WO2013035799A1 - ペリオスチンの特定領域に結合する抗体及びこれを用いたペリオスチンの測定方法 - Google Patents
ペリオスチンの特定領域に結合する抗体及びこれを用いたペリオスチンの測定方法 Download PDFInfo
- Publication number
- WO2013035799A1 WO2013035799A1 PCT/JP2012/072774 JP2012072774W WO2013035799A1 WO 2013035799 A1 WO2013035799 A1 WO 2013035799A1 JP 2012072774 W JP2012072774 W JP 2012072774W WO 2013035799 A1 WO2013035799 A1 WO 2013035799A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- periostin
- region
- antibody
- measurement
- sample
- Prior art date
Links
Images
Classifications
-
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
Definitions
- the present invention relates to an antibody that binds to a specific region of periostin (also referred to as osteoblast-specific factor 2 or OSF2) that can be a marker for allergic diseases and other diseases, a method for measuring periostin contained in a sample using this antibody, and a measuring reagent And a method for improving accuracy, and a method for examining pulmonary fibrosis or interstitial pneumonia.
- periostin also referred to as osteoblast-specific factor 2 or OSF2
- the present invention is useful in the field of life science such as clinical examination, clinical pathology, immunology and medicine, and in the field of chemistry such as analytical chemistry.
- Periostin is an extracellular matrix protein, and is composed of an EMI region, an R1 region, an R2 region, an R3 region, an R4 region, and a C-terminal region in this order from the N-terminal side to the C-terminal side. Idemitsu found that the measurement of the expression level of the periostin gene was useful as a test method for allergic diseases, and completed the invention of the test method for allergic diseases (Patent Document 1 and Non-Patent Documents). 1). In addition, Izuhara has found that measurement of the expression level of the periostin gene is also useful as a test method for idiopathic interstitial pneumonia (see Patent Document 2).
- a polyclonal antibody, a monoclonal antibody against OSF2 (periostin), a diagnostic method using these antibodies, etc. are disclosed (see Patent Document 3), and a novel osteoblast-specific transcription factor named Osf2 / Cbfa1
- An immunoassay method using an anti-OSF2 (periostin) antibody is disclosed (see Patent Document 4), a purified antibody that specifically binds to human periostin, and breast cancer bone using this antibody
- a diagnostic assay method for examining metastasis and the like is disclosed (see Patent Document 5), and an antibody against periostin having anti-cell adhesion activity and a method for quantifying periostin using this antibody are disclosed (see Patent Document 6). .
- an object of the present invention is to provide a measurement method and a measurement reagent with improved accuracy in measurement using an antigen-antibody reaction of periostin contained in a sample, and to improve measurement accuracy. It is to provide a method and a method for examining pulmonary fibrosis or interstitial pneumonia with improved accuracy.
- the inventors of the present invention have studied the measurement of periostin contained in a sample, and as a result, have found that the above problem can be solved by detecting a specific region of periostin, and have completed the present invention.
- this invention consists of the following invention.
- the antibody according to (1) above which is an antibody that binds to a degradation product of periostin.
- the amino acid sequence of the heavy chain variable region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence of the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 18
- the antibody according to 4 comprises (6) Hybridoma SS16A strain with an accession number of NITE BP-1281, Hybridoma SS18A strain with an accession number of NITE BP-1282, Hybridoma SS19C strain with an accession number of NITE BP-1283, and an accession number of NITE BP-1068 Any one selected from the group consisting of a hybridoma SS19D strain, a hybridoma SS20A strain whose accession number is NITE BP-1284, a hybridoma SS25A strain whose accession number is NITE BP-1285, and a hybridoma SS27A strain whose accession number is NITE BP-1286 A monoclonal antibody produced by the hybridoma.
- a method for measuring periostin contained in a sample which comprises detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin.
- periostin is a periostin degradation product.
- 10 The method according to any one of (7) to (9) above, wherein periostin is not a multimer.
- periostin In measuring the amount or concentration of periostin contained in a sample, at least one region selected from the group consisting of the EMI region, R1 region, R2 region and R3 region of periostin is detected. How to improve the accuracy of (16) The method according to (15) above, wherein the antibody according to any one of (1) to (6) above is used. (17) The method according to (15) or (16) above, wherein periostin is a periostin degradation product. (18) The method according to any one of (15) to (17) above, wherein periostin is not a multimer. (19) A method for examining pulmonary fibrosis or interstitial pneumonia, comprising the following steps.
- an antibody that binds to at least one region selected from the group consisting of the EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof of the present invention (hereinafter, also referred to as “anti-periostin specific region antibody”).
- This antibody is an antibody that can be used to detect at least one region selected from the group consisting of the EMI region, R1 region, R2 region, and R3 region of periostin.
- This antibody is an antibody that can bind to a degradation product of periostin.
- the antibody that does not bind to a periostin multimer is at least selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof. It is an antibody that has the specificity that it can bind to one region and does not bind to a periostin multimer. This antibody is an antibody that can bind to a degradation product of periostin and does not bind to a multimer of periostin.
- the method for measuring periostin, the reagent for measuring periostin, the method for improving the accuracy of measuring periostin, and the method for examining pulmonary fibrosis or interstitial pneumonia in the measurement of periostin contained in a sample By detecting at least one region selected from the group consisting of region, R1 region, R2 region and R3 region, the sensitivity of the measurement [positive rate in affected group in the measurement (true positive rate)], or Specificity [negative rate in the unaffected group in the measurement (true negative rate; ie, 1-false positive rate)] and the like can be improved, and the accuracy of the measurement can be improved.
- the periostin measurement method, the periostin measurement reagent, the periostin measurement accuracy improvement method, and the pulmonary fibrosis or interstitial pneumonia testing method of the present invention can obtain accurate periostin measurement values.
- Anti-periostin specific region antibody The antibody in the present invention is an antibody (anti-periostin specific region antibody) that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof.
- the anti-periostin specific region antibody of the present invention can improve the accuracy of measurement by using it for the measurement utilizing the antigen-antibody reaction of periostin contained in a sample.
- the anti-periostin specific region antibody is an antibody that can bind to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product. There is no particular limitation.
- anti-periostin specific region antibody for example, a monoclonal antibody, a polyclonal antibody capable of binding to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof,
- examples include antisera, antibody fragments (such as Fab and F (ab ′) 2 ), and single chain antibodies (scFv).
- This anti-periostin specific region antibody is an antibody (such as a chimeric antibody, a humanized antibody, or a fully humanized antibody) that has been changed to an amino acid sequence of an animal species different from the animal that immunizes the immunogen by genetic recombination technology or the like. There may be.
- the anti-periostin specific region antibody is preferably a monoclonal antibody.
- two or more anti-periostin specific region antibodies may be used.
- the anti-periostin specific region antibody of the present invention is an antibody that binds to a degradation product of periostin (also referred to as “periostin degradation product”).
- periostin degradation product means a polypeptide in which the C-terminal region having at least the amino acid sequence represented by SEQ ID NO: 14 is deleted from periostin having the amino acid sequence represented by SEQ ID NO: 2.
- the periostin degradation product of the present invention lacks at least the C-terminal region having the amino acid sequence represented by SEQ ID NO: 14 from periostin having the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: 12
- the periostin degradation product of the present invention lacks at least the C-terminal region having the amino acid sequence represented by SEQ ID NO: 14 from periostin having the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: 12
- a polypeptide lacking all of the R4 region having the amino acid sequence represented by SEQ ID NO: 10 and a portion of the R3 region having the amino acid sequence represented by SEQ ID NO: 10 are also included.
- the periostin degradation product of the present invention is a polypeptide that the present inventors have found for the first time.
- the presence of the periostin degradation product can be clearly confirmed, for example, by detecting a polypeptide contained in a sample derived from a living body by an immunological technique using the antibody of the present invention (FIGS. 5 and 9). , FIG. 26 etc.).
- Examples of periostin degradation products include, but are not limited to, those having a molecular weight of about 40,000 Da (about 40 kDa).
- the periostin degradation product is preferably included in a sample derived from a living body.
- the anti-periostin specific region antibody of the present invention binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof, and is a dimer or trimer of periostin. It is preferably an antibody that does not bind to a multimer of periostin, tetramer, or higher. That is, the anti-periostin specific region antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or its degradation product in the present invention and does not bind to a periostin multimer. Is preferable because the degree of improvement in measurement accuracy is higher.
- immunogen for producing the anti-periostin specific region antibody in the present invention will be described below.
- the immunogen for producing the anti-periostin specific region antibody in the present invention all or part of periostin can be used. That is, all or part of periostin such as periostin derived from human or the like or periostin obtained by gene recombination can be used.
- the immunogen for producing this anti-periostin specific region antibody is preferably all or part of the EMI region, R1 region, R2 region and / or R3 region of periostin.
- the immunogen for producing an anti-periostin specific region antibody that does not bind to the periostin multimer is all or part of the periostin EMI region, R1 region, R2 region and / or R3 region. Is preferred.
- the anti-periostin specific region antibody in the present invention can be obtained.
- the immunogen for producing this anti-periostin specific region antibody is 1 to several (usually 1 to 8, preferably 1 to 6) amino acids in the whole or part of the amino acid sequence of periostin. It may be a peptide or protein containing an amino acid sequence obtained by performing deletion, substitution, insertion, addition or modification of residues.
- the minimum unit of the amino acid sequence of the immunogen of the anti-periostin specific region antibody in the present invention is 1 in all or part of the amino acid sequence of periostin, or all or part of the amino acid sequence of these amino acid sequences.
- the tripeptide consisting of an amino acid sequence consisting of these three consecutive amino acid residues, or the addition of another amino acid or peptide to this, etc. can be identified as anti-periostin in the present invention. It can be considered as the smallest unit of region antibody immunogen.
- the method for obtaining a peptide or protein containing all or part of the amino acid sequence of periostin is not particularly limited, and any method may be used, for example, obtaining by a known method. Can do.
- periostin As a method for obtaining periostin, the following method (“G. Takayama et al., J. Allergy Clin. Immunol., 118, No. 1, pages 713 to 723, issued in 2006”) and the like can be mentioned.
- periostin base sequence of polynucleotide: Accession Number D13666 of nucleic acid database GenBank (SEQ ID NO: 1); amino acid sequence: Accession Number BAA02837 (SEQ ID NO: 2) of nucleic acid database GenBank)
- Periostin protein is expressed in S2 cells, which are insect cells, and then purified.
