WO1998016825A1 - PROCEDE DE DOSAGE D'UNE AGALACTO-IgG ET TROUSSES DE DOSAGE, POLYPEPTIDES DE LECTINES ET ADN CODANT CEUX-CI - Google Patents
PROCEDE DE DOSAGE D'UNE AGALACTO-IgG ET TROUSSES DE DOSAGE, POLYPEPTIDES DE LECTINES ET ADN CODANT CEUX-CI Download PDFInfo
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- WO1998016825A1 WO1998016825A1 PCT/JP1997/003723 JP9703723W WO9816825A1 WO 1998016825 A1 WO1998016825 A1 WO 1998016825A1 JP 9703723 W JP9703723 W JP 9703723W WO 9816825 A1 WO9816825 A1 WO 9816825A1
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- xaa
- lectin
- igg
- amino acid
- gly
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4726—Lectins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
- C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
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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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
- G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Definitions
- the present invention relates to a method for measuring agaract IgG using a lectin fixed to a solid phase and a kit for measuring agaract IgG.
- the present invention provides a method for detecting rheumatism, which comprises measuring agaract IgG using a method for measuring agaract IgG using a lectin fixed to a solid phase. It relates to the G measurement kit and the diagnostic kit for rheumatism.
- the present invention also relates to a polypeptide having the same effect as P. musculature lectin (PsathyreUa velutina lectin, PVL), and a DNA encoding the same as Muzina lectin lectin or its equivalent.
- P. musculature lectin PsathyreUa velutina lectin, PVL
- Rheumatoid arthritis is a disease with polyarthritis as the main symptom and widespread inflammation of organ tissues throughout the body. It is known that the serum of a patient with rheumatoid arthritis contains an autoantibody that recognizes an epitope at the Fc site of immunoglobulin G (IgG) called rheumatoid factor. This suggests that the IgG ability of rheumatoid patients producing rheumatoid factor has a different structure from that of healthy human IgG.
- IgG immunoglobulin G
- the sugar chain portion at the Fc site of IgG in healthy human serum is basically a complex double-stranded structure, with or without Ga 1 residue, with or without fucose residue, ⁇ - It is a mixture of 16 types of oligosaccharides depending on the presence or absence of ⁇ -acetylglucosamine (G1cNAc) residue bound to mannose residues, and the relative proportion of these 16 types of oligosaccharides is healthy.
- G1cNAc ⁇ -acetylglucosamine
- the current method of diagnosing rheumatoid arthritis is mainly to measure rheumatoid factor (autoantibodies to agaract IgG), and to measure rheumatoid factor that reacts to denatured IgG by latex agglutination.
- Rheumatoid factors can be classified into three types: IgG, IgM, and IgA.Recently, by measuring each rheumatoid factor, sensitivity and specificity in diagnosis have been discussed. ing.
- the causative agent of these rheumatoid factors is thought to be agaract IgG, which results from abnormal sugar chains in IgG. Therefore, direct measurement of the causative substance, agaract IgG, is considered important in diagnosing rheumatoid arthritis.
- Examples of a method for measuring agaract IgG include a technique described in Japanese Patent Application Laid-Open No. Hei 5-87814. This technique involves the use of a lectin (RCA; ricinascominisagglutinin) that reacts with an anti-human IgG antibody and a normal human IgG (herein referred to simply as “human IgG”).
- RCA ricinascominisagglutinin
- human IgG human IgG
- the basic principle is to measure the amount of normal IgG by sandwiching normal IgG in human serum using the above method.
- an anti-human IgG antibody immobilized on a solid phase insolubilizing carrier
- a lectin immobilized on a solid phase as in the present invention is used. It is not disclosed.
- the anti-human I g G antibody immobilized on a solid phase to bind the I g G arsenide Bok serum (a mixture of normal I g G and Agaraku preparative I g G), therein Normal Ig Only G is recognized by lectin (RCA) (ie, normal IgG is sandwiched by anti-human IgG antibody and lectin (RCA)), and agaracta is detected based on the measured value of normal IgG.
- DNA encoding PVL can be obtained, it is expected to be used for lectin production and lectin property modification by a recombinant expression system. Therefore, another problem to be solved by the present invention is to provide a DNA encoding PVL. Another object is to provide a novel lectin polypeptide based on the DNA. Disclosure of the invention
- the present inventors can solve the problems of lectin aggregation and the stability of labeled lectin. And succeeded in providing a low-cost and simple method for measuring agaract IgG, and completed the present invention. That is, the present invention provides a method for reacting agaract IgG in a sample with lectin fixed to a solid phase to form a complex of the agaract IgG and lectin fixed to the solid phase.
- the present invention relates to a method for measuring agaract IgG in a sample, a method for detecting rheumatism, particularly rheumatoid arthritis using the measurement method, an agaract IgG measurement kit and a diagnostic kit for rheumatism.
- the complex formed is detected with an anti-IgG antibody.
- the step of Agaraku Bok I g G in the sample is reacted with a lectin which is fixed to a solid phase, to form a complex with a lectin which has fixed to Agara click preparative I g G and a solid phase, Reacting the agaract IgG with an anti-IgG antibody. More preferably, it is reacted with an anti-IgG antibody after the step of forming a complex.
- the lectin is preferably a lectin that specifically binds to a / S—N-acetylglucosamine residue, and particularly preferably is a mushroom (Ayre a velutina) lectin (PVL).
- the present invention relates to a method for measuring agaract IgG in a sample, which comprises a step of sandwiching agaract IgG with a lectin and an anti-IgG antibody.
- Lectin is preferably immobilized.
- the lectin is preferably a lectin which specifically binds to a / 3-N-acetyl darcosamine residue, and particularly preferably is Psathyrella veJuii / ja lectin (PVL).
- PVL Psathyrella veJuii / ja lectin
- the present inventors have succeeded in cloning cDNA encoding PVL, and have obtained a lectin polypeptide having a specific amino acid sequence and a partial polypeptide thereof (hereinafter collectively referred to as “the polypeptide of the present invention”. ), And an antibody that binds thereto, a lectin polypeptide containing PVL, and a DNA encoding the partial polypeptide thereof (hereinafter, also referred to as “DNA of the present invention”).
- the polypeptide of the present invention includes a lectin polypeptide comprising at least an amino acid sequence represented by the following formula (1).
- the polypeptide of the present invention has at least a part of the amino acid sequence shown in SEQ ID NO: 2, and has a sugar chain (preferably, a sugar chain having an N-acetyldarcosamine residue; A sugar chain having a glucosamine residue, particularly preferably the N-acetylglucosamine residue of a sugar chain having an N-acetylglucosamine residue at the terminal, very preferably an N-acetylglucosamine residue-mannose residue at the terminal Substitution, deletion or insertion of one or more amino acid residues which does not substantially impair the binding activity of the sugar chain having a group (GlcNAc ⁇ Mari) to the N-acetylglycosamine residue.
- lectin polypeptides that may be present.
- it is a polypeptide having at least a part of the amino acid sequence shown in SEQ ID NO: 2.
- polypeptide of the present invention may be a polypeptide comprising a part of the above polypeptide. Contains tide.
- fusion polypeptide comprising the above polypeptide and another polypeptide in a polypeptide chain, and an antibody which binds to the polypeptide of the present invention.
- the DNA of the present invention includes DNA encoding a lectin having the following properties.
- Sugar chain preferably a sugar chain having an N-acetyl glucosamine residue, more preferably a sugar chain having an N-acetyl glucosamine residue at the terminal, particularly preferably an N-acetyl ester at the terminal It binds to the N-acetylglucosamine residue of the sugar chain having a glucosamine residue, very preferably to the N-acetylglucosamine residue of the sugar chain having GlcNAc ⁇ Man at the terminal.
- SDS Sodium dodecyl sulfate
- the DNA of the present invention contains DNA encoding at least a part of the polypeptide of the present invention.
- it has a base sequence encoding an amino acid sequence represented by amino acid numbers 1 to 402 in SEQ ID NO: 2, and more preferably, a base sequence represented by base numbers 41 to 1246 in SEQ ID NO: 1. It has at least a part or all of the base sequence to be obtained. Furthermore, there are provided a recombinant DNA containing the DNA of the present invention, a recombinant vector, and a transformed cell obtained by introducing the vector into a host cell.
- the lectin containing the polypeptide of the present invention can be used as a lectin in the method for measuring agaract IgG described above.
- Figure 1A is a graph showing the results of the ELISA method for the reactivity of the prepared anti-PVL antibody with PVL.
- Figure 1B shows the reactivity specificity of the prepared anti-PVL antibody by Western blot.
- 6 is a schematic diagram showing the results of analysis by the G method.
- FIG. 2 shows the basic strategy (sequencing strategy) for determining the nucleotide sequence of the PVL cDNA clone, SKPVL231.
- FIG. 3 shows the correspondence between the nucleotide sequence of PVL cDNA clone, the amino acid sequence, and the amino acid sequence of the PVL partial.
- FIG. 4 shows repeats in the PVL amino acid sequence.
- FIG. 6 shows an example of expression of recombinant PVL in E. coli (electrophoresis photograph).
