- the powder was recovered by lyophilization. The weight of the recovered CSE-PE powder was 50 mg.
- Example 2 Obtaining a hybridoma producing a monoclonal antibody that reacts with CSE (1) Preparation of antigen for immunization CSE-PE prepared in Example 1 was dissolved in PBS to a final concentration of 2 mg / ml. It was heated to 37 ° C. to a concentration of 0.1 mg / ml. To this, 1136 ⁇ l of a solution obtained by suspending the acid-treated Salmonella minnesota cell membrane in 1 mg / ml in PBS in advance was added and allowed to stand at 37 ° C. for 10 minutes. This mixed solution was dispensed into 200 ⁇ l and stored at ⁇ 20 ° C.
- 1% human serum albumin-PBS ⁇ ⁇ ⁇ was added as a blocking solution to 200 ⁇ l / well and left at room temperature for 1 hour. After removing the blocking solution, the plate was washed once with PBS 200 ⁇ l / well.
- the mouse serum prepared in the above (1) diluted with PBS was added at 50 ⁇ l / well and reacted at room temperature for 1 hour. Serum was removed and washed 3 times with PBS 200 ⁇ l / well.
- Horse-radish peroxidase -labeled goat anti-mouse IgG + IgM diluted 2000-fold with 1% humanerserum albumin-PBS was added at 50 ⁇ l / well and allowed to react at room temperature for 1 hour. After removing the secondary antibody solution, the plate was washed 3 times with PBS 200 ⁇ l / well.
- Color development and measurement were performed by dissolving 2 mg of o-phenylenediamine in 5 ml of a color development solution [80 mM citrate-phosphate buffer (pH 5.6), and then adding 2 ⁇ l of 30% hydrogen peroxide solution. ] was added at 100 ⁇ l / well.
- color development was observed after standing in the dark, color development was stopped with 100 ⁇ l / well of 1 N HCl solution.
- PAI cells (JCRB cell bank, JCRB0113) were cultured at 37% with 10% FBS-RPMI [final medium concentration 10% FBS, 1% PSG, 10 mM HEPES, 1 mM sodium pyruvate]. The cells were cultured at 5 ° C. in the presence of 5% CO 2 .
- mice were aseptically removed from the mice 32 days after the start of immunization (7 days after the final immunization) and loosened on a petri dish containing 10% FBS-RPMI medium. This was centrifuged at 1300 rpm for 5 minutes and then resuspended in 10% FBS-RPMI medium, and the tissue in the suspension was removed with a Celestor strainer. The precipitate centrifuged at 1300 rpm for 5 minutes was resuspended in 20 ml of 10% FBS-RPMI medium, and spleen cells were collected.
- the colonies with positive culture supernatant and fast growth were cultured in 6-well plates with different well areas. Up to 6-well plates, four screening tests were performed to determine antibody titers.
- Cloning The cells whose culture supernatant was positive in the above (6) were cloned by the limiting dilution method.
- cloning medium CM-B (410022517, Adia) was used.
- the number of cells is diluted to 1 / well (5.0 cells / ml) and 0.5 / well (2.5 cells / ml), respectively, seeded in a 96-well microplate, and 2 per clone.
- the cells were cultured for 10 to 14 days in a 96-well plate. Similar to the above (5), clones that were positive after 4 screening tests were selected.
- Example 3 Characteristics of Monoclonal Antibody (1) Production of Monoclonal Antibody A monoclonal antibody that reacts with the target CSE was obtained by culturing the above hybridoma and removing the cells from the culture solution by centrifugation (culture supernatant). E-12C antibody was obtained from hybridoma E-12C, and E-18H antibody was obtained from hybridoma E-18H.
- a biobind strip assembly (95029-293, Thermo Fisher Scientific), which is a streptavidin-coated plate, was added with 1 ⁇ g / ml of various biotin-labeled GAGs diluted with PBS, and allowed to stand at room temperature for 1 hour for immobilization. Similarly, Biotin solution and PBS of the same concentration were added to the plate so as to be 50 ⁇ l / well, respectively, and used as a negative control and a blank.
- Example 4 Measurement of antibody subtypes Using the IsoQuick (TM) mouse monoclonal antibody isotype determination kit (ISOQ5, Sigma) and the IsoQuick (TM) mouse monoclonal antibody isotype determination strip (i9410-25EA, Sigma), the E-12C antibody and the E- The 18H antibody subtype was determined.
- TM mouse monoclonal antibody isotype determination kit
- TM mouse monoclonal antibody isotype determination strip
- the tip of the IsoQuick® kit was immersed in the E-12C antibody and E-18H antibody (culture supernatant) obtained from Example 3 (1) for 5 minutes, and the procedure was completed when a band was confirmed.
- the type to which the antibody belongs was determined from the position of the appearing band.
- both the E-12C antibody and the E-18H antibody were a single band of IgM, and the L chain was a ⁇ chain.
- Example 5 Production of CSE detection kit A CSE detection kit having the following constitution was produced. 1. Monoclonal antibody obtained from Example 3 (1) 2.2 Secondary antibody (horse-radish peroxidase labeled goat anti-mouse IgG + IgM) 3. Coloring solution set (80 mM citrate-phosphate buffer solution (pH 5.6), o-Phenylenediamine, 30% hydrogen peroxide solution) 4). Reaction stop solution (1 N HCl)
- the CSA-TBA salt was dissolved in a solvent mixed with formamide / dimethylformamide 1: 4, and sulfur trioxide pyridine complex as a sulfating agent was added.
- the amount of sulfating agent added was 8 equivalents per disaccharide unit.