- a transformant of S2 cells is prepared as follows.
- a cDNA encoding the above-mentioned part of periostin is inserted into the pMT / Bip / V5-HisA plasmid (Invitrogen, Carlsbad, Calif., USA), and this is designated as pMT / Bip / periostin-V5-HisA.
- PAcHygro Invitrogen, Carlsbad, Calif., USA
- a transformant is selected with hygromycin to obtain a stable transformant.
- periostin in which V5 epitope / His tag is bound is expressed at the carboxy terminus.
- S2 recombinant periostin protein Purification of the S2 recombinant periostin protein is performed as follows. The expression of S2 recombinant periostin protein is induced by adding copper sulfate to the medium of periostin gene stable transformant S2 cells. As a result, the S2 recombinant periostin protein is expressed and secreted into the culture supernatant. The culture supernatant is dialyzed against phosphate buffered saline (PBS) and then mixed with nickel resin (Ni-NTA Agarose, Qiagen, Hilden, Germany) to bind S2 recombinant periostin protein to the resin.
- PBS phosphate buffered saline
- the resin is washed to remove impurities, and the S2 recombinant periostin protein is eluted with an imidazole-containing buffer.
- the eluted S2 recombinant periostin protein is dialyzed against PBS or the like to obtain purified periostin protein.
- Periostin can also be obtained by the following method. That is, periostin cDNA is incorporated into a GEX-KG vector (“KL. Guan et al., Anal. Biochem., 192, 262-267, published in 1991”) and transfected into E. coli BL21. This is cultured in an LB medium containing ampicillin, and periostin to which glutathione S-transferase (GST) has been added is purified from the cells by glutathione sepharose 4B (GE Healthcare, Little Chalfont, UK). To this, GST is cleaved with thrombin, and periostin without GST is obtained. This can be quantified by the Bradford method to obtain periostin whose amount (concentration) has been clarified.
- GST glutathione S-transferase
- periostin can also be obtained by the method described in “I. Takayama et al., J. Biochem., 146, 5, 713-723, 2009”.
- amino acid sequence of periostin is represented by SEQ ID NO: 2
- nucleotide sequence of the polynucleotide encoding the amino acid sequence is represented by SEQ ID NO: 1.
- EMI region of periostin is described in, for example, “I. Kii et al., J. Biol. Chem., 285, No. 3, pages 2028-2039, 2010” or “T. Maruhashi et al., J. Biol. Chem., 285, No. 17, 13294-13303, published in 2010 ”and the like.
- the amino acid sequence of the periostin EMI region is represented by SEQ ID NO: 4, and the nucleotide sequence of the polynucleotide encoding the amino acid sequence is represented by SEQ ID NO: 3.
- the R1 region, R2 region or R3 region of periostin is obtained by the method described in “I. Takayama et al., J. Biochem, Vol. 146, No. 5, pages 713 to 723, published in 2009”, etc. be able to.
- the amino acid sequences of the R1 region, R2 region, and R3 region of periostin are represented by SEQ ID NOs: 6, 8, and 10, respectively.
- the nucleotide sequences of the polynucleotides that encode the amino acid sequences are represented by SEQ ID NOs: 5, 7, and 9, respectively.
- amino acid sequences of the R4 region and C-terminal region of periostin are represented by SEQ ID NOs: 12 and 14, respectively, and the nucleotide sequences of the polynucleotides encoding the amino acid sequences are represented by SEQ ID NOs: 11 and 13, respectively.
- the immunogen can be synthesized by a peptide synthesis method such as a liquid phase method and a solid phase method, and an automatic peptide synthesizer may also be used.
- Chemistry IV Tokyo Kagaku Doujin, 1975, Izumiya et al.“ Peptide Synthesis Fundamentals and Experiments ”, Maruzen, 1985, edited by the Japanese Biochemical Society,“ Sequel Biochemistry Laboratory 2 Under Protein Chemistry ”, Tokyo Kagaku Doujin, 1987 It can be synthesized according to the method described in the year etc., and it is also easy to produce a mutant in which the amino acid sequence is deleted, substituted, inserted or added.
- modifications such as introduction of unnatural amino acids, chemical modification of each amino acid residue, or cyclization of the interior of the molecule by introduction of cysteine residues to stabilize the structure may be performed.
- the immunogen may be prepared from DNA or RNA having a corresponding nucleobase sequence by using genetic engineering technology, edited by the Japanese Biochemical Society, “Second Life Chemistry Experiment Course 1 Gene Research Method I”, Tokyo Chemical. Doujin, 1986, Japan Biochemical Society, “Sequential Biochemistry Experiment Course 1 Gene Research Method II”, Tokyo Chemical Doujin, 1986, Japan Biochemical Society, “Sequence Biochemistry Experiment Course 1 Gene Research Method III”, Tokyo Chemistry Doujin, What is necessary is just to prepare with reference to 1987 grade
- the immunogen when the immunogen is a low-molecular substance, it is common to immunize an animal or the like with a carrier (carrier) bound to the immunogen, but a peptide having 5 amino acids is used as an immunogen. Since there is also a report (Kiyama et al., “Abstract 3 of the 112th Annual Meeting of the Japanese Pharmaceutical Society”, page 122, published in 1992) that a specific antibody was produced, it is not essential to use a carrier.
- the carriers include mussel hemocyanin (KLH), bovine serum albumin (BSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, Known carriers such as dipalmityl lysine, tetanus toxoid or polysaccharide can be used.
- the immunogen and carrier binding methods include glutaraldehyde method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, maleimidobenzoyl-N-hydroxysuccinimide ester method, bisdiazotized benzidine method or N-succimidyl- Known coupling methods such as the 3- (2-pyridyldithio) propionic acid method can be used.
- suck to carriers such as a nitrocellulose particle
- anti-periostin specific region antibody which is polyclonal antibody
- the anti-periostin specific region antibody which is a polyclonal antibody can be prepared by the following operation.
- the immunogen described above can be used as an immunogen for production of the anti-periostin specific region antibody which is this polyclonal antibody.
- the immunogen or the conjugate of the immunogen and a carrier can be used for mammals (mouse, guinea pig, hamster, rabbit, rat, sheep, goat, cow, horse, donkey, camel, etc.) or birds ( Immunize chicken, duck or ostrich).
- a gene related to periostin production in the body is inactivated or deleted, that is, related to periostin production. More preferred are animals in which the gene has been knocked out.
- periostin produced in the animal's body binds to the anti-periostin specific region antibody produced in the body by immunization with an immunogen such as periostin, so that the antibody activity of the anti-periostin specific region antibody is increased.
- an immunogen such as periostin
- mice in which a gene involved in periostin production is inactivated or deleted include, for example, knockout mice for periostin (“H. Rios et al., Molecular and Cellular Biology, Vol. 25, No. 24, 11131-11144, 2005”. Year issue ").
- the immunization amount of the immunogen or the conjugate of the immunogen and the carrier is determined by the immunogen, the carrier, the type of the immunized animal, the immunization injection site, and the like.
- 0.1 ⁇ g to 5 mg of the immunogen or a combination of the immunogen and a carrier is injected once per animal.
- the immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection.
- an adjuvant known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, chemical synthesis adjuvant or pertussis adjuvant can be used. Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
- booster injections of the immunogen or the conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 1 to 2 weeks.
- the number of booster injections is generally 2 to 6 times.
- the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
- the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, whole blood is collected, and the serum is separated to obtain an antiserum containing the antibody.
- the antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.
- the polyclonal antibody obtained here is a polyclonal antibody that can bind to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product (polyclonal antibody).
- the anti-periostin specific region antibody which is a polyclonal antibody is brought into the solid phase via a ligand such as this column (protein or peptide containing the periostin EMI region, R1 region, R2 region and / or R3 region amino acid sequence). Combined and collected.
- a ligand such as this column (protein or peptide containing the periostin EMI region, R1 region, R2 region and / or R3 region amino acid sequence).
- a polyclonal antibody that does not bind to any of the periostin EMI region, R1 region, R2 region, and R3 region is a ligand such as this column (periostin EMI region, R1 region, R2 region, or R3 region amino acid sequence).
- This column and the like are passed through without being bound to the protein and / or peptide containing.
- Anti-periostin specific region antibody which is a polyclonal antibody bound to ligand (protein or peptide containing amino acid sequence of periostin EMI region, R1 region, R2 region and / or R3 region) such as salt concentration or pH
- the anti-periostin specific region antibody which is a polyclonal antibody can be obtained by separating the ligand from the ligand by collecting and collecting the ligand.
- the obtained anti-periostin specific region antibody, which is a polyclonal antibody is an antibody that can bind to a degradation product of periostin.
- EMI region when “all or part of the EMI region, R1 region, R2 region and / or R3 region of periostin” is used as an immunogen, it binds to any of the EMI region, R1 region, R2 region and R3 region of periostin. No treatment for separating polyclonal antibodies is required.
- a polyclonal antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof and does not bind to a periostin multimer polyclonal antibody, Anti-periostin specific region antibody that does not bind to multimers of periostin
- the anti-periostin specific region antibody that is a polyclonal antibody obtained here is a polyclonal antibody, an anti-periostin specific region antibody that does not bind to a periostin multimer, and a polyclonal antibody, a periostin multimer.
- Anti-periostin specific region antibody ".
- the anti-periostin specific region antibody which is the obtained polyclonal antibody, is passed through and contacted with an affinity chromatography column or the like in which a periostin multimer is immobilized on a solid phase as a ligand.
- an anti-periostin specific region antibody that binds to a periostin multimer that is a polyclonal antibody binds to a solid phase via a ligand (periostin multimer) such as this column.
- a polyclonal anti-periostin specific region antibody that does not bind to a periostin multimer passes through this column without binding to a ligand (periostin multimer) such as this column.
- a ligand periostin multimer
- an anti-periostin specific region antibody that is a polyclonal antibody and does not bind to a periostin multimer can be obtained.
- an anti-periostin specific region antibody that binds to a periostin multimer that is a polyclonal antibody can be removed.
- an anti-periostin specific region antibody that does not bind to a periostin multimer that is a polyclonal antibody can be obtained from the residue.
- the obtained “anti-periostin specific region antibody that is a polyclonal antibody that does not bind to a periostin multimer” is an antibody that can bind to a degradation product of periostin and does not bind to a periostin multimer.