- FIG. 7 shows a structural schematic diagram of the PVL expression plasmid (PPICPVL231).
- FIG. 8 shows a standard curve of agaract IgG of Comparative Example 1.
- FIG. 9 shows a standard curve of agaract IgG of Example 3.
- FIG. 10 shows the effects of various monosaccharides of Example 4 on the measurement system.
- FIG. 11 shows the concentration of agalacto IgG in the serum of healthy subjects, OA patients, RA patients, and liver disease patients of Example 5.
- the measurement method of the present invention is characterized by including at least a step of forming a complex between lectin fixed to a solid phase and agaract IgG in a sample.
- the solid phase on which the lectin is immobilized includes a plate, a tube, a bead, a membrane, a gel, and the like.
- the material include polystyrene, polypropylene, nylon, latex, glass, crosslinked dextrin, agarose, crosslinked agarose, and polyacrylamide.
- the lectin can be immobilized on these solid bodies by a general method for preparing immobilized enzymes such as a physical adsorption method, a covalent binding method, and an inclusive method (immobilized enzyme, published in Kodansha, 1975, Kodansha, No. 9). Pp. 75) can be applied.
- immobilized enzyme published in Kodansha, 1975, Kodansha, No. 9.
- Pp. 75 Pp. 75
- physical adsorption is preferred in that the operation is simple.
- a surface portion where these are not fixed there may be a surface portion where these are not fixed, and if agaract IgG or other molecular species in the sample is fixed thereto.
- a blocking substance to cover the portion where the lectin is not fixed before the sample is brought into contact with the solid phase body.
- blocking substances include serum albumin, casein, milk protein, lactic acid fermentation products, collagen, and their decomposed substances that can be collected from mammals, for example, pests, etc., and commercially available as blocking substances. Those that have been used can also be used.
- washing solution examples include a buffer solution containing a surfactant such as a Tween-based surfactant (eg, a phosphate buffer solution, a phosphate buffered saline (PBS), a Tris-HCl buffer solution). ) And the like.
- a surfactant such as a Tween-based surfactant
- PBS phosphate buffered saline
- Tris-HCl buffer solution Tris-HCl buffer solution
- IgG makes up about 80% of the immunoglobulins in the blood, is present at a concentration of about 10 to 15 mg m 1, and is a glycoprotein with a half-life of 16 to 23 days.
- the complex-type double sugar chain is bound to asparagine at position 297 of the CH2 region, and contains one to two molecules of G a1 residues in healthy individuals.
- G a1 was significantly reduced, and the G a1 NAc molecules were exposed at both ends of the complex double sugar chain. ing. It is thought that this sugar abnormality causes the antibody to show antigenicity and produce autoantibodies (rheumatic factor).
- An antibody having this sugar chain abnormality (G a1 deficiency) is agaratato IgG.
- the lectin is not particularly limited as long as it reacts with agaract IgG, but is preferably a lectin that specifically binds to a / 3-N-acetylglucosamine (/ 3-GlcNAc) residue. is there.
- Such lectins include lectino from 73 ⁇ 4iiciwn vu4 ris, lectin from Datu stramoiizuii, lectin from Lycopersicon escuenium, lectin from oza iun tiiberosiwH, lectin from Laburnum a or _ ⁇ , and lectin from Oyza saira.
- Mudtaketake lectin is particularly preferable because it has been sufficiently confirmed that it reacts with agaract IgG and does not react with normal IgG.
- Lectins containing the polypeptide of the present invention described in 5> below can also be used.
- PVL is a protein with a molecular weight of about 40,000 that is present in the fruiting body of Mushroom mushroom.
- This lectin shows higher affinity for G1cNAc than the conventional lectin, especially GlcNAc / 31 ⁇ 6 binding and G1cNAc / 31 ⁇ 4 binding rather than G1cNAc / 31 ⁇ 4 binding. It has high affinity for 1 c NA c ⁇ 1 ⁇ 3 binding.
- this PVL It has been reported that it reacts with G a1 Nac at the terminal of the complex type double sugar chain present in -1 o-ct IgG molecules.
- Examples of the PVL used in the present invention include commercially available PVL, PVL extracted and purified from fruiting bodies of Mushroom lectin by a known method (Japanese Patent Application Laid-Open No. 11-35999) or known genetic engineering. Recombinant PVL may be used by using a standard method.
- the method for detecting the complex of the formed agaract IgG and lectin is not particularly limited. Since the anti-IgG antibody reacts with the agaract IgG, it is preferable to detect the complex of the agaract IgG and the lectin using the anti-IgG antibody.
- the complex of agaract IgG and lectin can be detected by forming the complex and reacting the formed complex with an anti-IgG antibody.
- the reaction may be performed by reacting gG with an anti-IgG antibody, and then forming a complex of agalactoIgG and lectin for detection.
- detection is performed by reacting with an anti-IgG antibody after forming a complex between agaract IgG and lectin.
- the anti-IgG antibody is preferably an anti-IgG antibody labeled or capable of being labeled with a labeling substance. Further, an anti-human IgG antibody is preferable.
- Anti-human IgG antibodies that can be used in the present invention include commercially available anti-human IgG antibodies, commercially available human IgG, and mice, rats, guinea pigs, hamsters, egrets, goats, sheep, and the like. Or a monoclonal antibody obtained by immunizing an animal to be immunized.
- the above-mentioned commercially available anti-human IgG antibodies are preferred because they are easily available.
- the recognition binding sites for protein G and PVL are both concentrated in the Fc portion of the agaract IgG molecule, but the anti-human IgG anti-reactivity recognizes sites other than Fc as well. This is preferable for forming a sandwich-like complex with agaract IgG.
- the present invention is a Agaraku bets I g G by a lectin and anti-I g G antibodies in Sandi Tutsi, Agaraku WINCH I g G the direct detection technique, i.e. Agaraku preparative I g by the lectin and anti-I g G antibodies It also includes a method for measuring agaract IgG in a sample, which includes a step of sandwiching G.
- Lectins are preferably anchored to a solid phase.
- Preferred and preferred lectins, preferred anti-IgG antibodies, and other preferred embodiments are the same as described above.
- PVL and an anti-human IgG antibody are used as an example.
- Other lectins and other anti-IgG antibodies can be used as well.
- a so-called sandwich method (see Japanese Patent Publication No. 6-41952) is usually used. That is, by utilizing the affinity between the sugar chain in the agaract IgG molecule and PVL and the affinity of the anti-human IgG antibody to the protein portion in the agaract IgG molecule,? This is a method of forming a sandwich-like complex and measuring the complex. Usually, PVL is fixed to a solid phase, and the sandwich-like complex is separated and measured.
- a sample containing an agaract IgG is brought into contact with a PVL immobilized on a solid phase body, and then contacted with an anti-human IgG antibody labeled with a labeling substance, whereby the agaract IgG in the sample is obtained.
- PVL and a labeled anti-human IgG antibody to react with each other to form a complex consisting of solid-phase resting-PVL-agaracto IgG-labeled anti-human IgG antibody.
- a method of separating a human IgG antibody and detecting a labeling substance in the complex to measure agaract IgG in the sample can be mentioned.
- the anti-anti-human IgG antibody that can be used in this method is an antibody that recognizes immunoglobulin of an animal of the same species as an animal immunized with human IgG in order to produce an anti-human IgG antibody.
- Labeling substances used for labeling antibodies include enzymes (peroxidase, alkaline phosphatase, / 3-galactosidase, nolesipherase, acetylcholinesterase) etc.), iso Tope C 2 5 I, 1 3 ' ], 3 H , etc.), fluorescent dyes (luminol, full O receptacle fin isothiocyanate Xia ne one Bok (FITC), ⁇ down Berifuweron, 7- amino-4-methylcoumarin - 3-acetic acid, etc.), chemiluminescent substances, haptens, biotin, avidin (eg, streptavidin, etc.).
- enzymes peroxidase, alkaline phosphatase, / 3-galactosidase, nolesipherase, acetylcholinesterase) etc.
- the labeling substance is a method that uses a substance that does not directly detect itself, such as biotin, but combines a substance that has a specific binding ability (for example, avidin) with a detectable label.
- a substance that does not directly detect itself such as biotin
- combines a substance that has a specific binding ability for example, avidin
- the substances used for the above are also included.
- Known methods suitable for labeling the antibody include, for example, glutaraldehyde, periodic acid cross-linking, maleimide cross-linking, calposimid, and activated ester when labeling an enzyme.
- a radioisotope such as the method, the chloramine T method, lactoperoxidase method, etc. (refer to Seismic Chemistry Laboratory Lecture 2 “Chemistry of Proteins (Bottom)”, Tokyo Chemical Dojin, 1987) You can choose.
- a method for detecting a labeling substance different powers are used depending on the labeling substance used.For example, when biotin is used as a labeling substance, an enzyme to which streptavidin or the like is bound is added, and the streptavidin or the like is added.
- An enzyme such as peroxidase is bound to a complex containing biotin as a labeling substance through the reaction, a chromogenic substrate such as tetramethylbenzidine and a hydrogen peroxide solution are added as a substance of the enzyme, and the product of the enzymatic reaction is added.