- Distilled water was added to the solution after the sulfation reaction to stop the reaction, and neutralized with sodium hydroxide.
- the solution was dialyzed overnight with a MWCO 12000 to 14000 dialysis membrane, and then freeze-dried to obtain 160 mg of sulfated CSA.
- a sulfated CSA can be prepared by introducing a sulfate group into the 6 position of GalNAc of CSA (Carbohydr. Res. 158, 183-190 (1986)).
- CSB was sulfated in the same manner to obtain 160 mg of sulfated CSB.
- the content of the ⁇ Di- (4,6) S structure of CSA-8S sulfated at the 6-position of CSA whose basic disaccharide unit is [GlcA ⁇ 1-3GalNAc (4S)] was 59.4%.
- a sulfated CSA mainly containing CSE basic disaccharide unit [GlcA ⁇ 1-3GalNAc (4S, 6S)] (hereinafter also referred to as E structure) was prepared.
- the content of the ⁇ Di- (4,6) S structure of CSB-8S in which the 6-position of CSB having [IdoA ⁇ 1-3GalNAc (4S)] as the basic disaccharide unit is sulfated was 66.0%.
- a sulfated CSB mainly containing the basic disaccharide unit [IdoA ⁇ 1-3GalNAc (4S, 6S)] was prepared.
- the sulfated CSA and sulfated CSB prepared above were biotinylated by the method described in “Preparation of biotin-labeled GAG of Reference Example 5” to prepare biotinylated sulfated CSA and biotinylated sulfated CSB.
- E-12C antibody and the E-18H antibody react not only with squid-derived CSE but also with CSE (sulfated CSA) chemically synthesized from CSA.
- CSE having different molecular weights can be prepared by enzyme treatment and column chromatography (J. Biol. Chem. 236, 983-987 (1961), J. Biol. Chem. 252). 4570-4576 (1977)). Five types of CSE having different molecular weights were prepared with reference to the literature.
- CSE having a weight average molecular weight (Mw) of 68.0 kDa and an E structure content of 61.4% was designated as CSE-68K.
- CSE having a weight average molecular weight (Mw) of 56.7 kDa and an E structure content of 61.0% was designated as CSE-56K.
- CSE having a weight average molecular weight (Mw) of 24.1 kDa and an E structure content of 63.0% was designated as CSE-24K.
- CSE having a weight average molecular weight (Mw) of 14.6 kDa and an E structure content of 70.4% was designated as CSE-14K. This CSE-14K is the same as that used for the immunizing antigen for antibody production.
- CSE having a weight average molecular weight (Mw) of 8.7 kDa and an E structure content of 66.7% was designated as CSE-8K. These five types of CSE were used.
- the KD values of CSE-14K, E-12C antibody, and E-18H antibody were 3.6 ⁇ 10 ⁇ 10 (M) and 2.5 ⁇ 10 ⁇ 10 (M), respectively, indicating strong interaction as an antigen-antibody reaction. Indicated. The reactivity of the E-12C antibody and the E-18H antibody with de6S-CSE selectively desulfated at position 6 of the antigen CSE was not detected (ND).
- the KD values of E-12C antibody and CSE-68K are 1.7 ⁇ 10 ⁇ 11 (M), CSE-56K is 3.4 ⁇ 10 ⁇ 11 (M), and CSE-24K is 2.3 ⁇ 10 ⁇ 10. (M) and CSE-14K were 3.6 ⁇ 10 ⁇ 10 (M), and CSE-8K was 3.7 ⁇ 10 ⁇ 10 (M).
- the KD value of the E-18H antibody and CSE-68K is 1.5 ⁇ 10 ⁇ 11 (M), 2.5 ⁇ 10 ⁇ 11 (M) with CSE-56K, and 1.2 ⁇ 10 ⁇ 10 with CSE-24K.
- CSE-14K was 2.6 ⁇ 10 ⁇ 10 (M)
- CSE-8K was 2.9 ⁇ 10 ⁇ 10 (M).
- Each KD value was 1 ⁇ 10 ⁇ 9 (M) or less, and it was confirmed that a strong interaction was exhibited.
- CSA-2S a product prepared by adding 2 equivalents of a sulfur trioxide pyridine complex as a sulfating agent
- CSA-2S a product prepared by adding 6 equivalents
- CSA-4S a product prepared by adding 6 equivalents
- CSA-4S Indicated as ⁇ 6S.
- the E structure content of CSA-2S is 14.1%
- the E structure content of CSA-4S is 32.7%
- the E structure content of CSA-6S is 45.9%.
- an E structure was generated.
- Table 9 The results of CSA, CSA-8S, CSB and CSB-8S described in “Table 4 of Example 6” are shown together in Table 9.
- E-12C antibody with CSA and CSA-2S Reactivity of E-12C antibody with CSA and CSA-2S was not detected.
- the KD value of E-12C antibody and CSA-4S is 1.0 ⁇ 10 ⁇ 8 (M), CSA-6S is 3.5 ⁇ 10 ⁇ 9 (M), and CSA-8S is 7.8 ⁇ 10 ⁇ 10. (M).
- Reactivity of E-18H antibody with CSA and CSA-2S was not detected.
- the KD values of E-18H antibody and CSA-4S are 9.4 ⁇ 10 ⁇ 9 (M), 1.8 ⁇ 10 ⁇ 9 (M) for CSA-6S, and 4.3 ⁇ 10 ⁇ 10 for CSA-8S. (M).
- Example 8 The tissue staining property of the anti-CSE antibody was examined as follows. (1) Immune tissue staining in frozen sections Squid cartilage was fragmented, and tissue sections were embedded in tissue-tech OCT compound (Sakura Finetech Japan Co., Ltd.) and frozen in liquid nitrogen to prepare frozen sections. .