- a carrier is added to the obtained polyclonal antibody solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography. Can do.
- Anti-periostin specific region antibody which is a monoclonal antibody Monoclonal antibody capable of binding to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product,
- the anti-periostin specific region antibody which is a monoclonal antibody can be prepared by the following operation.
- This monoclonal antibody can be used to produce antibodies such as hybridomas by the cell fusion method of Keller et al. (G. Koehler et al., Nature, Vol. 256, pages 495-497, issued in 1975) or tumorigenic cells by viruses such as Epstan-Barr virus. It can be obtained by cells.
- preparation of a monoclonal antibody by a cell fusion method can be performed by the following operation.
- the immunogen or the combined immunogen and carrier is used for mammals (mouse, hamster, rat, rabbit, etc., for example, inbred mouse BALB / c) or birds (chicken, etc.) Immunize).
- a gene related to periostin production in the body is inactivated or deleted, that is, related to periostin production. More preferred are animals in which the gene has been knocked out.
- periostin produced in the animal's body binds to the anti-periostin specific region antibody produced in the body by immunization with an immunogen such as periostin, so that the antibody activity of the anti-periostin specific region antibody is increased.
- an immunogen such as periostin
- mice in which the gene involved in periostin production is inactivated or deleted include, for example, knockout mice for periostin (“H. Rios et al., Mol. Cell. Biol., 25, 24, 11131-11144). , 2005 ”)).
- the immunity of the immunogen or the conjugate of the immunogen and the carrier is appropriately determined according to the type of immunized animal, the site of immunization, etc.
- 0.1 ⁇ g to 5 mg of the immunogen or a combination of the immunogen and a carrier is immunized at a time.
- the immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing.
- adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, chemical synthesis adjuvant, and pertussis adjuvant can be used as the adjuvant. Immunization may be carried out at sites such as subcutaneous, intravenous, intraperitoneal, footpad or back.
- booster injections of the immunogen or the conjugate of the immunogen and the carrier are given to sites such as subcutaneous, intravenous, intraperitoneal, footpad or back at intervals of 1 to 2 weeks. .
- the number of booster injections is generally 2 to 6 times.
- the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.
- the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA, etc.
- the immunogen or the combined immunogen and carrier A solution dissolved in physiological saline (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization.
- Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
- a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ)
- PEG polyethylene glycol
- HVJ Sendai virus
- fusion of cells having antibody-producing ability and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine Only cells (hybridomas) can be selectively cultured and propagated.
- the hybridoma culture supernatant thus obtained is used as the periostin EMI region, R1 region, R2 of human or other animal (preferably derived from human when used for measurement of human periostin).
- the “EMI region, R1 region, R2 region of periostin or its degradation product” By measuring by immunoassay such as ELISA or Western blotting using a protein or peptide comprising the region and / or the R3 region, the “EMI region, R1 region, R2 region of periostin or its degradation product”
- the production cell line of the anti-periostin specific region antibody which is a monoclonal antibody in the present invention can be isolated and obtained.
- the monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the antiperiostin specific region antibody that is a monoclonal antibody in the present invention can be obtained from the culture supernatant.
- a low-concentration serum medium or the like may be used, and in this case, it is preferable in terms of easy purification of the antibody, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.
- this monoclonal antibody-producing cell line is a monoclonal antibody according to the present invention, which is injected into the abdominal cavity of a mammal that is compatible therewith and previously stimulated with pristane, etc. Anti-periostin specific region antibodies can also be obtained.
- the anti-periostin specific region antibody which is a monoclonal antibody thus obtained, can be obtained by methods such as salting-out using ammonium sulfate, sodium sulfate, ion exchange chromatography, gel filtration, affinity chromatography, or the like.
- region antibody which is a purified monoclonal antibody can be obtained by combining these methods.
- region antibody which is the obtained monoclonal antibody is an antibody which can be couple
- the culture supernatant of the obtained hybridoma is treated with a protein or peptide comprising the EMI region, R1 region, R2 region and / or R3 region of human or other animal periostin.
- the hybridoma producing the anti-periostin specific region antibody which is a monoclonal antibody can be selected by using an immunoassay method such as ELISA or Western blotting, etc.
- EMI region By measuring by an immunoassay such as ELISA or Western blot using a multimer, “at least selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product” Can bind to one region and not to periostin multimers Monoclonal antibodies can be selected hybridomas producing "(which is a monoclonal antibody, an anti-periostin specific region antibody that does not bind to multimers of periostin).
- the obtained “antiperiostin specific region antibody that does not bind to a periostin multimer that is a monoclonal antibody” is an antibody that can bind to a degradation product of periostin and does not bind to a periostin multimer.
- the amino acid sequence of the heavy chain variable region of the antibody includes the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 18.
- the monoclonal antibody of the present invention includes, for example, a hybridoma SS16A strain having a deposit number of NITE BP-1281, a hybridoma SS18A strain having a deposit number of NITE BP-1282, and a deposit number of NITE BP-1283.
- hybridoma SS19C strain with the accession number NITE BP-1068, the hybridoma SS20A strain with the accession number NITE BP-1284, the hybridoma SS25A strain with the accession number NITE BP-1285, and the accession number NITE BP Examples include, but are not limited to, monoclonal antibodies produced by any hybridoma selected from the group consisting of the hybridoma SS27A strain -1286.
- Method for measuring periostin is a method for measuring periostin contained in a sample, and detects at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin. It is a feature.
- Periostin is composed of an EMI region, an R1 region, an R2 region, an R3 region, an R4 region, and a C-terminal region in that order from the N-terminal side to the C-terminal side.
- the EMI region, the R1 region The method for measuring periostin of the present invention is characterized by detecting at least one region selected from the group consisting of R2 region and R3 region.
- the measurement method of the present invention may be a method for measuring a periostin degradation product contained in a sample. More specifically, the method for measuring a periostin degradation product contained in a sample, comprising detecting at least one region selected from the group consisting of an EMI region, an R1 region, an R2 region, and an R3 region of a periostin degradation product. It may be.
- the measurement method of the present invention may be a method for measuring periostin that is not a multimer.
- the method for measuring periostin that is not a multimer means a method that does not detect (or measure) periostin that is a multimer.
- the measurement method of the present invention is a method for measuring periostin or a periostin degradation product contained in a sample, and is from the group consisting of the EMI region, R1 region, R2 region, and R3 region of periostin or periostin degradation product. It may be a method for measuring periostin or a periostin degradation product contained in a sample, wherein at least one region selected is detected and a periostin multimer is not detected (or measured).
- the method for measuring periostin of the present invention is a method for measuring periostin by detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin in the measurement of periostin contained in a sample. This is a measurement method that can improve accuracy.
- detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin means that the EMI region, R1 region, R2 region of periostin And finding the presence of at least one region selected from the group consisting of the R3 region or finding its abundance.
- this “detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin” will be described in detail: antigen and antibody, sugar and lectin, nucleotide When measuring periostin contained in a sample using a reaction between a chain and a substance having specific affinity such as a ligand and a receptor, EMI region, R1 region, R2 region of periostin And a substance capable of specifically binding to at least one region selected from the group consisting of R3 region (specific binding substance), etc., from the EMI region, R1 region, R2 region and R3 region of periostin
- the specific binding substance can bind to at least one region selected from the group consisting of Therefore periostin EMI region R1 region, for at least one region selected from the group consisting of R2 region and R3 regions can be found by finding the presence or the abundance. That is, at least one region selected from the group consisting of periostin EMI region, R1 region, R
- the specific binding substance is an antibody
- an antibody capable of binding to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof (anti-periostin specific
- anti-periostin specific The presence of, or the abundance of, at least one region selected from the group consisting of the EMI region, R1 region, R2 region and R3 region of periostin. . That is, at least one region selected from the group consisting of periostin EMI region, R1 region, R2 region and R3 region can be detected.
- the method for measuring periostin of the present invention may detect any one of the EMI region, R1 region, R2 region or R3 region of periostin, or may be the EMI region, R1 region, R2 region of periostin or Two or more members in the R3 region may be detected.
- the method for measuring periostin according to the present invention is a method for measuring periostin contained in a sample by using an antigen-antibody reaction, which comprises an EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof. It is preferable to use an antibody that binds to at least one region selected from the group (anti-periostin specific region antibody).
- Anti-periostin specific region antibody the antibody described in the above section “[1] Anti-periostin specific region antibody”, for example, the following antibodies can be used.
- an antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof (ii) an antibody that binds to a degradation product of periostin, (iii) an antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof, and that does not bind to a periostin multimer;
- an antibody that binds to a degradation product of periostin and does not bind to a periostin multimer (v) a monoclonal antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of
- periostin specific region antibodies In the method for measuring periostin of the present invention, for example, when two molecules of antibody are reacted with one molecule of periostin by antigen-antibody reaction, all of these antibodies need to be anti-periostin specific region antibodies.
- an enzyme-labeled antibody and a solid-phased antibody both the enzyme-labeled antibody and solid-phased antibody to be bound to periostin contained in the sample are anti-periostin specific region antibodies. There must be.
- an anti-periostin specific region antibody that does not bind to a periostin multimer is used in a measurement method for measuring periostin contained in a sample using an antigen-antibody reaction.
- the degree of improvement in accuracy is further high, in the method for measuring periostin of the present invention, for example, when two molecules of an antibody are reacted with one molecule of periostin, one antibody does not bind to a periostin multimer.
- the other antibody does not necessarily need to be an anti-periostin specific region antibody that does not bind to a periostin multimer, and may be an anti-periostin specific region antibody.
- an enzyme-labeled antibody or a solid-phased antibody that binds to periostin contained in a sample does not bind to a periostin multimer.
- the other antibody does not necessarily need to be an anti-periostin specific region antibody that does not bind to a periostin multimer, and may be an anti-periostin specific region antibody.
- the above-mentioned anti-periostin specific region antibody is not limited to one type, and a plurality of types may be used simultaneously.
- the method for measuring periostin of the present invention can improve the accuracy of the measurement, and is suitable for measurement for examining the presence or absence of a disease or its degree (such as a medical condition).
- the method for measuring periostin of the present invention is more suitable for measurement for examining whether or not cancer or lung disease is present or its degree (such as a medical condition).
- the method for measuring periostin of the present invention is more suitable for measurement for examination of the presence or absence of bile duct cell carcinoma, pulmonary fibrosis or interstitial pneumonia, or its degree (such as a disease state).