- a method of measuring the degree of color development by a change in absorbance can be used. Further, for example, when a fluorescent substance or a chemiluminescent substance is used as the labeling substance, a method of measuring the fluorescence or luminescence of the solution after the reaction may be used.
- the concentration of agaract IgG in a sample is determined by using a previously known concentration of an agaract IgG standard solution to determine the relationship between the concentration of agaract IgG and the detection result of a labeled substance.
- a calibration curve is created, and quantification can be performed by a method using the detection result of a sample having an unknown concentration and the above-mentioned calibration curve.
- the sample in the measurement method of the present invention is not particularly limited, but is preferably It is a liquid sample, more preferably a liquid sample such as synovial fluid, blood, serum, plasma, or a cell culture solution.
- the sample is preferably a body fluid such as synovial fluid, blood, serum, or plasma.
- the body fluid is of human origin.
- the sample containing agaract IgG used in the measurement method of the present invention does not need to be purified in advance. That is, even if contaminants such as other serum proteins are mixed in the sample, the agaract IgG can be selectively measured, so that the measurement result is not affected.
- PVL is fixed (coated) to the solid phase.
- the solution is dissolved in a buffer (eg, phosphate buffer, PBS, carbonate buffer, etc.) containing 10% glycerol at a pH of about 7 to 9 and added to the solid phase (eg, a microplate well).
- the method can be left standing at about ° C for 1 to 2 hours, or left standing at about 4 ° C for fixing.
- a blocking substance such as serum albumin is added, and the mixture is allowed to stand at about 37 ° C for 30 minutes to 2 hours or at room temperature (15 to 25 ° C) for 1 to 2 hours. It is preferable to cover a portion where PVL is not fixed.
- a sample sample containing agaract IgG is added to the solid phase to which the PVL is fixed, and the sample is left standing or stirred at 37 ° C for an optimal time of 20 to 80 minutes, for example. Agaract IgG is bound.
- the solid phase to which the complex is bound is washed with a buffer solution (eg, phosphate buffer solution, PBS, Tris-HCl buffer solution, etc.) to which a Tween-based surfactant has been added; .
- a buffer solution eg, phosphate buffer solution, PBS, Tris-HCl buffer solution, etc.
- an anti-human IgG antibody or an anti-human IgG antibody labeled with a labeling substance and an anti-anti-human IgG antibody labeled with a labeling substance are added to the solid phase, for example, at 37 ° C. And agitated for 20 to 80 minutes to bind the anti-human IgG antibody (or anti-human IgG antibody-anti-anti-human IgG antibody) to agaract IgG.
- the solid phase-PVL-agalacto IgG-anti-human IgG antibody or the solid phase-PVL-agalacto-IgG-anti-human IgG antibody-anti-anti-human IgG antibody- Complex).
- the labeled substance of the complex is detected and Measure lact IgG.
- a calibration curve was created for the relationship between the concentration of the agaract IgG standard and the detection result (for example, absorbance) of the labeled substance, and the detection results for the unknown sample and the calibration curve were used to determine the agaract in the unknown sample. Quantify the IgG.
- rheumatism can be detected by measuring agaract IgG using the method for measuring agaract IgG described above. This detection includes determination of the degree of rheumatism as well as the presence or absence of rheumatism.
- the agaract IgG ”t as a criterion for the detection of rheumatism is appropriately determined depending on the type of the specimen, and the detection can be performed based on the measured amount of the agaract IgG.
- the amount of agaract IgG used as a criterion for rheumatism detection is the amount of agaract IgG determined using a calibration curve created for the relationship between the concentration of the agaract IgG standard and the detection result of the labeled substance.
- the ratio may be a ratio to the amount of agaract IgG in a sample of a healthy human (a human who does not have rheumatoid arthritis) without using the calibration curve.
- the present invention also relates to a kit for use in a method for measuring agaract IgG by forming a complex of agaract IgG and lectin, wherein a lectin fixed to a solid phase as a component is provided.
- the present invention relates to a kit for measuring agaract IgG characterized by including:
- the kit for measuring agaract IgG of the present invention further comprises an anti-IgG antibody, and the anti-IgG antibody is labeled with or can be labeled with a labeling substance.
- the lectin mainly consists of a solid phase to which a lectin (particularly preferably PVL) that specifically binds to 3-N-acetylglucosamine residue is immobilized, and an anti-human IgG antibody labeled with a labeling substance. .
- PVL is fixed to a solid phase in advance, in the measurement method described above, The operation of sticking to the solid phase can be omitted. Also, unwanted aggregation of the lectin can be avoided.
- the kit further contains a standard concentration of an agaract IgG standard, a detection reagent for a labeling substance, a reagent for labeling an anti-human IgG antibody, or an anti-human IgG antibody as a standard for preparing a calibration curve.
- Reagents labeled anti-anti-human IgG antibody etc.
- the blocking substance, the washing solution, the sample diluting solution, the enzyme reaction stopping solution, and the like may be contained.
- these components can be stored in separate containers, respectively, and can be stored as a kit that can be used according to the above-described method when used.
- Agaraku Bok I g G measuring kit Bok of the present invention the analyte, e.g. synovial fluid, blood, serum, plasma, and specifically measure child the Agaraku bets I g G in samples such as a culture solution of cells It has a wide measurement range and high sensitivity.
- an agaract IgG measurement kit containing PVL immobilized on a solid phase and a labeled anti-human IgG antibody is used as follows. That is, by contacting a sample containing agaract IgG with the PVL immobilized on the solid phase, and then contacting with a labeled anti-human IgG antibody, the solid phase—PVL—agagalt IgG—labeled anti-human A complex consisting of an IgG antibody is formed, and a labeling substance contained in the complex is detected, whereby the concentration of IgG in the sample is measured.
- the kit for measuring agaract IgG of the present invention which can easily measure agarat IgG at a low cost, is a very effective diagnostic kit that can specifically and easily diagnose rheumatoid arthritis. That is, it can be used as a diagnostic agent.
- the polypeptide of the present invention includes a lectin polypeptide comprising at least the amino acid sequence represented by the above formula (1).
- the polypeptide of the present invention preferably contains at least one round of the sequence starting from any position of the amino acid cyclic sequence represented by the following formula (2).
- amino acid generally means 20 kinds of amino acids (aranine, arginine, asparagine, aspartic acid, cystine, glutamine, glutamic acid, glycine, histidine) constituting the protein. , Isoleucine, oral isin, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and non-phosphorus, but may contain other amino acids.
- the amino acids other than glycine are preferably L-amino acids.
- the “cyclic sequence” means the first amino acid (Xaa) in the above formula (2) —— the last amino acid ((Xaa)) — the first amino acid—
- the “array for one round starting from an arbitrary position in the circular array” is, for example, assuming that the starting position is the eighth Phe from the beginning in the above formula (2), and “Phe-Gly -Xaa-Xaa- -Asp Xaa- Xaa Gly '.
- This arrangement is merely an example, and the present invention is not limited to this arrangement.
- the polypeptide of the present invention comprises at least an amino acid sequence represented by the following formula (3).
- Water-soluble amino acid and more preferably" Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, Trp, Cys ", respectively.
- it contains at least the amino acid sequence represented by the following formula (4).
- Xaa represents an arbitrary amino acid
- Xaal represents a hydrophobic amino acid
- the amino acids shown in parentheses do not need to be present.
- the following formula (5 ) Is included at least. Asp XII XII Gly Phe Gly Xaa Xaa Xaa X12 Xaa Xaa Xaa Asn (Xaa) (Gly) (Xaa) Xaa Xaa X13 (Xaa) Xaa X14 X15 Xaa (Xaa) XI 6 XI 1 Xaa Xaa XI 3 Xaa Xaa (Xaa) Xaa Xaa Gly Trp Xaa Xl l Xaa Xaa X17 Arg Xaa X18 X19.
- Formula (5) Formula (5)
- Xaa is any amino acid
- XI I is lie ⁇ Val or Leu
- X12 is lie or Val
- X13 is Phe or Leu
- X14 is Pro, Ala or Val
- X15 is Pro
- X16 is Ala, Val or Leu
- X17 is Pro, Val, Leu or lie
- X18 is Ala, VaU Leu.
- X19 is Ala ⁇ Val or Gly
- the amino acids shown in parentheses indicate that they do not need to exist. ') Further, they more preferably include at least the amino acid sequence represented by the following formula (6).
- Xaa is any amino acid, XII is lie ⁇ Val or Leu, X12 is lie or Val, X13 is Phe or Leu, X14 is Pro, Ala or Val, X15 is Pro, Ala, Val or Met, X16 is Ala, Val or Leu, X17 is Pro, Val, Leu or lie, X18 is Ala, Val, Leu, lie or Met, X19 is Ala, Val And amino acids shown in parentheses indicate that they do not need to be present.
- the amino acids of SEQ ID NOs: 3 to 9 represented by the following formulas (7) to (13) are preferred. It contains one or more amino acid sequences selected from the sequences.