- the prepared sections were fixed with 10% neutral buffered formalin solution at room temperature for 10 minutes, and then washed twice with 50 mmol / L Tris-HCl buffer.
- the plate was washed 3 times with 0.01 mol / L phosphate buffered saline and blocked with 0.3% hydrogen peroxide-containing methanol at room temperature for 10 minutes.
- the plate was washed 3 times with 0.01 mol / L phosphate buffered saline and blocked with a 0.1% casein solution (Wako Pure Chemical Industries, Ltd.) at room temperature for 60 minutes.
- Anti-CSE antibody (E-12C or E-18H) diluted to 12 ⁇ g / ml with 0.1% casein solution was reacted at 37 ° C. for 60 minutes. At this time, mouse ⁇ IgM (Sigma) was used as a negative control.
- biotin-labeled anti-mouse IgG + IgA + IgM antibody Histfine SAB-PO (M) kit, Nichirei Bioscience
- Histfine SAB-PO (M) kit Nichirei Bioscience
- peroxidase-labeled streptavidin Histofine SAB-PO (M) kit, Nichirei Bioscience
- the plate was washed 3 times with 0.01 mol / L phosphate buffered saline, immersed in 50 mmol / L Tris-HCl buffer for 5 minutes, and then developed with DAB (Dojindo Laboratories) for 5 minutes. After washing with ultrapure water, dehydration and penetration operations were performed according to a conventional method and sealed.
- the prepared sections were deparaffinized (xylene) and hydrophilized (alcohol descending series: 99.9%, 95%, 80%, 70% ethanol) and then washed twice with 50 mmol / L Tris-HCl buffer. .
- the plate was washed 3 times with 0.01 mol / L phosphate buffered saline and blocked with 0.3% hydrogen peroxide-containing methanol at room temperature for 10 minutes.
- the plate was washed three times with 0.01 mol / L phosphate buffered saline, and blocked with an avidin solution (Histofine, endogenous avidin / biotin blocking kit, Nichirei Bioscience) at room temperature for 10 minutes.
- the plate was washed 3 times with 0.01 mol / L phosphate buffered saline, and blocked with a biotin solution (Histofine Endogenous Avidin / Biotin Blocking Kit, Nichirei Bioscience) at room temperature for 10 minutes.
- the plate was washed 3 times with 0.01 mol / L phosphate buffered saline and blocked with a 0.1% casein solution (Wako Pure Chemical Industries, Ltd.) at room temperature for 60 minutes.
- Anti-CSE antibody (E-12C or E-18H) diluted to 12 ⁇ g / ml with 0.1% casein solution was reacted at 37 ° C. for 60 minutes. At this time, mouse ⁇ IgM (Sigma) was used as a negative control.
- FIG. Staining was observed for both E-12C antibody and E-18H antibody. No staining was seen with mouse IgM used as a negative control.
- the squid cartilage was cut into small pieces, the tissue piece was embedded in Tissue Tech OCT compound (Sakura Finetech Japan Co., Ltd.), and frozen with liquid nitrogen to prepare a frozen section.
- the prepared section was fixed with 10% neutral buffered formalin for 10 minutes at room temperature, washed twice with 50 mmol / L Tris-HCl buffer, and then diluted to 250 mU / ml with 50 mmol / L Tris-HCl buffer. Digested with ABC at 37 ° C for 120-180 minutes. C-ABC untreated control sections were soaked in 50 mmol / L Tris-HCl buffer at 37 ° C. for 120-180 minutes. The plate was washed 3 times with 0.01 mol / L phosphate buffered saline, and blocked with 0.3% hydrogen peroxide-containing methanol at room temperature for 10 minutes. The plate was washed 3 times with 0.01 mol / L phosphate buffered saline and blocked with a 0.1% casein solution (Wako Pure Chemical Industries, Ltd.) at room temperature for 60 minutes.
- Anti-CSE antibody (E-12C) diluted to 12 ⁇ g / ml with 0.1% casein solution was reacted at 37 ° C. for 60 minutes. At this time, mouse ⁇ IgM (Sigma) was used as a negative control.
- biotin-labeled anti-mouse IgG + IgA + IgM antibody Histfine SAB-PO (M) kit, Nichirei Bioscience
- Histfine SAB-PO (M) kit Nichirei Bioscience
- peroxidase-labeled streptavidin Histofine SAB-PO (M) kit, Nichirei Bioscience
- the plate was washed 3 times with 0.01 mol / L phosphate buffered saline, immersed in 50 mmol / L Tris-HCl buffer for 5 minutes, and then developed with DAB (Dojindo Laboratories) for 5 minutes. After washing with ultrapure water, dehydration and penetration operations were performed according to a conventional method and sealed.