- the method for measuring periostin of the present invention is more suitable for measurement for examining the presence or absence of pulmonary fibrosis or interstitial pneumonia or its degree (such as a medical condition).
- the method for measuring periostin of the present invention is particularly suitable for measurement for examining the presence or absence of interstitial pneumonia or its extent (such as a medical condition).
- the method for measuring periostin of the present invention is a measurement method for measuring periostin contained in a sample using an antigen-antibody reaction, and preferably uses an anti-periostin specific region antibody.
- an anti-periostin specific region antibody the measurement principle is not particularly limited, and the desired effect is achieved.
- Examples of the measurement method for measuring periostin contained in this sample using an antigen-antibody reaction include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence.
- Immunoassay (LIA) enzyme antibody method, fluorescent antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination reaction measurement method, erythrocyte agglutination reaction method, particle agglutination reaction method, JP-A-9-229936 And a target substance (test substance) having a surface coated with a specific binding substance to the target substance (test substance) described in Japanese Patent Application Laid-Open No. 10-13819 and the like.
- any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method) can be applied to the measurement in the method for measuring periostin of the present invention.
- the measurement in the method for measuring periostin of the present invention may be performed by a method, or may be performed using an apparatus such as an analyzer.
- Samples in the present invention include periostin such as blood, serum, plasma, urine, semen, spinal fluid, saliva, ascites or amniotic fluid, or extracts of organs, tissues, or cells such as blood vessels or liver. If it is a sample such as a biological sample that may be removed, it will be the target.
- periostin such as blood, serum, plasma, urine, semen, spinal fluid, saliva, ascites or amniotic fluid, or extracts of organs, tissues, or cells such as blood vessels or liver. If it is a sample such as a biological sample that may be removed, it will be the target.
- the sample used for measurement is preferably a liquid
- pretreatment such as extraction or solubilization may be performed according to a known method to obtain a liquid sample.
- the sample may be subjected to concentration treatment as necessary.
- the sample may be diluted by adding a diluent before the measurement.
- a dilution treatment may be performed by adding a diluent to the sample.
- a dilution treatment may be performed by adding a diluent to the sample.
- Various aqueous solvents can be used as the diluent.
- an aqueous solvent such as water, physiological saline, or various buffers such as tris (hydroxymethyl) aminomethane buffer [Tris buffer], phosphate buffer, or phosphate buffered saline can be used.
- Tris buffer tris (hydroxymethyl) aminomethane buffer
- phosphate buffer phosphate buffered saline
- the pH of this buffer is preferably in the range of pH 5 to pH 10.
- the whole blood sample is mixed with a hypotonic solution such as water or an aqueous solvent containing a surfactant, and a treatment for rupturing red blood cells is performed. It is preferable for performing the measurement without hindrance.
- the substance to be measured is periostin.
- This periostin includes periostin monomer, periostin dimer, trimer, tetramer or higher periostin multimer, or periostin degradation product (for example, periostin degradation product having a molecular weight of about 40 KDa) ) And the like, but all of them are substances to be measured in the present invention.
- periostin which is a measuring object substance in this invention
- the monomer of said periostin or the decomposition product of periostin is suitable. That is, in other words, periostin other than a periostin multimer (also referred to as “periostin that is not a multimer” in the present specification) is suitable as the measurement target substance.
- periostin which is a measurement target substance in the present invention
- the above-described degradation product of periostin is particularly suitable. That is, in other words, periostin other than a periostin monomer and a periostin multimer is particularly suitable as the measurement target substance.
- Immunological measurement method using labeled antibody Immunoassay using labeled antibody such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay, or luminescent immunoassay is performed in the method for measuring periostin of the present invention.
- the measurement method that is, the measurement method using an antigen-antibody reaction using a labeled antibody
- the sandwich method or the competition method
- the periostin contained in the sample can be obtained by the sandwich method.
- Both the immobilized antibody and the labeled antibody to be bound to the antibody need to be anti-periostin specific region antibodies.
- the enzyme-labeled antibody or the immobilized antibody when either the enzyme-labeled antibody or the immobilized antibody is an anti-periostin specific region antibody that does not bind to a periostin multimer, the other antibody is not necessarily periostin. It is not necessary to be an anti-periostin specific region antibody that does not bind to any multimer, and any anti-periostin specific region antibody may be used.
- a solid support in the form of a microcapsule, a bead, a microplate (microtiter plate), a test tube, a stick, or a test piece made of a material such as cellulose, sepharose, glass, metal, ceramics, or a magnetic material can be used.
- the antibody such as the anti-periostin specific region antibody and the solid phase carrier are adsorbed and bound by a known method such as a physical adsorption method, a chemical binding method or a combination thereof, and the antibody is immobilized on the solid phase carrier. can do.
- an anti-periostin specific region in which an antibody such as an anti-periostin specific region antibody and a solid phase carrier are mixed and contacted in a solution such as a buffer solution or dissolved in a buffer solution or the like according to a known method It can be carried out by bringing an antibody such as an antibody into contact with a solid phase carrier.
- a solution such as a buffer solution or dissolved in a buffer solution or the like
- an antibody such as an antibody into contact with a solid phase carrier.
- the Japanese Society of Clinical Pathology “Special Issue on Extraordinary Clinical Pathology No. 53 Immunoassay for Clinical Examinations—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with known methods described in ed.
- an antibody such as an anti-periostin specific region antibody and a solid phase carrier are mixed with glutaraldehyde, carbodiimide, imidoester, maleimide This is carried out by mixing and contacting with a bivalent cross-linking reagent, and reacting with an amino group, a carboxyl group, a thiol group, an aldehyde group, a hydroxyl group or the like of an antibody such as an anti-periostin specific region antibody and a solid phase carrier. Can do.
- the surface or inner wall surface of the solid phase carrier to which an antibody such as an anti-periostin specific region antibody is immobilized is used.
- a known method such as bovine serum albumin (BSA), human serum albumin (HSA), casein, gelatin, ovalbumin or a salt thereof, a surfactant, skim milk powder or the like.
- the solid phase carrier may be subjected to blocking treatment (masking treatment).
- peroxidase POD
- alkaline phosphatase ALP
- ⁇ -galactosidase urease
- catalase glucose oxidase
- lactate dehydrogenase or amylase can be used in the case of enzyme immunoassay.
- fluorescence immunoassay fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, or the like can be used.
- a radioimmunoassay tritium, iodine 125, iodine 131, or the like can be used.
- a luminescence immunoassay method NADH-FMNH 2 -luciferase system, luminol-hydrogen peroxide-POD system, acridinium ester system or dioxetane compound system can be used.
- the operation method of the measurement is a known method or the like (Japan Clinical Pathology Society, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination -Technology and Applications”, published by Clinical Pathology, 1983; edited by Yuji Ishikawa et al. “Enzyme Immunoassay”, 3rd edition, Medical School, published in 1987; edited by Kitagawa Tsuneki et al., “Protein Nucleic Acid Enzyme Separate Volume No.31, Enzyme Immunoassay”, Kyoritsu Shuppan, published in 1987).
- an enzyme labeled with an antibody is reacted with a substrate under the optimum conditions, and the amount of the enzyme reaction product is measured by an optical method or the like.
- the fluorescence immunoassay the fluorescence intensity by the fluorescent substance label is measured
- the radioimmunoassay the radiation dose by the radioactive substance label is measured.
- the luminescence immunoassay the amount of luminescence by the luminescence reaction system is measured.
- Immunological measurement method by agglutination method The measurement in the measurement method of periostin of the present invention is performed by immunocomplex aggregation such as immunoturbidimetry, latex turbidimetry, latex agglutination method, erythrocyte agglutination method or particle agglutination method.
- immunocomplex aggregation such as immunoturbidimetry, latex turbidimetry, latex agglutination method, erythrocyte agglutination method or particle agglutination method.
- the anti-periostin specific region antibody may be an anti-periostin specific region antibody that does not bind to a multimer of periostin, and is preferable because accuracy is improved.
- a phosphate buffer a glycine buffer, a tris (hydroxymethyl) aminomethane buffer [Tris buffer], a Good buffer, or the like
- Tris buffer a tris (hydroxymethyl) aminomethane buffer
- a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor may be included.
- the solid phase carrier may be polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, chloride.
- Vinyl-acrylic acid ester copolymer vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid Particles made of materials such as polymers, acrylic acid polymers, latex, gelatin, liposomes, microcapsules, erythrocytes, silica, alumina, carbon black, metal compounds, metals, ceramics, and magnetic materials can be used.
- a method for immobilizing an antibody such as an anti-periostin specific region antibody on a solid phase carrier a known method such as a physical adsorption method, a chemical binding method, or a combination thereof can be used.
- an anti-periostin specific region in which an antibody such as an anti-periostin specific region antibody and a solid phase carrier are mixed and contacted in a solution such as a buffer solution or dissolved in a buffer solution or the like according to a known method It can be carried out by bringing an antibody such as an antibody into contact with a solid phase carrier.
- antibodies such as anti-periostin specific region antibody and solid phase carrier are glutaraldehyde, carbodiimide, imide ester or maleimide This is carried out by mixing and contacting with a bivalent cross-linking reagent, and reacting with an amino group, a carboxyl group, a thiol group, an aldehyde group, a hydroxyl group or the like of an antibody such as an anti-periostin specific region antibody and a solid phase carrier. Can do.
- the surface or inner wall surface of the solid phase carrier to which an antibody such as an anti-periostin specific region antibody is immobilized is used.
- a known method such as bovine serum albumin (BSA), human serum albumin (HSA), protein such as casein, gelatin, ovalbumin or a salt thereof, a surfactant or nonfat dry milk, etc.
- the solid phase carrier may be subjected to blocking treatment (masking treatment).
- the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are bonded via the measurement target substance (periostin) and aggregates are formed.
- the latex particles preferably have an average particle size of 0.04 to 1 ⁇ m.
- the concentration of latex particles on which an antibody such as an anti-periostin specific region antibody is immobilized includes the concentration of periostin in the sample, an antibody such as an anti-periostin specific region antibody, etc. Since the optimum concentration differs depending on various conditions such as the distribution density on the surface of the latex particles, the particle size of the latex particles, and the mixing ratio of the sample and the measuring reagent, it cannot be generally stated.