- Gly Arg Ala Asp lie Val Gly Phe GLy Glu Ala Gly lie Leu Val Ser Leu Asn
- Gly Ala Gly Gly Trp Thr lie Ala Ser His Pro Arg Val lie Ala Asp Leu Thr Gly Asp-formula (10) [SEQ ID NO: 6]
- Lys Arg Ala Asp lie Leu Gly Phe Gly Gly Ala Gly Val Tyr Thr Ser Leu Asn
- amino acids represented by each symbol may be the same or different.
- the above-mentioned polypeptide of the present invention is usually a sugar chain (preferably, a sugar chain having an N-acetyl dalcosamine residue, more preferably, a sugar chain having an N-acetyl glucosamine residue at the terminal, particularly preferably at the terminal.
- a sugar chain preferably, a sugar chain having an N-acetyl dalcosamine residue, more preferably, a sugar chain having an N-acetyl glucosamine residue at the terminal, particularly preferably at the terminal.
- GlcN'Ac-Man at the N-acetylglycosamine residue of the sugar chain having the N-acetylglycosamine residue, and most preferably at the terminal
- the polypeptide of the present invention has at least a part of the amino acid sequence shown in SEQ ID NO: 2, and has a sugar chain (preferably a sugar chain having an N-acetylglycosamine residue, more preferably a N-acetyl ester at the terminal).
- a sugar chain having a glucosamine residue particularly preferably the N-acetylglucosamine residue of a sugar chain having an N-acetylglucosamine residue at the terminal, and very preferably a sugar chain having a GlcMc ⁇ Man at the terminal.
- Lectin polypeptides which may have one or more amino acid residue substitutions, deletions or insertions which do not substantially impair the activity of binding to N-acetyl glucosamine residues.
- it is a lectin polypeptide having at least a part of the amino acid sequence shown in SEQ ID NO: 2 without substitution, deletion or insertion of amino acid residues.
- Such substitution, deletion or insertion of an amino acid residue may involve substitution, deletion or insertion of one or more amino acid residues in DNA encoding at least a part of the amino acid sequence shown in SEQ ID NO: 2. It can be obtained by expressing DNA obtained by introducing nucleotide substitutions, deletions or insertions that cause such occurrences. Nucleotide substitutions, deletions or insertions into the DNA sequence are introduced by synthesizing a sequence that has both ends of the restriction enzyme at both ends and that includes both sides of the mutation point, and replaces the corresponding part of the unmutated DNA sequence. be able to. In addition, site-directed mutagenesis (Kramer. W.
- sugar chain binding activity (lectin activity) of a lectin polypeptide can be measured, for example, by the lectin activity measurement method described below.
- lectin activity measurement method One of ordinary skill in the art knows one or more amino acid residues that do not substantially impair the sugar chain binding activity. Substitutions, deletions or insertions can be readily identified.
- the polypeptide of the present invention also includes a polypeptide having a structure corresponding to a homologous variant of the polypeptide having at least a part of the amino acid sequence shown in SEQ ID NO: 2.
- having at least a part of the amino acid sequence shown in SEQ ID NO: 2 means having a necessary and minimum polypeptide amino acid sequence having sugar chain binding activity. is there.
- polypeptide of the present invention includes a polypeptide containing a part of the above-mentioned polypeptide.
- the “part” preferably means a part having some activity or function such as having a sugar chain binding activity or having antigenicity. It is easy for those skilled in the art to identify such a part.
- polypeptide of the present invention a polypeptide having an amino acid sequence represented by amino acid numbers 18 to 402 in SEQ ID NO: 2 is preferable, and amino acids 1 to 4 in SEQ ID NO: 2 are preferable.
- Polypeptides having the amino acid sequence represented by 02 are particularly preferred.
- polypeptide of the present invention is not necessarily a single polypeptide, and may be a part of a fusion polypeptide (fusion protein), if necessary.
- Other polypeptides constituting this fusion polypeptide include, for example, a polypeptide to be biotinylated, glutathione-S-transferase or a part thereof, and a protein encoded by genelO of T7 phage ( T7 phage genelO product) or a part thereof.
- the amino acid sequence of the polypeptide of the present invention has been clarified by the present invention, it can be synthesized based on the amino acid sequence.However, it can be obtained by expressing using the DNA of the present invention described later. Possible and preferred. That is, the cells of the present invention are cultured in a suitable medium to produce and accumulate the polypeptide of the present invention in the medium, and the polypeptide of the present invention is collected from the medium to produce the polypeptide of the present invention. can do.
- a host-vector system usually used for production of protein can be used for expression of protein can be used. Mutants and expression vectors (PinPointXa ⁇ pGEMEX, pET-5 (Promega), pGEX-5X (Pharma -1-Shea (Pharmacia)) is preferred.
- the polypeptide of the present invention can be expressed from the DNA of the present invention using a yeast such as Pichia ptorz's as a host. .
- Expression may be performed directly using the DNA of the present invention, or may be expressed as a fusion protein with another protein. Further, the DNA of the present invention may be expressed in full length, or may be partially expressed as a partial peptide.
- the antibody of the present invention is an antibody that binds to the polypeptide of the present invention, and may be either a polypeptide or a monoclonal.
- the antibody of the present invention can be prepared by using a polypeptide of the present invention produced as described above or a partial polypeptide thereof or a fusion protein of these with other proteins as an antigen in the usual manner, for example, as follows. It is possible to manufacture it.
- the polyclonal antibody of the present invention can be obtained, for example, by immunizing an animal to be immunized, such as a mouse, rat, guinea pig, rabbit, goat, or sheep, with the above antigen, and collecting serum from these animals. be able to.
- an adjuvant adjuvant
- an immunoglobulin fraction may be purified by a conventional method.
- the monoclonal antibody of the present invention can be obtained, for example, as follows. That is, the antigen is administered to the intraperitoneal, subcutaneous, or footpad of an animal to be immunized, such as a mouse, rat, guinea pig, rabbit, goat, or sheep, and then the spleen or popliteal lymph node is removed. The cells collected from these cells are fused with myeloma cells, which are tumor cell lines, to establish hybridomas, and the obtained hybridomas are continuously grown. From the obtained hybridomas, specificity for the polypeptide of the present invention is obtained. Select cell lines that continuously produce antibodies. By culturing the selected strain in a suitable medium, the monoclonal antibody of the present invention can be obtained in the medium.
- the monoclonal antibody of the present invention can be produced in a large amount by culturing the hybridoma in a living body such as the abdominal cavity of a mouse.
- a living body such as the abdominal cavity of a mouse.
- lymph node cells, lymph cells in peripheral blood, and the like can be used in addition to spleen cells.
- the myeoma cell line is derived from the same cell type as compared to the cell type derived from a different cell type, and a stable antibody-producing hybridoma can be obtained.
- Purification of the obtained polyclonal and monoclonal antibodies includes salting out using sodium sulfate, ammonium sulfate, etc., low-temperature alcohol precipitation, selective precipitation fractionation using polyethylene glycol or isoelectric point, and electrophoresis. , DEAE (getylaminoethyl)-derivatives, CM (carboxymethyl)-ion-exchange chromatography using ion exchangers such as derivatives, affinity chromatography using protein A or protein G, high Examples include droxy-pattern chromatography, immunosorbent chromatography in which an antigen is immobilized, gel filtration, ultracentrifugation, and the like.
- the antibody of the present invention may be
- Fab-containing fragments obtained by treating (Fab) with a protease that does not degrade may be used.
- a protease that does not degrade eg, plasmin, pepsin, papain, etc.
- the nucleotide sequence of the gene encoding the antibody of the present invention or the amino acid sequence of the antibody is determined, the fragment chimeric antibody containing Fab of the antibody of the present invention (for example, the present invention) Chimeric antibodies containing the Fab portion of the above-mentioned antibodies), and such fragments ⁇ chimeric antibodies are also included in the antibodies of the present invention.
- the DNA of the present invention includes a DNA encoding a lectin having the following properties.
- Sugar chain preferably a sugar chain having an N-acetylglucosamine residue, more preferably a sugar chain having an N-acetylglucosamine residue at the terminal, particularly preferably at the terminal The N-acetylglycosamine residue of the sugar chain having an N-acetylglycosamine residue;
- SDS The molecular weight estimated by polyacrylamide gel electrophoresis is about 40 kilodaltons.
- the DNA of the present invention includes a DNA encoding at least a part of the above-described polypeptide of the present invention.
- the lectin has 402 amino acid residues in addition to the above properties.
- the nucleotide sequence of the DNA of the present invention is not particularly limited as long as it encodes at least a part of the lectin having the above properties or the polypeptide of the same effect.
- the DNA of the present invention also includes at least all of the DNA encoding the polypeptide of the present invention.
- a sugar chain having at least a part of the amino acid sequence shown in SEQ ID NO: 2 and having a sugar chain (preferably a sugar chain having an N-acetyl glucosamine residue, more preferably a sugar chain having an N-acetyl glucosamine residue at a terminal) Chain, particularly preferably the N-acetylglycosamine residue of a sugar chain having an N-acetylglycosamine residue at the terminal, very preferably the N-acetylglucosamine residue of a sugar chain having a GlcNAc ⁇ Man at the terminal.