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Abstract
Description
CSA:コンドロイチン硫酸A
CSB:コンドロイチン硫酸B
CSC:コンドロイチン硫酸C
CSD:コンドロイチン硫酸D
CSE:コンドロイチン硫酸E
DS:デルマタン硫酸
Hep:ヘパリン
HS:ヘパラン硫酸
HPN:ヘパロサン
KS:ケラタン硫酸
HA:ヒアルロン酸
deS-CSC:完全脱硫酸化コンドロイチン硫酸C
de6S-CSE:6-O-脱硫酸化コンドロイチン硫酸E
CS:コンドロイチン硫酸
GAG:グリコサミノグリカン
GlcA:グルクロン酸
IdoA:イズロン酸
GalNAc: N-アセチルガラクトサミン
PE:ジパルミトイル-L-(α-ホスファチジル)エタノールアミン
C-ABC:コンドロイチナーゼABC
PBS:リン酸緩衝生理食塩水
CSE-PE:ジパルミトイル-L-(α-ホスファチジル)エタノールアミン結合コンドロイチン硫酸E
本発明者は鋭意検討の結果、得られた抗体がCSEに反応し、CSA、CSB及びCSDに反応しないことを見出し、CSEと特異的に反応する抗CSE抗体を発明するに至った。
(1)コンドロイチン硫酸Eに反応し、コンドロイチン硫酸A、コンドロイチン硫酸B及びコンドロイチン硫酸Dに反応しない抗体。
(2)コンドロイチン硫酸C、完全脱硫酸化コンドロイチン硫酸C、ヘパリン、ヘパラン硫酸、ヘパロサン、ケラタン硫酸、ヒアルロン酸及び6-O-脱硫酸化コンドロイチン硫酸Eのいずれにも反応しない、(1)に記載の抗体。
(3)硫酸化コンドロイチン硫酸Bに反応しない、(1)又は(2)に記載の抗体。
(4)脂質とコンドロイチン硫酸Eとが結合してなる物質を抗原として免疫することによって得られる(1)~(3)のいずれかに記載の抗体。
(5)脂質がジパルミトイル-L-(α-ホスファチジル)エタノールアミンである、(4)に記載の抗体。
(6)モノクローナル抗体である、(1)~(5)のいずれかに記載の抗体。
(7)受領番号がNITE ABP-01808又はNITE ABP-01809であるハイブリドーマにより産生されるモノクローナル抗体。
(8)(6)に記載のモノクローナル抗体を産生するハイブリドーマ。
(9)脂質とコンドロイチン硫酸Eとが結合してなる物質を抗原として免疫した動物由来の抗体産生細胞と腫瘍細胞との細胞融合により形成される(8)に記載のハイブリドーマ。
(10)脂質がジパルミトイル-L-(α-ホスファチジル)エタノールアミンである、(9)に記載のハイブリドーマ。
(11)受領番号がNITE ABP-01808又はNITE ABP-01809であるハイブリドーマ。
(12)(1)~(7)のいずれかに記載の抗体を試料に接触させる工程を少なくとも含むことを特徴とする、当該試料中に存在するコンドロイチン硫酸Eの検出方法。
(13)(1)~(7)のいずれかに記載の抗体を少なくとも含む、コンドロイチン硫酸Eの検出キット。
(14) 脂質とコンドロイチン硫酸Eとが結合してなる物質を抗原として免疫する工程を少なくとも含むことを特徴とする、コンドロイチン硫酸Eに反応し、コンドロイチン硫酸A、コンドロイチン硫酸B及びコンドロイチン硫酸Dに反応しない抗体の製造方法。
(15) 脂質がジパルミトイル-L-(α-ホスファチジル)エタノールアミンである、(14)に記載の製造方法。
本発明はCSEに反応し、CSA、CSB及びCSDに反応しない抗体を提供する。
ここで、硫酸化CSBとは、後述する実施例6(1)に記載の方法により得られたものを指す。
1.抗原の調製
2.免疫方法
3.ポリクローナル抗体の取得
4.モノクローナル抗体の取得
5.ハイブリドーマの作製
6.ハイブリドーマの選択とクローニング
本発明の抗体は、例えば、CSEの抗原性を高めるために、脂質とCSEとが結合してなる物質を抗原として抗体を産生しうる動物を免疫することにより得ることができる。CSEは特に限定されないが、イカ由来であるものが好ましい。脂質は、CSEと結合するものであれば特に限定されないが、リン脂質が好ましく、PEであることがさらに好ましい。脂質とCSEとの結合は特に限定されないが、化学的な結合であることが好ましく、共有結合であることがより好ましく、アミノアルキル結合であることがさらに好ましい。CSEとPEの結合に用いることができるGAGとPEの結合方法は、特開平4-80201に記載の還元末端をラクトン化する方法、特開平4-80202に記載の還元末端の限定酸化方法、特開2003-335801に記載のGAG-テトラブチルアンモニウム(GAG-But4N+)塩の中間体を経た調製方法等が挙げられる。
免疫される動物は、CSEに反応する抗体を産生しうる動物であれば特に限定されないが、ブタ、ウシ、ウサギ、ヒツジ、ニワトリ、ラット、マウス等の動物であることが好ましく、マウスであることが好ましい。
免疫のアジュバントは、特に使用しなくてもよいが、使用する場合は、ミョウバン、グラム陽性菌、グラム陰性菌、核酸等、アジュバント効果が期待されるものが好ましく、サルモネラ菌体膜がより好ましい。
免疫は、例えば、抗原とアジュバントを動物に静脈注射することで行うことができる。注射方法は、これに限らず、皮下注射、腹腔内注射でもよい。通常、投与は数回に分けて実施する。