- concentration of latex particles on which “antibody such as anti-periostin specific region antibody” is immobilized is generally 0.005 to 1% (w / v) in the reaction mixture.
- the measurement reagent contains “latex particles on which an antibody such as an anti-periostin specific region antibody is immobilized” having such a concentration in the reaction mixture.
- the particle size of the particles used as the solid phase carrier is not particularly limited, but the average particle The diameter is preferably in the range of 0.01 to 100 ⁇ m, and more preferably in the range of 0.5 to 10 ⁇ m.
- the specific gravity of these particles is preferably in the range of 1 to 10, and more preferably in the range of 1 to 2.
- a container used for the measurement when an indirect agglutination reaction method such as a latex agglutination reaction method an erythrocyte agglutination reaction method or a particle agglutination reaction method is used as a measurement principle, for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc.
- a test tube, a microplate (microtiter plate), a tray, and the like for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc.
- a test tube, a microplate (microtiter plate), a tray, and the like a test tube, a microplate (microtiter plate), a tray, and the like.
- the bottom surface of the solution storage portion (such as a well of a microplate) of these containers preferably has a shape having an inclination from the center to the periphery of the bottom, such as U-type, V-type, or
- the measuring operation can be performed by a known method.
- the sample and an antibody such as an anti-periostin specific region antibody, or the sample and “immobilized on a solid phase carrier” are used.
- the transmitted light and scattered light are measured by the endpoint method or the rate method.
- the sample is reacted with “an antibody such as an anti-periostin specific region antibody immobilized on a solid phase carrier” to determine the state of aggregation. Measure visually.
- a measurement reagent containing “a solid phase carrier on which an anti-periostin specific region antibody is immobilized” or a measurement reagent containing “a solid phase carrier on which an anti-periostin specific region antibody is immobilized” and an “aqueous solvent” Prepare and prepare the measurement reagent to be contained.
- a measurement reagent containing “a solid phase carrier on which an anti-periostin specific region antibody is immobilized” and a sample are mixed, whereby a “solid phase carrier on which an anti-periostin specific region antibody is immobilized” is obtained.
- a sample are mixed, whereby a “solid phase carrier on which an anti-periostin specific region antibody is immobilized” is obtained.
- the measurement of the generated complex aggregate should be performed by measuring the absorbance of the reaction mixture, such as transmitted light or scattered light, in the measurement reaction in which the complex aggregate is present by the endpoint method or the rate method. To implement.
- the measured value such as absorbance obtained by measuring the sample was compared with the measured value such as absorbance obtained by measuring the standard substance (sample with known periostin concentration), and was included in the sample.
- concentration (quantitative value) of periostin is calculated.
- the measurement of absorbance such as transmitted light or scattered light may be performed by measuring transmitted light or scattered light, and may be one-wavelength measurement or two-wavelength measurement (two It may be a difference or ratio depending on the wavelength.
- the measurement wavelength is generally selected from 340 nm to 1,000 nm.
- the measurement of periostin in the present invention may be performed by a method, or may be performed using an apparatus such as a measuring apparatus.
- the measuring device may be a general-purpose automatic analyzer or a dedicated measuring device (dedicated machine).
- the measurement of periostin in the present invention may be performed by a one-step method (one-reagent method) or a method performed by a plurality of operation steps such as a two-step method (two-reagent method).
- Second reagent Buffer solution containing “latex particles immobilized with anti-periostin specific region antibody”
- a certain amount of a sample such as serum and a certain amount of the first reagent are mixed and allowed to stand at a certain temperature for a certain time.
- the temperature at the time of standing is preferably a constant temperature within a range of room temperature (1 ° C. to 30 ° C.) or low temperature (30 ° C. to 40 ° C.). (For example, 37 ° C.)
- the temperature at the time of standing is preferably a constant temperature within a range of room temperature (1 ° C. to 30 ° C.) or low temperature (30 ° C. to 40 ° C.). (For example, 37 ° C.)
- the standing time is preferably a fixed time of 1 minute or more and 10 minutes or less, and more preferably a fixed time of 3 minutes or more and 5 minutes or less.
- the antigen-antibody reaction between the anti-periostin specific region antibody immobilized on the latex particles and the periostin contained in the sample is performed by adding and mixing the second reagent to the mixed solution of the sample and the first reagent.
- the reaction mixture is irradiated with light, and a decrease in transmitted light intensity at an appropriate wavelength, which is a signal generated by complex aggregates of latex particles generated (absorbance) Or the increase in scattered light intensity, the amount of the complex aggregate produced, that is, the amount of periostin contained in the sample is determined.
- aqueous solvents can be used as the solvent.
- the aqueous solvent include purified water, physiological saline, various buffer solutions such as tris (hydroxymethyl) aminomethane buffer [Tris buffer], phosphate buffer, or phosphate buffered saline.
- Tris buffer tris (hydroxymethyl) aminomethane buffer
- phosphate buffer or phosphate buffered saline.
- the pH of the buffer solution may be appropriately selected and used as appropriate. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3 to pH 12.
- an antibody such as the above-mentioned anti-periostin specific region antibody, a solid phase carrier on which an antibody such as the above anti-periostin specific region antibody is immobilized, and / or the above anti-periostin specific region
- a labeling substance such as an enzyme, proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or salts thereof; various salts; Non-specific reaction inhibitor; sugar; skim milk powder; various animal sera such as normal rabbit serum; various preservatives such as sodium azide or antibiotics; activator; reaction accelerator; sensitivity increasing substance such as polyethylene glycol; 1 type of various surfactants such as nonionic surfactants, amphoteric surfactants or anionic surfactants Or two or more may be used as appropriate.
- the concentration when these are used for measurement is not particularly limited, but is preferably 0.001 to 10% (W).
- surfactant examples include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether vinegar Anionic surfactants such as salts, and the like.
- the measurement reagent for periostin or its degradation product of the present invention is a measurement reagent for periostin or its degradation product contained in a sample, and is at least selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin One area is detected.
- Periostin is composed of an EMI region, an R1 region, an R2 region, an R3 region, an R4 region, and a C-terminal region in that order from the N-terminal side to the C-terminal side.
- the EMI region, the R1 region The measurement reagent for periostin or its degradation product of the present invention is characterized by detecting at least one region selected from the group consisting of the R2 region and the R3 region.
- the measurement reagent for periostin or its degradation product of the present invention is specific to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product.
- the specific binding substance means a substance that specifically binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof. Examples of such substances include lectins, substances that specifically bind to nucleotide chains (for example, aptamers), antibodies or fragments thereof, receptors, and other polypeptides or oligopeptides that specifically bind to each region.
- the specific binding substance is an antibody
- examples of such an antibody include the antibodies (i) to (ix) described in the above section “[2]. Method for measuring periostin 1. General remarks”. However, it is not limited to these.
- examples of such a peptide include peptides obtained from a peptide phage display library.
- the measurement reagent of the present invention may be a measurement reagent for a periostin degradation product contained in a sample. More specifically, measurement of a periostin degradation product contained in a sample containing a substance that specifically binds to at least one region selected from the group consisting of the EMI region, the R1 region, the R2 region, and the R3 region of the periostin degradation product. It may be a reagent.
- the measurement reagent of the present invention may be a periostin measurement reagent that is not a multimer.
- the measurement reagent for periostin that is not a multimer means a reagent that does not detect (or measure) periostin that is a multimer.
- the measurement reagent of the present invention is a measurement reagent for periostin or a periostin degradation product contained in a sample, and is from the group consisting of the EMI region, R1 region, R2 region, and R3 region of periostin or periostin degradation product.
- It may be a reagent for measuring periostin or a periostin degradation product contained in a sample, which contains a substance that specifically binds to at least one selected region and does not bind to a periostin multimer.
- the description of the substance that specifically binds to each region (specific binding substance) is as described above.
- the measurement reagent for periostin or its degradation product of the present invention comprises at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin in the measurement of periostin or its degradation product contained in a sample. By detecting, it is a measuring reagent that can improve the accuracy of periostin measurement.
- detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin means EMI region of periostin, R1 For at least one region selected from the group consisting of the region, the R2 region and the R3 region, this means finding its presence or finding its abundance.
- this “detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin” will be described in detail: antigen and antibody, sugar and lectin, nucleotide When measuring periostin contained in a sample or a degradation product thereof using a reaction between a chain and a substance having specific affinity such as a ligand and a receptor, an EMI region of periostin, R1 By using a specific binding substance capable of specifically binding to at least one region selected from the group consisting of the region, R2 region and R3 region, etc., such as EMI region, R1 region, R2 of periostin or its degradation product Finding the presence of at least one region selected from the group consisting of region and R3 region; It may find its abundance. That is, at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or its degradation product can be detected.
- the specific binding substance is an antibody
- an antibody capable of binding to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof (anti-periostin specific
- the presence of, or the abundance of, at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product Can be found. That is, at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or its degradation product can be detected.
- the measurement reagent for periostin or its degradation product of the present invention may be one that detects any one of the EMI region, R1 region, R2 region or R3 region of periostin or its degradation product, or periostin.
- the EMI region, R1 region, R2 region, or R3 region of the degradation product may be detected.
- the measurement reagent for periostin or its degradation product is a measurement reagent for measuring periostin or its degradation product contained in a sample using an antigen-antibody reaction, and the EMI region, R1 region of periostin or its degradation product It preferably contains an antibody that binds to at least one region selected from the group consisting of the R2 region and the R3 region (anti-periostin specific region antibody).
- anti-periostin specific region antibody examples include, but are not limited to, the antibodies (i) to (ix) described in the above section “[2]. Method for measuring periostin 1. General remarks”.
- the measurement reagent for periostin or its degradation product of the present invention when measuring periostin contained in a sample using this measurement reagent, for example, when reacting two molecules of antibody with one molecule of periostin, these antibodies Any of the antibodies must be anti-periostin specific region antibodies.
- the measurement of periostin is performed by an ELISA sandwich method using an enzyme-labeled antibody and a solid-phased antibody, both the enzyme-labeled antibody and the solid-phased antibody to be bound to periostin contained in the sample are used. This anti-periostin specific region antibody is required.
- an anti-periostin specific region antibody that does not bind to a periostin multimer is contained in a measurement reagent that measures periostin contained in a sample using an antigen-antibody reaction.