- a lectin polypeptide which may have a substitution, deletion or insertion of an amino acid residue, which does not substantially impair the binding activity to the amino acid sequence shown in SEQ ID NO: 2.
- Lectin polypeptides having at least a part thereof, and DNAs encoding each of the polypeptides containing a part of these polypeptides are all included in the DNA of the present invention. It is.
- the DNA of the present invention includes a DN having a base sequence encoding the amino acid sequence represented by amino acid number 18 to 402 or 1 to 402 in SEQ ID NO: 2.
- A is preferred and preferred.
- a DNA having at least a part or all of the base sequence (base numbers 92 to 1246 or 41 to 1246) shown in SEQ ID NO: 1 is mentioned, and is particularly preferable. .
- DNA of different nucleotide sequences due to degeneracy of the genetic code is also included in the DNA of the present invention.
- the deviation of these DNAs is also included in the DNA of the present invention.
- the chromosome-derived PVL gene has a small amount of PVL polypeptide even if it is a DNA fragment that is predicted to contain an intron in the coding region. Both are included in the DNA of the present invention as long as they are partially coded. That is, in the present specification, the term "encode” also includes having a base sequence capable of undergoing processing or the like at the time of transcription to finally produce a target polypeptide.
- encoding at least a portion of a polypeptide preferably refers to a sugar chain (preferably a sugar chain having an N-acetyltylcolasamine residue, and more preferably a sugar chain having an N- ⁇ A sugar chain having a cetyl glucosamine residue, particularly preferably the N-acetyl dalcosamine residue of a sugar chain having an N-acetyl glucosamine residue at the terminal, and very preferably a sugar chain having GlcNAc ⁇ Man at the terminal; A portion having some activity or function, such as having binding activity to an N-acetylglycosamine residue) or having antigenicity, or a base sequence corresponding to the portion is specific to a PVL gene. It means coding a part that can be used as a primer or a probe.
- the present invention includes DNA or RNA complementary to the DNA of the present invention.
- the DNA of the present invention may be a single strand of only the coding strand encoding lectin, or a double strand consisting of this single strand and a DNA or RNA strand having a sequence complementary thereto. It may be a main chain.
- the DNA of the present invention may have the entire length of the coding region encoding the entire PVL, or may encode a part of the peptide of the PVL.
- the sequence The present invention is based on the PCR method (polymerase-chain reaction method) using oligonucleotide primers synthesized based on or based on the sequence, from the DNA of mushroom chromosome DNA or mRNA. It can also be obtained by amplifying DNA.
- the DNA of the present invention was obtained for the first time by cDNA cloning comprising the following steps, as described in Examples below.
- the method for producing the DNA of the present invention is not limited to this, and cDNA using DNA probe synthesized based on partial amino acid sequence
- the DNA of the present invention can also be produced by a screening method using a library, an expression cloning method using lectin activity as an index, or other known cDNA cloning techniques.
- an example of the method for obtaining the DNA of the present invention will be specifically described.
- PVL can be purified from the fruiting body of Mushroom mushroom by combining ordinary protein purification methods. Specifically, it is preferably performed according to the method described in J. Biol. Chera., 264, 173-177 (1989).
- the sugar chain binding activity (lectin activity) of PVL can be determined by the ELI SA method using a biotinylated FVL such as Kodiibe (J. Biol. Chem., 264, 173-177 (1989)), or the agaract Ig by Tsuchiya et al. It can be measured by a binding method to G (J. Immunology, 151, 1137 1146 (1993)).
- the lectin's sugar chain binding activity can also be measured by the hemagglutination method or the hemagglutination inhibition method described in Examples below.
- the presence of natural PVL and recombinant PVL polypeptide can be detected by Western blot using an anti-PVL antibody described later.
- An anti-PVL antibody can be prepared by a known method by immunizing a egret with PVL.
- the immunization method and the detection method in general are described in detail, for example, in Ed Harlow and David Lane (Antibodies, A Laboratory Manual, Cold. Spring Harbor Laboratory, 1988).
- Total RNA can be obtained by a known method (Kingston, RE, (1991) in Current Protocols in Molecular Biology, Suppl. 14, Unit 4.2, Greene Publishing Associates and Wiley Interscience, New York, etc.). the material is, ? Otsu's! The material is not limited as long as it expresses ⁇ NA, but fresh mujinatake fruiting bodies are preferable for obtaining full-length mRNA. Since there may be local variants and subspecies, it is particularly preferable that the mushrooms that inhabit the ground at an altitude of 500 m to 60 Om at Mt. Haruna and Mt. Akagi in Gunma Prefecture.
- total RNA can be obtained from the fruiting body of Mushroom mushroom by a commonly used method for preparing total RNA, but the guanidine thiosineto ZCsCl method (Kingston, RE, (1991) in Current Protocols in Molecular Biology, Suppl. 14, Unit 4.2, Greene Publishing Associates and Wiley Interscience, New York).
- poly (A) + RNA is obtained from the total RNA obtained as described above by a known method, for example, Rigo d-T (oligo- (dT)) It is preferable to use a mRNA isolation system using a magnetism that can be purified by cellulose column chromatography or the like, for example, a PolyATtract mRNA isolation system (promega).
- cDNA can be synthesized by the reverse transcriptase reaction using poly (A RNA as type I. It is convenient to use a commercially available kit for cDNA synthesis. For example, RiboClone cDNA synthesis systems (Promega) ) Can be used to synthesize cDNA and ligate cDNA to a cloning vector (eg, EcoRI-digested gt11) .In the present invention, it is preferable to use EcoRI-digested gt11. Although oligo (dT) n can be used as a primer in the reverse transcriptase reaction, it is preferable to use a random oligonucleotide primer.
- the recombinant DNA can be directly introduced into E. coli treated with calcium chloride.
- a method for efficiently infecting E. coli is used, and kits for this purpose are also commercially available (Gigapack II packaging extracts, manufactured by Stratagene). It is preferable to use this method also in the present invention.
- a mushroom mushroom cDNA library can be created.
- the recombinant DNA that has been packaged infects E. coli infects E. coli, but it is necessary to select the E. coli strain to be used depending on the cloning vector used.
- the E. coli strain it is possible to select an E. coli EscAei! ZWacom) Y1088 or other colon strain which does not express a 3- / 3-galactosidase activity and is suitable as a phage host. Good.
- Igt11 is used as a vector, it is only necessary to suspend it in a soft agar medium with a precautionary statement and overlay it on an agar medium to form plaque.
- the mushroom mushroom cDNA fragment is inserted into the EcoRI cleavage site downstream of the / 3-galactosidase gene, so that the mushroom gene fragment is fused with the -galactosidase gene. And expression of proteins from Mushroom mushroom Is done.
- a phage clone having PVL cDNA can be selected using the reactivity to the anti-PVL antibody as an index.
- Preparation and selection of a phage clone that reacts with the anti-PVL antibody may be performed according to a conventional method, for example, gci on Sambrook et al. (Sambrook, J. et al, Molecular cloning, Cold Spring Harbor lab. Press. 1989). .
- the use of the ProtBlot II AP system is preferred.
- a candidate cDNA By preparing phage DNA from the selected positive clones and cutting with an appropriate restriction enzyme, a candidate cDNA can be cut out. The obtained cDNA is directly or subcloned into an appropriate plasmid to determine the nucleotide sequence.
- SEQ ID NO: 1 shows the open reading frame portion of the nucleotide sequence of the cDNA coding for PVL determined as described above, and SEQ ID NO: 2 shows the amino acid sequence. From a single open reading frame beginning with the first ATG codon, a protein consisting of 402 amino acid residues and having a molecular weight of 43 kDa is expected.
- the polypeptide encoded by the DNA is a sugar chain (preferably, a sugar chain having an N-acetylglycosamine residue, and more preferably, a sugar chain having an N-acetyl A sugar chain having a cetyl glucosamine residue, particularly preferably the N-acetyl glucosamine residue of a sugar chain having an N-acetyl dalcosamine residue at the terminal, very preferably a sugar chain having a GlcNAc-Man at the terminal!
- N-acetylglucosamine residue ⁇ N-acetylglucosamine residue
- Substitution, deletion, or insertion of nucleotides into the DNA sequence is performed by synthesizing a sequence that has restriction enzyme-cleaved ends at both ends and that includes both sides of the mutation point, and replacing it with the corresponding portion of the unmutated DNA sequence. can do.
- site-directed mutagenesis (Kramer, W.
- Any method can introduce substitutions, insertions or deletions into the DNA sequence.
- the activity of binding to the sugar chain of the polypeptide can be measured, for example, by the lectin activity measurement method described above, and those skilled in the art can substitute one or more amino acid residues that do not substantially impair the activity, Deletions or insertions can be easily identified.
- the method of purifying the purified PVL fragmentation is not particularly limited, but it is preferable to use a protein analyzing enzyme such as Lysyl Endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) for sequence analysis.