ポリクローナル抗体は、免疫した動物より、血液を採取することで取得することができる。採取した血液中にポリクローナル抗体が存在するか否かは、抗体反応を指標に評価することができる。抗体反応の測定方法としては、ELISA法、RIA法、プラーク法、凝集反応法、フローサイトメトリー法、組織染色法、ウェスタンブロット法等が挙げられる。ポリクローナル抗体は採取された血液そのものでもよく、遠心分離によって得られる血清でもよく、精製されたものでもよい。抗体の精製は、塩析、イオン交換、ゲルろ過、アフィニティークロマトグラフィー、電気泳動等、生化学的手法を適宜組み合わせることにより達成することができる。
モノクローナル抗体を取得する方法としては、得られたハイブリドーマを、in vitroあるいはin vivoで培養する方法が挙げられ、目的に応じて選択できる。in vitroの場合、ハイブリドーマを培養した際の細胞培養液を回収することで得られ、in vivoの場合、ハイブリドーマを移植したマウスの腹水から得ることができる。
モノクローナル抗体は、細胞培養液や腹水をそのまま使用してもよく、遠心分離し細胞等を除いたものを使用してもよく、精製したものを使用してもよい。
抗体の精製は、塩析、イオン交換、ゲルろ過、アフィニティークロマトグラフィー、電気泳動等、生化学的手法を適宜組み合わせることにより達成することができる。
E-12C抗体およびE-18H抗体は、CSEに反応し、他の各種GAG(CSA、CSB、CSC、CSD、deS-CSC、Hep、HS、HPN、KS、HA、de6S-CSE、硫酸化CSB)には反応しない特徴を有する。
ハイブリドーマは免疫した動物から、リンパ節又は脾臓を摘出することで得られる抗体産生細胞と腫瘍細胞とを細胞融合することにより作製される。抗体産生細胞はリンパ球が好ましい。腫瘍細胞はミエローマ細胞が好ましく、由来は哺乳動物由来が好ましく、マウス由来がより好ましい。
ハイブリドーマの選択は、例えば、ハイブリドーマの増殖速度と抗体反応を指標に行うことができる。抗体反応の測定方法としては、ELISA法、RIA法、プラーク法、凝集反応法、フローサイトメトリー法、組織染色法、ウェスタンブロット法、表面プラズモン共鳴法等が挙げられる。
上記の「試料」とは、CSEを含むか、含む可能性を有する試料であれば特に限定されないが、試料の由来となるものとしては、例えば、尿、血液、血漿、血清、関節液、髄液等の体液、分泌物、動物細胞若しくは植物細胞等の細胞、組織、臓器、又は細胞若しくは微生物の培養物等が例示される。
CSEは、イカ軟骨よりプロテアーゼ処理、アルコール沈殿を経て抽出され(J.Biochem.60,317-321(1966))、酵素処理、カラムクロマトグラフィ法により調製することができる(J.Biol.Chem.236,983-987 (1961)、J.Biol.Chem.252,4570-4576(1977))。
(1)分子量解析
抗原CSEの分子量は、CSEの0.2%溶液50μlをHPLC (HLC-8220GPC, 東ソー)によるゲルろ過で分析した。ゲルろ過カラムは、TSKgel PWXLG4000(0008020, 7.8mm I.D.×30cm, 東ソー)、TSKgel PWXLG3000(0008021, 7.8mm I.D.×30cm, 東ソー)、TSKgel PWXLG 2500(0008022, 7.8mm I.D.×30cm, 東ソー)を連結させて用いた。移動相に0.2M NaClを使用して、カラム温度 40℃、流速 0.6 ml/minの条件で展開した。CSEの検出には示唆屈折計を用いた。平均分子量はCSの分子量標準品を対照にして求めた。尚、標準CSの分子量は光散乱法により測定した。当該標準品を対照として、算出されたCSEの重量平均分子量(Mw)と数平均分子量(Mn)はそれぞれ、14.6kDaと7.7kDaで、分子量分散度(Mw/Mn)は1.8であった。
抗原CSEにおける硫酸基の置換位置を確認する二糖分析は、C-ABCにより分解された二糖画分を、ポストカラム微量蛍光二糖分析HPLCシステム(Toypda H,et al.,J.Biol.Chem.(2000)275,2269-2275)を用い定量した。
上記手法により算出された二糖組成を次の表1に示す。
ΔDi-0S:ΔHexA1-3HexNAc
ΔDi-4S:ΔHexA1-3HexNAc(4S)
ΔDi-6S:ΔHexA1-3HexNAc(6S)
ΔDi-2S:ΔHexA(2S)1-3HexNAc
ΔDi-(4,6)S:ΔHexA 1-3HexNAc(4S, 6S)
ΔDi-(2,4)S:ΔHexA (2S)1-3HexNAc(4S)
ΔDi-(2,6)S:ΔHexA(2S)1-3HexNAc(6S)
ΔDi-(2,4,6)S:ΔHexA(2S)1-3HexNAc(4S, 6S)
上記のΔHexAは不飽和ウロン酸、HexNAcはN-アセチルヘキソサミン、括弧内は硫酸基の結合位置を示す。
上記により得られたCSEの6位硫酸基を選択的に脱硫酸化し、de6S-CSEを調製した。以下に工程を記す。プロトンフォームとしたCSEを無水ピリジンで中和し、CSEピリジニウム塩を得た。50mgのCSEピリジニウム塩と[ビス(トリエチルシリル)アセトアミド](BTSA)を無水ピリジン中で60℃、2時間反応させた。反応液に蒸留水を加え反応を停止させてから、透析により塩を除去した。透析後、溶液を30分間煮沸し、室温に戻してから0.5M NaOHでpHを9.5に調整した。再び透析した溶液を凍結乾燥させ、粉体として、40mg回収した。
CSCをメタノール/塩化アセチル中で反応させ、脱硫酸化させる方法 (Thomas G. Kantor, Maxwell Schubert.(1957)J.Am.Chem.Soc. 79,152-153)に従いdeS-CSCを調製した。