- a measurement reagent that measures periostin contained in a sample using an antigen-antibody reaction for example, when reacting two molecules of antibody with one molecule of periostin as an antigen-antibody reaction, when one antibody is an anti-periostin specific region antibody that does not bind to a periostin multimer, the other antibody does not necessarily need to be an anti-periostin specific region antibody that does not bind to a periostin multimer. I just need it.
- periostin specific region antibody that does not bind to a periostin multimer
- the other antibody need not necessarily be an anti-periostin specific region antibody that does not bind to a periostin multimer, and may be an anti-periostin specific region antibody.
- the anti-periostin specific region antibody may contain not only one type but also a plurality of types.
- the measurement reagent for periostin or a degradation product thereof according to the present invention can improve the accuracy of the measurement, and is suitable for measurement for examining the presence or absence of a disease or its degree (such as a medical condition). is there.
- the measurement reagent for periostin or a degradation product thereof according to the present invention is more suitable for measurement for examining whether or not cancer or lung disease is present or its degree (such as a medical condition).
- the measurement reagent for periostin or a degradation product thereof according to the present invention is more suitable for measurement for examination of the presence or absence of cholangiocellular carcinoma, pulmonary fibrosis or interstitial pneumonia, or its degree (such as disease state). .
- measuring reagent of periostin or its degradation product of this invention is still more suitable in the measurement for the test
- the measurement reagent for periostin or a degradation product thereof according to the present invention is particularly suitable for measurement for examining the presence or absence of interstitial pneumonia or its degree (such as a medical condition).
- Sample The sample in the present invention is as described in “3. Sample” in “[2]. Periostin Measuring Method”.
- Substance to be measured The substance to be measured in the present invention is as described in “4. Substance to be measured” in “[2]. Method for measuring periostin”.
- the measuring reagent for periostin or its degradation product of the present invention is a measuring reagent for measuring periostin or its degradation product contained in a sample using an antigen-antibody reaction, and is anti-periostin specific Those containing a region antibody are preferred, but any one containing this anti-periostin specific region antibody is not particularly limited to the measurement principle of the periostin measurement, and exhibits the desired effect.
- the measurement principle of the measurement reagent for periostin or its degradation product of the present invention is, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay ( LIA), enzyme antibody method, fluorescent antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination reaction measurement method, erythrocyte agglutination reaction method, particle agglutination reaction method, JP-A-9-229936 and special A specific binding substance to a measurement target substance (test substance) described in, for example, Kaihei 10-132919 is fixed, and a carrier having a surface coated with the substance, and a specific binding substance to the measurement target substance (test substance) A measurement method using particles to which a binding substance is immobilized, or an ELSA method (Enzyme-linked Ligand shown by Dahlbeack et al. orbent Assay) (Thr
- any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method) can be applied to the measurement of the periostin or its degradation product of the present invention with a measuring reagent.
- the measurement with the measuring reagent of periostin of this invention or its decomposition product may be performed by a method, or may be performed using an apparatus such as an analyzer.
- the measurement reagent for periostin or its degradation product of the present invention may be composed of one measurement reagent.
- the anti-periostin specific region antibody is contained in the one measurement reagent.
- the measurement reagent of periostin or its degradation product of the present invention may be composed of two or more measurement reagents.
- the anti-periostin specific region antibody may be contained in one measurement reagent of two or more measurement reagents, or may be contained in two or more measurement reagents. Also good.
- the measurement reagent for periostin or its degradation product of the present invention is composed of two measurement reagents, a first reagent and a second reagent
- the anti-periostin specific region antibody is contained only in the first reagent. It may be contained only in the second reagent, or may be contained in both the first reagent and the second reagent.
- aqueous solvents can be used as a solvent for the measurement reagent of periostin or a decomposition product thereof according to the present invention.
- aqueous solvent examples include water, physiological saline and the like, or tris (hydroxymethyl) aminomethane buffer [Tris buffer], phosphate buffer, or phosphate buffered saline. And various buffer solutions.
- an appropriate pH may be selected and used as appropriate. Although there is no particular limitation, it is general to select and use a pH within the range of pH 5 to pH 10.
- the measurement reagent for periostin or its degradation product of the present invention includes proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or a salt thereof;
- proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or a salt thereof;
- Various metal ions such as calcium salts, various salts such as calcium salts, various saccharides, skim milk powder, various animal sera such as normal rabbit serum, various preservatives such as sodium azide or antibiotics, activator, reaction accelerator, polyethylene glycol Sensitive substances such as non-specific reaction-suppressing substances; or one or more of various surfactants such as nonionic surfactants, amphoteric surfactants or anionic surfactants You may make it contain suitably.
- the concentration when these are contained in the measurement reagent for periostin or its degradation product of the present invention is not particularly limited, but is preferably 0.001 to 10% (W / V), particularly 0.01 to 5% (W / V) is preferable.
- surfactant examples include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether Anionic surfactants such as salts, and the like.
- the measuring reagent of periostin of this invention or its degradation product can be sold by itself, or can be used for the measurement of periostin or its degradation product contained in a sample.
- the measurement reagent for periostin or its degradation product of the present invention can be sold in combination with other reagents or used for measurement of periostin or its degradation product contained in a sample.
- Examples of the other reagent include a buffer solution, a sample diluent, a reagent diluent, a reagent containing a labeling substance, a reagent containing a substance that generates a signal such as color development, or calibration (calibration). And reagents containing these substances.
- the measurement reagent for periostin or its degradation product of the present invention may be a measurement reagent for periostin including a first reagent containing an aqueous solvent and a second reagent containing an anti-periostin specific region antibody.
- the measurement reagent for periostin or its degradation product of the present invention is preferably a measurement reagent kit.
- Measuring reagent based on immunological measuring method using labeled antibody as measurement principle is enzyme immunoassay, fluorescent immunoassay, radioimmunoassay, luminescence immunoassay, etc.
- the measurement principle is an immunological measurement method using a labeled antibody of the above, that is, a measurement method using an antigen-antibody reaction using a labeled antibody, the sandwich method or the competitive method can be used.
- the immobilized antibody and labeled antibody to be bound to periostin contained in the sample are anti-periostin specific region antibodies.
- the labeled antibody or the immobilized antibody when either the labeled antibody or the immobilized antibody is an anti-periostin specific region antibody that does not bind to a periostin multimer, the other antibody is not necessarily periostin.
- the antibody is not necessarily an anti-periostin specific region antibody that does not bind to a multimer, and may be an anti-periostin specific region antibody.
- the measuring reagent for periostin or its degradation product of the present invention is immunoturbidimetric method, latex turbidimetric method, latex agglutination method, hemagglutination method or particle
- the production of immune complex aggregates such as the agglutination reaction method is carried out by measuring the transmitted light or scattered light by an optical method or by a visual measurement method, that is, a complex by an antigen-antibody reaction
- the measurement method (aggregation reaction method) for measuring the formation of the aggregate is used as the measurement principle, the antibody to be bound to periostin contained in the sample needs to be an anti-periostin specific region antibody.
- the anti-periostin specific region antibody may be an anti-periostin specific region antibody that does not bind to a periostin multimer, and is preferable because accuracy is improved.
- the measurement reagent for periostin or a degradation product thereof according to the present invention is an “anti-periostin specific region antibody” or “a solid phase carrier on which an anti-periostin specific region antibody is immobilized”. contains.
- the “anti-periostin specific region antibody” or “solid phase carrier on which the anti-periostin specific region antibody is immobilized” It is preferable to make it contain in a reagent.
- the measurement reagent for periostin or a degradation product thereof according to the present invention is composed of two or more measurement reagents, “an anti-periostin specific region antibody” or “an anti-periostin specific region antibody immobilized solid phase carrier”
- the reagent containing neither the “anti-periostin specific region antibody” nor the “solid phase carrier on which the anti-periostin specific region antibody is immobilized” is, for example, a reagent containing the above-mentioned aqueous solvent. It may be.
- the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are bonded and aggregated via the measurement target substance (periostin).
- the average particle diameter of the latex particles is preferably 0.04 to 1 ⁇ m.
- the concentration of latex particles on which an antibody such as an anti-periostin specific region antibody is immobilized is about the concentration of periostin contained in the sample, the anti-periostin specific region antibody, etc. Since the optimal concentration differs depending on various conditions such as the distribution density of the antibody on the latex particle surface, the particle size of the latex particle, and the mixing ratio of the sample and the measuring reagent, it cannot be generally stated.
- concentration of latex particles on which “antibody such as anti-periostin specific region antibody” is immobilized is generally 0.005 to 1% (w / v) in the reaction mixture.
- the measurement reagent contains “latex particles on which an antibody such as an anti-periostin specific region antibody is immobilized” having such a concentration in the reaction mixture.
- the particle size of the particles used as the solid phase carrier is not particularly limited, but the average particle The diameter is preferably in the range of 0.01 to 100 ⁇ m, and more preferably in the range of 0.5 to 10 ⁇ m.
- the specific gravity of these particles is preferably in the range of 1 to 10, and more preferably in the range of 1 to 2.
- a container used for the measurement when an indirect agglutination reaction method such as a latex agglutination reaction method an erythrocyte agglutination reaction method or a particle agglutination reaction method is used as a measurement principle, for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc.
- a test tube, a microplate (microtiter plate), a tray, and the like for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc.
- a test tube, a microplate (microtiter plate), a tray, and the like a test tube, a microplate (microtiter plate), a tray, and the like.
- the bottom surface of the solution storage portion (such as a well of a microplate) of these containers preferably has a shape having an inclination from the center to the periphery of the bottom, such as U-type, V-type, or
- the measurement reagent for periostin or a degradation product thereof includes periostin or a second reagent containing a first reagent containing an aqueous solvent and a second reagent containing a solid phase carrier on which an anti-periostin specific region antibody is immobilized. It may be a reagent for measuring degradation products.
- the solid phase carrier is preferably latex particles.
- the measurement method for measuring the formation of complex aggregates due to the antigen-antibody reaction is related to the measurement reagent for periostin or its degradation product according to the present invention based on the measurement method for measuring the formation of complex aggregates due to the antigen-antibody reaction.
- the details of the method are as described in “6. Immunological measurement method by agglutination” in “[2]. Periostin measurement method”.
- the method for improving the accuracy of periostin measurement according to the present invention is a method for measuring the amount or concentration of periostin contained in a sample. At least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin Is detected.
- Periostin is composed of an EMI region, an R1 region, an R2 region, an R3 region, an R4 region, and a C-terminal region in that order from the N-terminal side to the C-terminal side.