- the cut gel may be contacted with a protease and then separated by SDS PAGE or the like, or by gel filtration or the like.
- electrophoresis is suspended by first turning off the power, performing enzyme digestion for about 30 minutes, and then restarting electrophoresis.
- This method is convenient because enzyme digestion and separation of peptide fragments after digestion can be performed in one step.
- the peptide formed by protease digestion is transferred to a PVDF membrane or a nitrocellulose membrane. The membrane is stained with a dye that stains proteins such as Kumashi 'Brilliant' Blue and Amid Black, and then the peptide band is cut out.
- Proteolytic enzymes Amino-terminal sequencing of peptides can be performed by known methods on PVDF membranes, nitrocellulose membranes, etc. containing peptides generated after digestion. By knowing the partial amino acid sequence of the peptide, the consistency with the base sequence can be confirmed.
- the PVL gene can be expressed by a well-known method using a commonly used or commercially available expression vector having Escherichia coli or yeast as a host.
- the expression vector may be one expressing the present PVL DNA directly from the initiation codon or one expressing it as a fusion protein.
- expression vectors PmPointXa, pGEMEX, pET-5 (manufactured by Promega), pGEX-5X (manufactured by Pharmacia), pPIC3K (manufactured by Invitrogen) which is Escherichia coli and yeast shuttle vector, etc. The use of is preferred.
- the recombinant DNA of the present invention can be obtained by incorporating a DNA encoding any other polypeptide into the DNA of the present invention by a conventional method.
- the DNA encoding any other polypeptide preferably has a nucleotide sequence encoding a peptide that undergoes biotinylation.
- the recombinant vector containing the DNA of the present invention can be obtained by incorporating the DNA of the present invention into an arbitrary vector by a conventional method.
- the vector is an expression vector.
- the expression vector is not particularly limited as long as it is a vector that can be usually used in the field of genetic engineering, but a nucleotide sequence encoding a peptide to be subjected to biotinylation, a nucleotide sequence encoding glutathione-1S-transferase, etc. It preferably has a sequence or a base sequence encoding a genelO product of T7 phage.
- the polypeptide produced in the host cell can be used as an avidin-binding resin. Purification can be easily performed by using such a method.
- the polypeptide produced in the host cell may use a glutathione-binding resin or the like. Makes it easier to purify.
- Transformed cells can be obtained by introducing the above-described recombinant vector into host cells in a conventional manner.
- the host cell is E. coli or yeast.
- Those skilled in the art can appropriately select the combination of the recombinant vector and the host cell.
- Hemagglutination Using a 96-well microtiter plate, mix the PBS solution serially diluted with PBS 251 and 2% type 0 human red blood cell suspension 251 in PBS. After standing at room temperature for 20 minutes, the minimum amount that causes aggregation is defined as the 1 aggregation position of PVL.
- Hemagglutination inhibition method Mix 25 ⁇ l of serially diluted inhibitor solution of known concentration with 8 agglutination units of ⁇ VL (25 ⁇ ), leave at 4 ° C for 2 hours, and suspend in 2% type 0 human erythrocyte PBS Add 50 ⁇ l of solution. After standing at room temperature for 20 minutes, the concentration (mM) of the inhibitor that completely inhibits aggregation is defined as the inhibitory concentration.
- Example 1 Purification of PVL from fruiting body of Mushroom mushroom and preparation of anti-PVL polyclonal antibody
- Mujinata mushroom fruit bodies collected on the ground at an altitude of 500 m to 60 Om at Mt.Akagi in Gunma Prefecture are collected by the method described in J. Biol. Chem., 264, 173-177 (1989). Approximately 40 mg of PVL showing an almost electrophoretically uniform band was obtained from 300 g of Mushroom fruit body.
- SDS-PAGE Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a partial modification of the method of Laemmli et al. (Lae Married Li, UK., (1970), Nature 227, 680-685).
- SDS-PAGE Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis
- the PVDF membrane was immersed in 100% methanol for 20 seconds, washed with distilled water for several seconds, and immersed in 25 mM Tris-192 mM glycine, 20% methanol (plot buffer) for 5 minutes.
- a filter paper (3MM chromatography paper; manufactured by Whatman) immersed in a plot buffer solution was overlaid, the membrane and the gel were sandwiched, and electricity was supplied at 180 mA for 1 hour at room temperature.
- the PVDF membrane was washed with TBST (10 mM Tris-HCl (pH 7.5), 150 mM aCl, 0.1% Tween 20) three times for 5 minutes. The membrane was transferred into TBST containing 3% BSA, and blocking was performed for 1 hour.
- the cells were transferred to TBS ⁇ containing anti-PVL antibody and shaken for 1 hour at room temperature. After similarly washing with TBS TB, the cells were transferred into TBST containing an alkaline phosphatase-conjugated anti-Peacock IgG (Fc) antibody (Promega) and allowed to stand at room temperature with shaking for 1 hour. After washing with TBST for 5 minutes, washing twice with TBS (without Tween 20 of TBST) for 5 minutes, transfer to Western Blue substrate (Promega) and perform color reaction. went. After confirming appropriate color development, the membrane was washed with distilled water to stop color development.
- Fc alkaline phosphatase-conjugated anti-Peacock IgG
- ELISA and Western blot were performed for the assay of the purified anti-PVL antibody (FIG. 1).
- ELIISA could be used at a 10000-fold dilution.
- no band other than the 40-kDa PVL was found in the mushroom mushroom extract.
- no band was confirmed in the stamp lot using an extract of Escherichia coli derived from the K12 strain, and it was considered that there was no cross-reacting protein.
- the purified FVL was fragmented using Lysyl Endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.).
- the fragmented polypeptide was separated by gel filtration.
- the partial amino acid sequences of the four peptide fragments were determined. These matched the amino acid sequence predicted from the nucleotide sequence, as described later ( Figure 3).
- the fruit bodies of Mujinatake are collected on the ground at 500 m to 60 Om above Mt.Akagi, and the fresh fruit bodies of Mujinatake are crushed with a polytron homogenizer in the presence of denatured protein.
- Total cellular RNA was extracted. After ethanol precipitation, total cellular RNA was stored at 180 ° C. Purification of inRNA from total RNA was performed using the PoLyATract mRNA isolation system (Promega, manufactured by Promega) according to the protocol provided with RiboClone cDNA Synthesis Systems AMV.
- RT Promega kit
- the reagents attached to the kit were used: 50 mM Tris-HCl (pH 8.5), 50 mM KC1, lOmM MgCl2, 500 M spermidine, lOmM
- DTT dithiothreitol
- ImM dNTPs 25 units (U) RNasin, 4m sodium phosphate, 30U AMV RT (Avian Myeloblastosis Virus Reverse Transcriptase) were added.
- the reaction solution was added with a final concentration of 40 mM Tris-HCl (pH 7.2), 90 mM KC1, 3 mM MgCl, 3 mM DTT, 50 g / ml BSA, and 0.8 U RNase was added.
- H 23U DNA polymerase I was added and incubated for 2 hours at 14'C.
- the reaction mixture was added to a final concentration of 20 mM ethylenediaminetetraacetic acid (EDTA).
- EDTA ethylenediaminetetraacetic acid
- the membrane was washed twice with TBST. Transfer to TBST containing 1% BSA and pro- I locked. After washing once with TBST, the plate was reacted with an anti-PVL antibody at room temperature for 30 minutes. After washing three times with TBST, incubate at room temperature for 30 minutes in a solution containing an alkaline phosphatase-conjugated anti-Peacock IgG antibody (Promega), wash three times with TBST, and further wash with TBS. Was washed once.
- the prepared mushroom cDNA library was screened, and 6 positive plaques were detected and purified from the plaques of 1.4 ⁇ 10 5 plaque forming units (pfu).
- cDNA was isolated from each clone and cut with EcoRI, the molecular weight of the insert was confirmed by agarose electrophoresis.
- the clones with the longest inserts were; IPVL231 and IPVL2111, and the insert lengths were almost the same and were approximately 1300 bp.
- the insert lengths of 1 PVL511, APVL3121, and PVL2221 were approximately 700 bp, 300 bp, and 250 bp, respectively.
- the inserts were removed from ⁇ PVL231 and PVL2111 by EcoRI treatment and inserted into the EcoRI site of OBluescript SK (+) (Stratagene), SKPVL231 and SKPVL2111 were created respectively. These were cloned into SJ with various restriction enzymes and confirmed by electrophoresis. As a result, the inserts of the two clones showed the same pattern (data not shown). Therefore, we decided to analyze mainly SKPVL231.
- the determination of the base system IJ was performed using ABI Prism Primer Cycle Sequencing reaction Kit (manufactured by Nokinkin Enolema Co., Ltd. (Perkin-Elmer Co.)). Also, the Samurai cycler
- Figure 3 shows the results of the nucleotide sequence analysis. Insert DKA had a total length of 1256 bp and did not include the polyA tail and its associated signal. This is probably because a random primer was used when preparing the library.
- ORF search for the base sequence of the obtained insert DNA only one ORF of 900 bp or more was confirmed, and only four RFs of 500 bp or less were confirmed for four of the six frames.