各種GAG(CSE、CSA、CSB、CSC、CSD、deS-CSC、Hep、HS、HPN、KS、HA、de6S-CSE)5 mgを2M NH4Cl溶液 1 mlに溶解した。その溶液にNaCNBH3 50 mgを添加し、70℃で48時間反応させた。更に、NaCNBH3 25 mg加え、70 ℃で48時間反応させた。反応溶液の塩を除去するため、透析膜(MWCO 12000~14000)で一晩透析した。透析後の溶液を凍結乾燥し、粉体として回収した。
CSE抗原の調製
(1)CSEテトラブチルアンモニウム塩(以下「CSE-But4N+塩」)の調製
参考例1で調製したCSE1.0gを蒸留水50mlに溶解し、Dowex 50W-X8 H+ form(217514-500g,SIGMA)カラム(2.5 cmφ × 6.5 cm)にアプライした。溶出した溶液の酸性画分としてナトリウム塩フリーとなったCSEを回収した。当該集積通過液にテトラブチルアンモニウム(10% in Water)(T0955,東京化成工業)を添加し、pHを酸性から中性に調整した(pH 7.15)。溶液を室温で3日間凍結乾燥して、CSE-But4N+塩を乾燥粉末として1.85g回収した。
CSE-But4N+乾燥粉末1.85 g を脱水メタノール25 mlに溶解し、PE(163-161193, Wako)104 mg(150μmol)を添加した。アルゴンガス雰囲気下、50℃で1時間撹拌した後、トリメチルアミンボラン(T1181,東京化成工業)を36mg加え、継続して24時間、50℃で攪拌した。更に、トリメチルアミンボラン 36mgを24時間おきに2回に分けて添加し、50℃で反応させ、CSE-PE反応溶液を得た。
上記(2)記載の反応溶液を減圧濃縮後、メタノールを加え減圧濃縮を繰り返し、トリメチルアミンボランをメタノールとの共沸で除去した。その残渣に0.2M 酢酸ナトリウム40 mlを加え、室温で2時間撹拌した後、遠心分離(6000 rpm、30分間)により不溶物を除去した。その上清に酢酸ナトリウム飽和エタノールを3倍量(120ml)加えて目的物を沈殿させた。生成した沈殿を蒸留水に溶解し凍結乾燥により、0.83gの粉体を回収した。
CSEに反応するモノクローナル抗体を産生するハイブリドーマの取得
(1)免疫用抗原の調製
実施例1で調製したCSE-PEを最終濃度2 mg/mlとなるようPBSに溶解させ、超音波処理後、最終濃度0.1 mg/mlとして、37℃に加温した。これに酸処理したSalmonella minnesota菌体膜をPBSで予め1 mg/mlに懸濁した溶液を1136μl加え、37℃、10分間静置させた。この混合溶液を200μl に分注し、-20℃に保存した。
上記(1)で作製した分注液を0、4、7、11、21、25日目にC3H/HeNマウス3匹(6週齢)に200μlずつ尾静注より注入した。また、抗体価をELISAにより確認するため、29日目に眼底採血をし、これを4℃、一晩静置後、5000 rpm、10分間遠心した後、血清を回収し、-80℃で保存した。
実施例1で調製したCSE-PEを70% EtOHで2.5μg/50 μlに溶解し、F底96穴マイクロタイタープレート(3355,サーモフィッシャーサイエンティフィック)に該溶液を50μl/well加えた。
PAI細胞(JCRB細胞バンク,JCRB0113)を10% FBS-RPMI [培地終濃度10% FBS,1% PSG,10mM HEPES,1 mM sodium pyruvate含有]で37℃、5%CO2存在下において培養した。
免疫開始後32日目(最終免疫7日後)のマウスから脾臓を無菌的に取り出し、10%FBS-RPMI培地を入れたシャーレ上で脾臓をほぐした。これを1300 rpm、5分間遠心後、10%FBS-RPMI培地で再懸濁し、懸濁液中の組織をセレストレーナーで取り除いた。1300 rpm、5分間遠心した沈殿を10% FBS-RPMI培地20 mlで再懸濁し、脾臓細胞を回収した。
上記で調製したPAI細胞と脾臓細胞をPAI細胞:脾臓細胞=1:5 cell/cellの比率で混合し、ポリエチレングリコール#4000にて融合させた。細胞融合の培養にはHATサプリメント(Gibco)を100倍希釈で加えた10%FBS-RPMI培地を使用した。HAT耐性コロニーの出現が観察できるまで、マウス1匹あたり2枚の96ウェルプレートで10~14日間培養した。
前述(6)で培養上清が陽性であった細胞を限界希釈法によりクローニング操作した。クローニング操作では、クローニングメディウムCM-B(410022517,エーディア)を使用した。細胞数をそれぞれ1個/well(5.0 cells/ml)、0.5個/well(2.5 cells/ml)となるように希釈し、96穴マイクロプレートに播種し、1クローンあたり2枚の96穴プレートで10~14日間培養した。前述(5)と同様に、4回のスクリーニング試験を経て陽性であったクローンを選択した。
前述(7)で選択したクローンのCSEに対する特異性を評価し、2つのクローンを得た。この2つのクローン名をそれぞれE-12C、E-18Hとした。上記のハイブリドーマクローンは、2014年2月26日(寄託日)、独立行政法人製品評価技術基盤機構 特許微生物寄託センター(NPMD)に寄託し、それぞれ受託番号NITE P-01809、NITE P-01808が付与された。また、平成27年2月17日付で、国際寄託に移管されている(受領番号NITE ABP-01809、NITE ABP-01808)。
モノクローナル抗体の特徴
(1)モノクローナル抗体の生産