- the EMI region, the R1 region The method for improving the accuracy of periostin measurement according to the present invention is characterized by detecting at least one region selected from the group consisting of the R2 region and the R3 region.
- the improvement method of the present invention provides at least one region selected from the group consisting of the EMI region, R1 region, R2 region and R3 region of the periostin degradation product in the measurement of the amount or concentration of the periostin degradation product contained in the sample. It may be characterized by detecting.
- the improvement method of the present invention is a method for measuring the amount or concentration of periostin that is not a multimer, and at least selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or periostin degradation product.
- One region may be detected, and a periostin multimer may not be detected (or measured).
- This method for improving the accuracy of periostin measurement is a method for measuring the amount or concentration of periostin contained in a sample.
- At least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin Is a method that can improve the accuracy of periostin measurement.
- detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin means that the EMI region of periostin, R1 For at least one region selected from the group consisting of the region, the R2 region and the R3 region, this means finding its presence or finding its abundance.
- this “detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin” will be described in detail: antigen and antibody, sugar and lectin, nucleotide When measuring periostin contained in a sample using a reaction between a chain and a substance having specific affinity such as a ligand and a receptor, EMI region, R1 region, R2 region of periostin And a specific binding substance capable of specifically binding to at least one region selected from the group consisting of R3 regions, etc., from the group consisting of periostin EMI region, R1 region, R2 region and R3 region This specific binding substance can bind to at least one selected region, and so on.
- EMI region of Riosuchin, R1 region for at least one region selected from the group consisting of R2 region and R3 regions can be found by finding the presence or the abundance. That is, at least one region selected from the group consisting of periostin EMI region, R1 region, R2 region and R3 region can be detected.
- the specific binding substance is an antibody
- an antibody capable of binding to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof (anti-periostin specific
- This anti-periostin specific region antibody can be bound to at least one region selected from the group consisting of the EMI region, R1 region, R2 region and R3 region of periostin, and the like.
- the presence or the abundance of at least one region selected from the group consisting of the EMI region, the R1 region, the R2 region, and the R3 region of periostin can be found. That is, at least one region selected from the group consisting of periostin EMI region, R1 region, R2 region and R3 region can be detected.
- one of the EMI region, R1 region, R2 region, and R3 region of periostin may be detected, or the EMI region of periostin. , R1 region, R2 region or R3 region may be detected.
- a method for improving the accuracy of the measurement of periostin of the present invention in the measurement using the antigen-antibody reaction of periostin contained in a sample, from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product It is preferable to use an antibody that binds to at least one selected region (anti-periostin specific region antibody).
- anti-periostin specific region antibody examples include, but are not limited to, the antibodies (i) to (ix) described in the above section “[2]. Method for measuring periostin 1. General remarks”.
- periostin specific region antibody when measuring periostin contained in a sample, for example, when two molecules of antibody are reacted with one molecule of periostin, all of these antibodies are It must be an anti-periostin specific region antibody.
- the measurement of periostin is performed by an ELISA sandwich method using an enzyme-labeled antibody and a solid-phased antibody, both the enzyme-labeled antibody and the solid-phased antibody to be bound to periostin contained in the sample are used. This anti-periostin specific region antibody is required.
- the measurement accuracy is improved.
- the method for improving the accuracy of periostin measurement of the present invention for example, when two molecules of an antibody are reacted with one molecule of periostin, one antibody does not bind to a periostin multimer.
- the other antibody does not necessarily need to be an anti-periostin specific region antibody that does not bind to a periostin multimer, and may be an anti-periostin specific region antibody.
- periostin specific region antibody that does not bind to a periostin multimer
- the other antibody need not necessarily be an anti-periostin specific region antibody that does not bind to a periostin multimer, and may be an anti-periostin specific region antibody.
- the above-mentioned anti-periostin specific region antibody is not limited to one type, and a plurality of types may be used simultaneously.
- the method for improving the accuracy of periostin measurement according to the present invention can improve the accuracy of periostin measurement, and is suitable for measurement for examining the presence or absence of a disease or its degree (such as a medical condition). It is.
- the method for improving the accuracy of periostin measurement according to the present invention is more suitable for measurement for examining whether or not cancer or lung disease is present or its degree (such as a medical condition).
- the method for improving the accuracy of periostin measurement according to the present invention is more suitable for measurement for examining the presence or absence of bile duct cell carcinoma, pulmonary fibrosis, or interstitial pneumonia, or its degree (such as a disease state). .
- the method for improving the accuracy of periostin measurement according to the present invention is even more suitable for measurement for examination of the presence or absence of pulmonary fibrosis or interstitial pneumonia or its degree (such as a medical condition).
- the method for improving the accuracy of periostin measurement according to the present invention is particularly suitable for measurement for examining the presence or absence of interstitial pneumonia or its degree (such as a medical condition).
- Sample The sample in the present invention is as described in “3. Sample” in “[2]. Periostin Measuring Method”.
- Substance to be measured The substance to be measured in the present invention is as described in “4. Substance to be measured” in “[2]. Method for measuring periostin”.
- an anti-periostin specific region antibody is preferably used.
- the measurement principle is not particularly limited, and the desired effect is achieved.
- periostin measurement in the method for improving the accuracy of periostin measurement of the present invention, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunity Measurement method (LIA), enzyme antibody method, fluorescent antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination measurement method, erythrocyte agglutination method, particle agglutination method, JP-A-9-229936 A carrier having a surface coated with a specific binding substance to a measurement target substance (test substance) described in Japanese Patent Laid-Open No.
- test substance a measurement target substance (test substance)
- test substance a measurement target substance (test substance)
- ELSA method Enzyme-li shown by Dahlbeack et al. ked Ligandsorbent Assay) (Thromb.Haemost, 79, pp. 767-772 pp., 1998 issue;. mention may be made of WO 98/23963 pamphlet), and the like.
- the periostin measurement in the method for improving the accuracy of periostin measurement according to the present invention can employ any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method).
- the periostin measurement in the method for improving the accuracy of periostin measurement according to the present invention may be performed by a technique, or may be performed using an apparatus such as an analyzer.
- the periostin measurement in the method for improving the accuracy of periostin measurement of the present invention may be one using only one measurement reagent (one reagent method or one step method).
- the anti-periostin specific region antibody is contained in the one measurement reagent.
- the periostin measurement in the method for improving the accuracy of periostin measurement of the present invention may be one using two or more measurement reagents (multi-reagent method or multi-step method).
- the anti-periostin specific region antibody may be contained in one measurement reagent of two or more measurement reagents, or may be contained in two or more measurement reagents. Also good.
- the anti-periostin specific region antibody is used only in the first reagent. It may be contained, may be contained only in the second reagent, or may be contained in both the first reagent and the second reagent.
- the above-mentioned various aqueous solvents can be used as the solvent used for periostin measurement in the method for improving the accuracy of periostin measurement of the present invention.
- periostin measurement in the method for improving the accuracy of periostin measurement of the present invention for details of the measurement method using an antigen-antibody reaction using a labeled antibody, refer to “5. Periostin measurement method” in “5. As described in “Immunological assay method using labeled antibody”.
- periostin measurement in the method for improving the accuracy of periostin measurement of the present invention, the details of “other components at the time of measurement” are described in “7. Other at the time of measurement” in “[2]. Method for measuring periostin”. As described in “Components of”.
- the pulmonary fibrosis or interstitial pneumonia testing method of the present invention includes the following steps. a) a step of measuring the amount or concentration of periostin in a sample derived from a subject, wherein at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin is detected. And b) a step of comparing the amount or concentration of periostin in a sample derived from a subject and the amount or concentration of periostin in a sample derived from a living body not affected by pulmonary fibrosis or interstitial pneumonia
- Periostin is composed of an EMI region, an R1 region, an R2 region, an R3 region, an R4 region, and a C-terminal region in that order from the N-terminal side to the C-terminal side.
- the method for examining pneumonia includes the step of detecting at least one region selected from the group consisting of the EMI region, the R1 region, the R2 region, and the R3 region in measuring periostin contained in the sample.
- the amount or concentration of periostin in a sample derived from a subject and a living body not affected by pulmonary fibrosis or interstitial pneumonia Comparing the amount or concentration of periostin in the derived sample.
- the method for examining pulmonary fibrosis or interstitial pneumonia of the present invention in the measurement of periostin contained in a sample, at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin is obtained. By detecting it, it is a test method that can improve the sensitivity or specificity of the measurement and improve the accuracy of the measurement. As a result, the method for examining pulmonary fibrosis or interstitial pneumonia according to the present invention can obtain an accurate measurement value of periostin, improve discrimination from healthy subjects and patients with other diseases, and misdiagnose. This is an inspection method that can prevent this.
- inspection method of this invention may include the process of measuring the periostin degradation product in the sample derived from a subject. More specifically, the inspection method of the present invention may include the following steps. a) a step of measuring the amount or concentration of a periostin degradation product in a sample derived from a subject, wherein at least one region selected from the group consisting of an EMI region, an R1 region, an R2 region, and an R3 region of the periostin degradation product; Step b) comprising detecting the amount or concentration of a periostin degradation product in a sample derived from a subject and the amount of a periostin degradation product in a sample derived from a living body not affected by pulmonary fibrosis or interstitial pneumonia Or the step of comparing the concentration
- the test method of the present invention may include detecting (or measuring) periostin that is not a multimer. More specifically, the inspection method of the present invention may include the following steps. a) a step of measuring the amount or concentration of periostin or periostin degradation product in a sample derived from a subject, at least selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or periostin degradation product Step b) detecting one region and not detecting periostin multimers b) suffering from either the amount or concentration of periostin or a periostin degradation product in a sample derived from a subject and pulmonary fibrosis or interstitial pneumonia Comparing the amount or concentration of periostin or a periostin degradation product in a non-biological sample
- detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin means that EMI of periostin For at least one region selected from the group consisting of a region, R1 region, R2 region and R3 region, it means finding its presence or finding its abundance.
- this “detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin” will be described in detail: antigen and antibody, sugar and lectin, nucleotide When measuring periostin contained in a sample using a reaction between a chain and a substance having specific affinity such as a ligand and a receptor, EMI region, R1 region, R2 region of periostin And a specific binding substance capable of specifically binding to at least one region selected from the group consisting of R3 regions, etc., from the group consisting of periostin EMI region, R1 region, R2 region and R3 region This specific binding substance can bind to at least one selected region, and so on.