- no start codon and no stop codon were identified in the remaining one frame, and it was considered to be a part of the larger 0RF, but the sequence obtained by analysis of the amino acid sequence was not included at all.
- the molecular weight becomes 43 kDa or more. It is difficult to imagine a longer ORF containing the amino acid sequence obtained by amino acid sequence analysis, and it is unlikely that it encodes the target substance.
- the ORF considered to encode PVL had a nucleotide count of 1206 bp and was 402 amino acids in terms of amino acid.
- the amino acid sequence predicted from the nucleotide sequence contained all of the partial amino acid sequences of the four peptides obtained from the amino acid sequence analysis of the fragment by enzymatic digestion of PVL (bold underline in FIG. 3).
- Amino acid Amino acid composition Amino acid composition
- GenBank GenBank, EMBL and protein databases (PIR, SWISS-PROT)
- PIR protein databases
- SWISS-PROT a search was performed not only for fungi but also for all species, but no significant homology was found.
- the fragment obtained by digesting SKPVL231 with Hindlll was inserted into the Hindi 11 site of Expression Point Pinpoint Xa-1 (Promega) to create pPPVL231.
- Escherichia coli JM109 was transformed with this plasmid.
- Pre-culture the transformed E. coli in LB (LBamp) containing 50 g / ml ampicillin add 1 ml of the preculture to 100 ml of LBamp, shake culture at 37 ° C for 1 hour, IPTG was added to a concentration of 100 M, and the cells were further cultured with shaking for 4 hours. 100 1 of the culture was removed, centrifuged at 10,000 xg for 5 minutes at room temperature, and the supernatant was discarded.
- sample buffer containing 50 mM Tris-HCl, 2% SDS, 20% glycerol, 5% 3_mercaptoethanol, and 0.002% BPB to the pellet, stir, and boil at 95 ° C for 5 minutes. did. Thereafter, analysis was performed by SDS-PAGE and Western blot.
- Expression vector Pinpoint Xa 1 expresses a peptide fusion protein that undergoes biotinylation in E. coli.
- the insert inserted into the Hindlll site downstream of the biotinylated peptide of this vector lacks 23 amino acids from the beginning of the ⁇ RF of PVL cDNA and 8 amino acid residues in terms of amino acids.
- Analysis by SDS PAGE shows that the E. coli extract transformed with PPPVL231 did not show L, 53 kDa, which was not observed in the E. coli extract transformed only with Becyu (Pinpoint Xa-1). I checked the band.
- This recombinant fusion tanno II. 3 ⁇ 4 ⁇ Use the avidin resin (Softlink SoftRelease Avidin Resin; manufactured by Promega) included in the PinPoint protein purification system (kit manufactured by Promega) and attach it to the kit. Those purified according to a known protocol exhibited lectin activity and agaract IgG binding by the above-mentioned method.
- avidin resin Softlink SoftRelease Avidin Resin
- Plasmid PGEMEXPVL231 was prepared by inserting the SacK EcoRI digested DNA fragment containing the PVL gene of PPPVL231 into the corresponding Sacl and EcoRI sites of plasmid pGEMEX-1 (Promega).
- Escherichia coli BL21 (DE3) pLysS was transformed with this PGEMEXPVL231.
- Escherichia coli transformed with each plasmid was cultured overnight on an LB ampicillin plate (50 g / ml), and the resulting single colony was inoculated into a LB-ampicillin (SO ⁇ g / ml) liquid medium and cultured in a 37 " Pre-incubated overnight at C.
- LB medium was inoculated with 1/100 volume of the pre-cultured solution, shake-cultured at 37 ° C for 1 hour, and then IPTG was added to a final concentration. The cells were collected by centrifugation after cultivation at 0 ° C.
- the cell pellet was mixed with 10 times the wet weight of solution A (50 mM Tris HCl (pH 8.0), 145 mM NaCl, 5 mM EDTA, 0.5 mM PMSF (phenyl methanesulfonyl fluoride) and 10% glycerol were added, and the mixture was sonicated
- solution A 50 mM Tris HCl (pH 8.0), 145 mM NaCl, 5 mM EDTA, 0.5 mM PMSF (phenyl methanesulfonyl fluoride) and 10% glycerol was added, and the mixture was sonicated
- the centrifuged cell suspension was centrifuged at 20,000 xg at 4 ° C for 30 minutes, and the supernatant was subjected to crude cell culture. The extract was used.
- the extract was analyzed by SDS-PAGE and Western blot, and as a result, bands of 40 kDa and 56 kDa, respectively, which specifically reacted with the anti-PVL antibody were detected. From these results, expression of the recombinant PVL from pGEMEXPVL231 was confirmed.
- Plasmid PETPVL231 was prepared by inserting a DNA fragment of PSKPVL231 that had been cut with the restriction enzyme Hindi IK EcoRI containing the PVL gene into the corresponding HindIII and EcoRI sites of plasmid pET5c (Promega).
- Escherichia coli BL21 (DE3) pLysS was transformed with this PETPVL231, and the same operation as in 3-1-2 was performed to obtain a crude cell extract.
- the extract was analyzed by SDS-PAGE and Western blot, and as a result, bands of 40 kDa and 56 kDa, respectively, which specifically reacted with the anti-PVL antibody were detected. From these results, expression of the recombinant PVL from PETPVL231 was confirmed.
- PETPVL231 contained the same sequence at the amino terminus as the genelO product of T7 phage, but its molecular weight was close to that of natural PVL as predicted from the sequence.
- Escherichia coli ABLEK was transformed with this PGSTPVL231, and the same operation as in 3-1-2 was performed to obtain a crude cell extract.
- the extract was analyzed by SDS-PAGE and Western blot, and as a result, a specific band of 68 kDa was confirmed. This band reacted specifically with the anti-PVL antibody by Western blot.
- This expression plasmid (PPICPVL231) has a replication initiation region ( ⁇ ; ⁇ 1 replication origin) in E. coli and an ampicillin resistance gene (bla) and can be propagated in E. coli.
- 5 'A0X1 is the promoter region of the alcohol oxidase gene A0X1, into which the 0RF (open reading frame) of the PVL gene is inserted.
- this plasmid does not contain the replication initiation region (ARS) in yeast, it is replicated as part of the chromosome when inserted into the chromosome.
- Plasmid DNA of PPICPVL231 was digested with restriction enzyme BspEI and introduced into yeast Pichia pastoris, SMD1m (his4 pep4) by electroporation. Gene transfectants that can grow without histidine were transferred to MD agar medium (Minimal Dextrose Medium); 1.35% Yeast Nitrogen Base, 4xl (T 5 % Pyotin, 2% Darcose, 2% agarose) These inductants were
- PPICPVL231DM is integrated as a part of the yeast chromosome. In these transfectants, the expression level is expected to increase due to gene duplication. I chose a loan (named # 20).
- protein A cell mouth fine 2 g was added and gently stirred at room temperature for 10 minutes. This suspension was centrifuged at 1 000 rpm for 1 minute to recover the protein A cell port fine. To the collected protein A cellulophene, 1-2 ml of protein A cellulophene elution buffer (0.1 M citrate buffer (pH 5)) was added and left at room temperature for 5 minutes. Next,
- Agaract IgG was measured using a known method for directly measuring agaract IgG (J. Immunol., 151, 1137 1146 (1993)). That is, commercially available protein G is diluted to 20 g / ml in 0.1 M carbonate buffer (pH 8.5 to 9), and 50 ⁇ ⁇ (1 ⁇ gZell) of this solution is added to each Nunc immunoplate (trade name). : Maxi Soap, Nunc (made by Nunc)), and allowed to stand at 4 ° C for 16 hours to uniformly coat.
- the plate is washed twice with PBS (—), and 3% serum albumin (BSA, sold by Seikagaku Corporation) is used as a blocking substance in order to cover the portion where protein G is not fixed. Was added and the mixture was allowed to stand at room temperature for 2 hours.
- PBS —
- serum albumin sold by Seikagaku Corporation
- the plate was washed three times with a washing solution (PBS (-) containing 0.05% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.)), and then the agaractate purified in Preparation Example 1 was washed.
- a washing solution PBS (-) containing 0.05% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.)
- Tween 20 manufactured by Wako Pure Chemical Industries, Ltd.
- 50 ⁇ of standard solutions of various concentrations of IgG each concentration of 1.25 to 20 ⁇ g / ml
- the solvent used for the agaract IgG standard solution was 1% 83% and 0.05%
- reaction diluent 20? 83 (—) (hereinafter referred to as “reaction diluent”) was used.
- the plate was washed three times with the above-mentioned washing solution, and diluted with the reaction diluent.
- the reaction was allowed to stand at 60 C for 60 minutes.
- TMB tetramethylbenzidine solution
- Example 1 The PVL purified in Example 1 or a commercially available PVL was diluted to 20 ⁇ g / ml with 0.1 M carbonate buffer (pH 8.5 to 9) containing 10% glycerol. 1 (1 gZ) is added to each well of Nunc Immunoplate (trade name: Maxi Soap, manufactured by Nunc), and left at 4 ° C for 16 hours to obtain uniform I did.