目的のCSEに反応するモノクローナル抗体は、上記ハイブリドーマを培養し、その培養液から遠心分離により細胞を除去し取得した(培養上清)。ハイブリドーマE-12CよりE-12C抗体、ハイブリドーマE-18HよりE-18H抗体をそれぞれ取得した。
E-12C抗体及びE-18H抗体について、各種GAG(CSE、CSA、CSB、CSC、CSD、deS-CSC、Hep、HS、HPN、KS、HA、de6S-CSE)に対する反応性をビオチン標識化GAG固相化プレートによるELISA法で検討した。CSEはイカ由来、CSAはクジラ由来、CSBはブタ由来、CSCはサメ由来、CSDはサメ由来、Hepはウシ由来、HSはブタ由来、HPNはE.coli K5株由来、KSはサメ由来、HAは鶏由来のものを使用した。以下の表2に使用した各種GAGの分子量、分散値及び硫黄含量を示す。
抗体のサブタイプの測定
IsoQuick(TM)マウスモノクローナル抗体アイソタイプ判定キット(ISOQ5,シグマ)およびIsoQuick(TM)マウスモノクローナル抗体アイソタイプ判定用ストリップ(i9410-25EA,シグマ)を用い、E-12C抗体及びE-18H抗体のサブタイプを判定した。
CSE検出キットの作製
以下の構成からなるCSE検出キットを作製した。
1.実施例3(1)より得られるモノクローナル抗体
2.2次抗体(Horse-radish peroxidase 標識goat anti-mouse IgG+IgM)
3.用時調製 発色溶液セット(80 mM クエン酸-リン酸緩衝液(pH 5.6)、o-Phenylenediamine 、30% 過酸化水素水)
4.反応停止液(1 N HCl)
硫酸化CSA及び硫酸化CSBに対するE-12C抗体及びE-18H抗体の反応性
(1)硫酸化CSA及び硫酸化CSBの調製
CSA100mgを蒸留水50mlに溶解し、陽イオン交換樹脂ダイヤイオン(PK220、三菱化学)(2.5 cmφ × 6.5 cm)にアプライした。溶出した溶液の酸性画分としてナトリウム塩フリーとなったCSAを回収した。当該集積通過液にテトラブチルアミン(TBA)を添加し、pHを酸性から中性に調整した。溶液を室温で凍結乾燥して、CSA-TBA塩を得た。
当該CSA-TBA塩をホルムアミド/ジメチルホルムアミドを1:4で混合させた溶媒に溶解させ、硫酸化剤である三酸化硫黄ピリジンコンプレックスを添加した。硫酸化剤の添加量は、二糖ユニット当たり8当量となるよう添加した。硫酸化反応後の溶液に、蒸留水を添加し反応を停止させ、水酸化ナトリウムで中和させた。この溶液をMWCO12000から14000の透析膜で一晩透析させた後に、凍結乾燥により160mgの硫酸化CSAを得た。このようにしてCSAのGalNAcの6位に硫酸基を導入することで硫酸化CSAを調製することが可能である(Carbohydr.Res.158,183-190(1986))。CSBも同様に硫酸化させ、160mgの硫酸化CSBを得た。
解析は「(参考例2)の(1)分子量解析、(2)二糖組成解析」に記載の方法で実施した。結果を表4に示す。表中では、硫酸化剤を8当量添加した硫酸化CSAをCSA-8S、同様に硫酸化剤を8当量添加した硫酸化CSBをCSB-8Sとしている。
ΔDi-0S:ΔHexA1-3HexNAc
ΔDi-4S:ΔHexA1-3HexNAc(4S)
ΔDi-6S:ΔHexA1-3HexNAc(6S)
ΔDi-2S:ΔHexA(2S)1-3HexNAc
ΔDi-(4,6)S:ΔHexA 1-3HexNAc(4S, 6S)
ΔDi-(2,4)S:ΔHexA (2S)1-3HexNAc(4S)
ΔDi-(2,6)S:ΔHexA(2S)1-3HexNAc(6S)
ΔDi-(2,4,6)S:ΔHexA(2S)1-3HexNAc(4S, 6S)
上記のΔHexAは不飽和ウロン酸、HexNAcはN-アセチルヘキソサミン、括弧内は硫酸基の結合位置を示す。
表面プラズモン共鳴法による相互作用解析
(1)アミンカップリング法によるリガンドGAGの固定化
センサーチップCM5表面にコーティングされている直鎖デキストランには負に帯電したカルボキシル基が導入されている。カルボキシル基をNHS(N-ヒドロキシスクシンイミド)により活性化し、pH5.5の10mM酢酸緩衝液に溶解させた50μg/mlのストレプトアビジンを固定化した。固定化後に残った活性NHS基をエタノールアミンによりブロッキングした。ブロッキング後、50RU(レゾナンスユニット)以上の固定化量が得られるようリガンドとなるビオチン化GAGを添加した。リファレンスセルも同様にビオチンを固定化させネガティブコントロールセルとした。ランニング緩衝液としてPBSを流速10μl/minで流し、温度25℃の条件で実施した。
0.22μmフィルターで透過させたE-12C抗体及びE-18H抗体の培養上清をアナライトとして、相互作用解析した。抗体培養上清はPBSにより希釈して、IgM濃度として2nMとなるように調製した。流速20μl/minで注入し、リガンドと相互作用させた。リファレンスセルのレスポンスを差し引いたセンサーグラムを表示させ、レスポンスがない場合は検出されないもの(ND)とした。解析可能なレスポンスが得られた場合は、1:1binding modelにより、結合速度定数(ka)、解離速度定数(kd)の値から解離定数(KD)値を算出した。KD値は、kd/kaにより求めることができる。
分子量の異なるCSEは、酵素処理、カラムクロマトグラフィ法により調製することができる(J.Biol.Chem.236,983-987 (1961)、J.Biol.Chem.252,4570-4576(1977))。当該文献を参考に分子量が異なる5種類のCSEを調製した。
ΔDi-0S:ΔHexA1-3HexNAc
ΔDi-4S:ΔHexA1-3HexNAc(4S)
ΔDi-6S:ΔHexA1-3HexNAc(6S)
ΔDi-2S:ΔHexA(2S)1-3HexNAc
ΔDi-(4,6):ΔHexA 1-3HexNAc(4S, 6S)
ΔDi-(2,4):ΔHexA (2S)1-3HexNAc(4S)
ΔDi-(2,6):ΔHexA(2S)1-3HexNAc(6S)
ΔDi-(2,4,6)S:ΔHexA(2S)1-3HexNAc(4S, 6S)
上記のΔHexAは不飽和ウロン酸、HexNAcはN-アセチルヘキソサミン、括弧内は硫酸基の結合位置を示す。
上記表6に記載の5種類のCSEとde6S-CSEをセンサーチップに固定化させて相互作用解析した。試験は、「実施例7の(1)アミンカップリング法によるリガンドの固定化、(2)相互作用測定」に記載の方法で実施した。結果を表7に示す。
CSA-TBA塩に対して硫酸化剤である三酸化硫黄ピリジンコンプレックスを2、4、6当量添加して反応させた。以降の工程は実施例6(1)硫酸化CSA及びCSBの調製に記載の方法に従い調製した。調製された硫酸化CSAの分子量および二糖組成解析を参考例2(1)分子量解析、(2)二糖組成解析に記載の方法で実施した。結果を表8に示す。
ΔDi-0S:ΔHexA1-3HexNAc
ΔDi-4S:ΔHexA1-3HexNAc(4S)
ΔDi-6S:ΔHexA1-3HexNAc(6S)
ΔDi-2S:ΔHexA(2S)1-3HexNAc
ΔDi-(4,6):ΔHexA 1-3HexNAc(4S, 6S)
ΔDi-(2,4):ΔHexA (2S)1-3HexNAc(4S)
ΔDi-(2,6):ΔHexA(2S)1-3HexNAc(6S)
ΔDi-(2,4,6)S:ΔHexA(2S)1-3HexNAc(4S, 6S)
上記のΔHexAは不飽和ウロン酸、HexNAcはN-アセチルヘキソサミン、括弧内は硫酸基の結合位置を示す。
抗CSE抗体の組織染色性を以下の通り検討した。
(1)凍結切片における免疫組織染色
イカ軟骨を小片化し、ティシュー・テックO.C.T.コンパウンド(サクラファインテックジャパン株式会社)に組織片を埋め、液体窒素で凍らせて凍結切片を作製した。
イカ軟骨を小片化し、10%中性緩衝ホルマリン液に固定後、パラフィン包埋し、パラフィン切片を作製した。
C-ABC処理し、CSE等のコンドロイチン硫酸が除去された組織の染色性を検討した。
本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書に参照により取り込まれる。
Claims (14)
- コンドロイチン硫酸Eに反応し、コンドロイチン硫酸A、コンドロイチン硫酸B及びコンドロイチン硫酸Dに反応しない抗体。
- コンドロイチン硫酸C、完全脱硫酸化コンドロイチン硫酸C、ヘパリン、ヘパラン硫酸、ヘパロサン、ケラタン硫酸、ヒアルロン酸及び6-O-脱硫酸化コンドロイチン硫酸Eのいずれにも反応しない、請求項1に記載の抗体。
- 脂質とコンドロイチン硫酸Eとが結合してなる物質を抗原として免疫することによって得られる請求項1又は2に記載の抗体。
- 脂質がジパルミトイル-L-(α-ホスファチジル)エタノールアミンである、請求項3に記載の抗体。
- モノクローナル抗体である、請求項1~4のいずれか1項に記載の抗体。
- 受領番号がNITE ABP-01808又はNITE ABP-01809であるハイブリドーマにより産生されるモノクローナル抗体。
- 請求項5に記載のモノクローナル抗体を産生するハイブリドーマ。
- 脂質とコンドロイチン硫酸Eとが結合してなる物質を抗原として免疫した動物由来の抗体産生細胞と腫瘍細胞との細胞融合により作製される請求項7に記載のハイブリドーマ。
- 脂質がジパルミトイル-L-(α-ホスファチジル)エタノールアミンである、請求項8に記載のハイブリドーマ。
- 受領番号がNITE ABP-01808又はNITE ABP-01809であるハイブリドーマ。
- 請求項1~6のいずれか1項に記載の抗体を試料に接触させる工程を少なくとも含むことを特徴とする、当該試料中に存在するコンドロイチン硫酸Eの検出方法。
- 請求項1~6のいずれか1項に記載の抗体を少なくとも含む、コンドロイチン硫酸Eの検出キット。
- 脂質とコンドロイチン硫酸Eとが結合してなる物質を抗原として免疫する工程を少なくとも含むことを特徴とする、コンドロイチン硫酸Eに反応し、コンドロイチン硫酸A、コンドロイチン硫酸B及びコンドロイチン硫酸Dに反応しない抗体の製造方法。
- 脂質がジパルミトイル-L-(α-ホスファチジル)エタノールアミンである、請求項13に記載の製造方法。
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EP3127922A4 (en) | 2017-11-08 |
JPWO2015152280A1 (ja) | 2017-04-13 |
EP3127922A1 (en) | 2017-02-08 |
US9745386B2 (en) | 2017-08-29 |
US20170107301A1 (en) | 2017-04-20 |
JP6659024B2 (ja) | 2020-03-04 |
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