- 0.1 M carbonate buffer pH 8.5 to 9
- 1 (1 gZ) is added to each well of Nunc Immunoplate (trade name: Maxi Soap, manufactured by Nunc), and left at 4 ° C for 16 hours to obtain uniform I did.
- the plate is washed twice with PBS (-), and PBS containing 3% serum albumin (BSA, sold by Seikagaku Corporation) as a blocking substance is used to cover the part of the well where PVL is not fixed. (-) The solution was added and left at room temperature for 2 hours.
- the plate was washed three times with a washing solution (PBS (0.05) containing 0.05% Tween 20), and then various concentrations of agaract IgG purified in Preparation Example 1 (0. 50 ⁇ 1 was added to the plate and allowed to react at 37 ° C for 60 minutes.
- a washing solution PBS (0.05) containing 0.05% Tween 20
- reaction diluent PBS (-) containing 1% BSA and 0.05% Tween 20
- the measurement method of the present invention enables quantification of 0.125 to 2 g / ml of agaract IgG. Compared to the measurement method shown in Comparative Example 1, it is about 10 times more sensitive, and it does not require biotin-labeled PVL, which is difficult to prepare due to the aggregation properties of expensive protein G and PVL itself.
- the measurement method of the present invention was found to be a highly sensitive and simple method for measuring agaract IgG.
- N-acetylgalactosamine GalNA
- G1cNAc N-acetylglucosamine
- OA knee osteoarthritis
- RA rheumatoid arthritis
- liver disease patients 15 cases
- the quantification of agaract IgG present in each serum was measured according to the method described in Example 3.
- the obtained measurement results are shown in Table 2 and FIG. According to Table 2 and FIG. 11, the average value of agaract IgG in the serum of RA patients was about twice as high as that of healthy individuals.
- the serum IgG of OA patients showed almost the same value as that of healthy individuals.
- the serum of patients with liver disease tended to be slightly lower than the sera of healthy subject agaract IgG.
- Example 6 (Agaract IgG measurement kit of the present invention :)
- the agaract IgG measurement kit consists of:
- the standard agaract IgG solution was prepared with a mixed solution of 1% BSA (dissolved in PBS), 0.05% Tween 20 and 0.05% procrine (manufactured by Iwaki, manufactured by Supelco Inc.), and then freeze-dried.
- this sample diluent stock solution is a mixed solution of 5% BSA (dissolved in 5 times concentration of PBS :), 0.25% Tween 20, and 0.25% procrine.
- the component of this undiluted washing solution is a 5-fold concentration of PBS (-) containing 0.25% Tween-20.
- agaract IgG contained in a sample such as serum can be measured directly, specifically, with extremely high sensitivity, at a low cost and easily, and in a lectin.
- the problem of labeling and lectin instability can be eliminated.
- the detection method based on this measurement method is an effective method as a specific detection method that can accurately distinguish between OA and RA in the diagnosis of arthritis.
- the kit of the present invention is extremely useful as a kit having similar advantages.
- the anti-experimental animal IgG antibody was used to It is possible to measure agalact IgG in Dell animals (eg, model animals in which RA is induced by drugs or model animals that develop RA spontaneously), which is useful for elucidating the causes of RA development and useful in drug development. Method.
- DNA encoding PVL or an equivalent thereof, and a polypeptide expressed from the DNA or a DNA fragment derived from the DNA can be obtained.
- DNA encoding PVL or an equivalent thereof has been obtained, and thus it is expected that PVL or an equivalent thereof can be mass-produced to the extent that it can be used industrially. It is expected to be used in applications where PVL has been used in the past and applications that utilize the binding specificity of PVL.
- the recombinant PVL obtained by the present invention is expected to be applied to diagnostic agents.
- abnormal IgG in serum that is, galactose (Gal) of sugar chains linked to the 297th asparagine residue is missing, and N-linked
- Asn—GlcNAc GldVAc—Man—GlcNAc is unusually increased in IgG (Agaract IgG) (Mullinax, F., Arthritis Rheum. , 18, 417 (1975); Parekh, B. et al., Nature, 316, 452 (1985)).
- PVL is a monomeric protein ⁇ and binds to a sugar chain having GlcNAc at the terminal such as / 3GlcNAcl ⁇ 6Man and l ⁇ 3Man (Kochibe, A. and Matta, ⁇ ⁇ , J. Biol. Chem., 264, 173 ⁇ 177 (1989)). It has been reported that PVL specifically recognizes agaract IgG and that it can be applied to the diagnosis of RA (Tsuchiya, N. and Kobata, A., J. Immunol., 151, 1137 1146 1993)). Specific increases in agaract IgG have also been reported for some other diseases. Therefore, application of the recombinant PVL to diagnostics for such diseases is greatly expected. It is also expected that lectins having novel activity will be created by genetic engineering techniques using the DNA of the present invention. Sequence listing
- Organism name Mushroom (Psa thyrella vel utina)
- Pro Pro Lys Met Val lie Ala Asn Phe Ala Tyr Asn Ala Gly Gly Trp
- Gly Asp lie Thr Gly Asp Gly Leu Leu Asp Val Val Gly Phe Gly Glu
- Lys Arg Val lie Asp Asn Phe Gly Tyr Asn Gin Gly Trp Arg Val Asp
- Lys Arg Ala Asp lie Leu Gly Phe Gly Gly Ala Gly Val Tyr Thr Ser
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Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002268038A CA2268038A1 (en) | 1996-10-15 | 1997-10-15 | Method for assaying agalacto-igg and assay kits, polypeptides of lectins, and dnas encoding the same |
EP97944128A EP0977036A1 (en) | 1996-10-15 | 1997-10-15 | METHOD FOR ASSAYING AGALACTO-IgG AND ASSAY KITS, POLYPEPTIDES OF LECTINS, AND DNAS ENCODING THE SAME |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8/272732 | 1996-10-15 | ||
JP8/272731 | 1996-10-15 | ||
JP27273196A JP3889463B2 (ja) | 1996-10-15 | 1996-10-15 | アガラクトIgGの測定法および測定キット |
JP27273296 | 1996-10-15 | ||
JP9/197628 | 1997-07-23 | ||
JP9197628A JPH10174588A (ja) | 1996-10-15 | 1997-07-23 | レクチンのポリペプチド及びそれをコードするdna |
Publications (1)
Publication Number | Publication Date |
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WO1998016825A1 true WO1998016825A1 (fr) | 1998-04-23 |
Family
ID=27327395
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PCT/JP1997/003723 WO1998016825A1 (fr) | 1996-10-15 | 1997-10-15 | PROCEDE DE DOSAGE D'UNE AGALACTO-IgG ET TROUSSES DE DOSAGE, POLYPEPTIDES DE LECTINES ET ADN CODANT CEUX-CI |
Country Status (3)
Country | Link |
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EP (1) | EP0977036A1 (ja) |
CA (1) | CA2268038A1 (ja) |
WO (1) | WO1998016825A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8409811B2 (en) | 2007-09-11 | 2013-04-02 | Galab Technologies Gmbh | Lectin-based glycan assay |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2591359B1 (en) * | 2010-07-07 | 2017-03-01 | Aethlon Medical Inc | Methods for quantifying exosomes |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH01139599A (ja) * | 1987-08-27 | 1989-06-01 | Nichirei Corp | 新規レクチン及びその製造方法 |
JPH0587814A (ja) * | 1991-09-30 | 1993-04-06 | Konica Corp | リウマチ診断方法及びリウマチ診断薬並びにアガラクト シルIgGの定量方法 |
JPH08220099A (ja) * | 1995-02-10 | 1996-08-30 | Morinaga Milk Ind Co Ltd | 物質の検出試薬及び慢性関節リウマチの検知法 |
-
1997
- 1997-10-15 WO PCT/JP1997/003723 patent/WO1998016825A1/ja not_active Application Discontinuation
- 1997-10-15 CA CA002268038A patent/CA2268038A1/en not_active Abandoned
- 1997-10-15 EP EP97944128A patent/EP0977036A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH01139599A (ja) * | 1987-08-27 | 1989-06-01 | Nichirei Corp | 新規レクチン及びその製造方法 |
JPH0587814A (ja) * | 1991-09-30 | 1993-04-06 | Konica Corp | リウマチ診断方法及びリウマチ診断薬並びにアガラクト シルIgGの定量方法 |
JPH08220099A (ja) * | 1995-02-10 | 1996-08-30 | Morinaga Milk Ind Co Ltd | 物質の検出試薬及び慢性関節リウマチの検知法 |
Non-Patent Citations (1)
Title |
---|
THE JOURNAL OF IMMUNOLOGY, Vol. 151, No. 2, (1993), N. TSUCHIYA et al., p. 1137-1146. * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8409811B2 (en) | 2007-09-11 | 2013-04-02 | Galab Technologies Gmbh | Lectin-based glycan assay |
Also Published As
Publication number | Publication date |
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CA2268038A1 (en) | 1998-04-23 |
EP0977036A1 (en) | 2000-02-02 |
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