- EMI region of Riosuchin, R1 region for at least one region selected from the group consisting of R2 region and R3 regions can be found by finding the presence or the abundance. That is, at least one region selected from the group consisting of periostin EMI region, R1 region, R2 region and R3 region can be detected.
- the specific binding substance is an antibody
- an antibody capable of binding to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or a degradation product thereof (anti-periostin specific
- This anti-periostin specific region antibody can be bound to at least one region selected from the group consisting of the EMI region, R1 region, R2 region and R3 region of periostin, and the like.
- the presence or the abundance of at least one region selected from the group consisting of the EMI region, the R1 region, the R2 region, and the R3 region of periostin can be found. That is, at least one region selected from the group consisting of periostin EMI region, R1 region, R2 region and R3 region can be detected.
- either one of EMI region, R1 region, R2 region or R3 region of periostin may be detected, or periostin.
- Two or more of the EMI region, the R1 region, the R2 region, or the R3 region may be detected.
- a method for examining pulmonary fibrosis or interstitial pneumonia in the measurement using the antigen-antibody reaction of periostin contained in a sample, from the EMI region, R1 region, R2 region and R3 region of periostin or its degradation product It is preferable to use an antibody that binds to at least one region selected from the group (anti-periostin specific region antibody).
- anti-periostin specific region antibody examples include, but are not limited to, the antibodies (i) to (ix) described in the above section “[2]. Method for measuring periostin 1. General remarks”.
- periostin contained in a sample for example, when two molecules of antibody are reacted with one molecule of periostin as an antigen-antibody reaction, Both need to be anti-periostin specific region antibodies.
- periostin specific region antibody is required.
- the measurement accuracy is improved.
- one antibody is a periostin multimer.
- the other antibody need not necessarily be an anti-periostin specific region antibody that does not bind to a periostin multimer, and may be an anti-periostin specific region antibody.
- periostin specific region antibody that does not bind to a periostin multimer
- the other antibody need not necessarily be an anti-periostin specific region antibody that does not bind to a periostin multimer, and may be an anti-periostin specific region antibody.
- the above-mentioned anti-periostin specific region antibody is not limited to one type, and a plurality of types may be used simultaneously.
- the method for examining pulmonary fibrosis or interstitial pneumonia is suitable for examining the presence or absence of pulmonary fibrosis or interstitial pneumonia or its degree (such as a medical condition).
- the method for examining pulmonary fibrosis or interstitial pneumonia according to the present invention is particularly suitable for examining the presence or absence of pulmonary fibrosis or its degree (such as a medical condition).
- the method for examining pulmonary fibrosis or interstitial pneumonia according to the present invention is particularly suitable for examining the presence or absence of interstitial pneumonia or its degree (such as a medical condition).
- Sample The sample in the present invention is as described in “3. Sample” in “[2]. Periostin Measuring Method”.
- Substance to be measured The substance to be measured in the present invention is as described in “4. Substance to be measured” in “[2]. Method for measuring periostin”.
- pulmonary fibrosis or interstitial pneumonia by measuring periostin contained in a sample using antigen-antibody reaction is used.
- the test method is preferably one using an anti-periostin specific region antibody, but if it uses this anti-periostin specific region antibody, it is not particularly limited to its measurement principle, and the desired effect can be obtained. It is what you play.
- Examples of the measurement principle of periostin measurement in the method for examining pulmonary fibrosis or interstitial pneumonia according to the present invention include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), and radioimmunoassay (RIA).
- Luminescence immunoassay (LIA) enzyme immunoassay
- enzyme antibody method fluorescent antibody method
- immunochromatography method immunoturbidimetric method
- latex turbidimetric method latex turbidimetric method
- latex agglutination measurement method erythrocyte agglutination method
- particle agglutination method JP-A-9 2-293636 and Japanese Patent Application Laid-Open No.
- a specific binding substance to the measurement target substance (test substance) is fixed and covered with the carrier, and the measurement target substance (test target)
- inspection method of the pulmonary fibrosis or interstitial pneumonia of this invention can apply any methods, such as a sandwich method, a competition method, or a homogeneous system method (homogeneous system method).
- the periostin measurement in the method for examining pulmonary fibrosis or interstitial pneumonia of the present invention may be performed by a method or using an apparatus such as an analyzer.
- the periostin measurement in the method for examining pulmonary fibrosis or interstitial pneumonia of the present invention may be one using only one measurement reagent (one reagent method or one step method).
- the anti-periostin specific region antibody is contained in the one measurement reagent.
- the periostin measurement in the method for examining pulmonary fibrosis or interstitial pneumonia of the present invention may be one using two or more measurement reagents (multi-reagent method or multi-step method).
- the anti-periostin specific region antibody may be contained in one measurement reagent of two or more measurement reagents, or may be contained in two or more measurement reagents. Also good.
- the anti-periostin specific region antibody is the first antibody. It may be contained only in the reagent, may be contained only in the second reagent, or may be contained in both the first reagent and the second reagent.
- the solvent used for periostin measurement in the method for examining pulmonary fibrosis or interstitial pneumonia of the present invention the above-mentioned various aqueous solvents can be used.
- periostin comprised of the EMI region, R1 region, R2 region, and R3 region of periostin is used. Measured by detecting at least one region selected from the group, and the amount or concentration of periostin in the sample derived from the subject, and from a living body not affected by pulmonary fibrosis or interstitial pneumonia The amount or concentration of periostin in each sample is compared.
- measurement is performed by detecting at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin.
- Periostin EMI region, R1 region, R2 region and R3 region for samples derived from living organisms not affected by pulmonary fibrosis or interstitial pneumonia
- the details of detecting at least one region selected from the group consisting of the EMI region, R1 region, R2 region and R3 region of periostin in measuring periostin contained in the sample are as described above.
- the amount or concentration of periostin in the sample derived from the subject is compared with the amount or concentration of periostin in a sample derived from a living body not suffering from either pulmonary fibrosis or interstitial pneumonia.
- the amount or concentration of periostin in the sample derived from the subject is higher than the amount or concentration of periostin in the sample derived from a living body not affected by pulmonary fibrosis or interstitial pneumonia, This indicates that the subject is likely to have pulmonary fibrosis or interstitial pneumonia.
- an increase in the amount or concentration of periostin in the sample derived from the subject indicates that there is a high possibility that the subject's pathology of pulmonary fibrosis or interstitial pneumonia is hated.
- the amount or concentration of periostin in a sample derived from a subject is compared with the amount or concentration of periostin in a sample derived from a living body (control sample) not affected by pulmonary fibrosis or interstitial pneumonia. If it is high, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, Or when it is about 100% or more high, it can be judged that there is a high possibility of having pulmonary fibrosis or interstitial pneumonia.
- the difference from the concentration is a critical value for detection or diagnosis of pulmonary fibrosis or interstitial pneumonia, and may be set as follows, for example.
- the amount or concentration of periostin in a sample derived from two or more patients with pulmonary fibrosis or interstitial pneumonia is measured, and the average value (A) is obtained.
- the number of target patients is 2 or more, for example, 5 or more, 10 or more, 50 or more, or 100 or more.
- the amount or concentration of periostin in two or more control samples is measured, and the average value (B) is obtained.
- the number of examples of the target control sample is 2 or more, for example, 5 or more, 10 or more, 50 or more, or 100 or more.
- [(A ⁇ B) / B] ⁇ 100 (%) is calculated from the obtained average values A and B, and the average amount or concentration of periostin in a biological sample derived from a patient with pulmonary fibrosis or interstitial pneumonia Determine what percentage the value is higher compared to the average amount or concentration of periostin in the control sample.
- the value thus determined is set as the difference (critical value) between (i) the amount or concentration of periostin in the subject-derived sample and (ii) the amount or concentration of periostin in the control sample.
- the measurement value of the amount or concentration of periostin in the sample is incorporated into the average values (A) and (B), and the number of patient cases and It is preferable to increase the number of subjects such as healthy subjects (healthy subjects, those who are not affected by pulmonary fibrosis or interstitial pneumonia, etc.). By increasing the number of cases, the accuracy of detection or diagnosis of pulmonary fibrosis or interstitial pneumonia can be increased.
- the average value (B) of the amount or concentration of periostin in the obtained control sample may be “(ii) the amount or concentration of periostin in the control sample”.
- the subject when the measured amount or concentration of periostin in a sample derived from a subject is equal to or higher than a predetermined protein amount, the subject suffers from pulmonary fibrosis or interstitial pneumonia. It may be determined that there is a high possibility of being. Then, the “predetermined protein amount” for determining that there is a high possibility of suffering from pulmonary fibrosis or interstitial pneumonia can be obtained, for example, as follows. First, the amount or concentration of periostin in a sample derived from two or more patients with pulmonary fibrosis or interstitial pneumonia is measured. At this time, the number of target patients is 2 or more, for example, 5 or more, 10 or more, 50 or more, or 100 or more.
- the amount or concentration of periostin in two or more control samples is also measured.
- the number of examples of the target control sample is 2 or more, for example, 5 or more, 10 or more, 50 or more, or 100 or more.
- the optimum threshold (cut-off value) is obtained by statistical processing.
- statistical processing For example, analysis using a Receiver-Operating-Characteristics (ROC) curve can be mentioned.
- periostin Periostin (nucleotide sequence: nucleotide sequence of nucleic acid database GenBank Accession Number D13666, amino acid sequence: nucleic acid database GenBank Accession Number BABA02837 amino acid sequence) V5 / His-tagged recombinant periostin protein in insect cells It was expressed in some S2 cells and purified.
- S2 cell transformants were prepared as follows. A cDNA encoding the above-mentioned part of periostin was inserted into pMT / Bip / V5-HisA plasmid (Invitrogen, Carlsbad, Calif., USA), and this was designated as pMT / Bip / periostin-V5-HisA.
- pAcHygro (Invitrogen, Carlsbad, Calif., USA), which is a plasmid expressing the pMT / Bip / peristin-V5-HisA and hygromycin resistance gene, was co-introduced into S2 cells by a known method and transformed.
- S2 recombinant periostin protein which 6 histidine couple
- the culture supernatant was then added to phosphate buffered saline (PBS) [137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, and 8.04 mM disodium hydrogen phosphate.
- PBS phosphate buffered saline
- Ni-NTA Agarose, Qiagen, Hilden, Germany nickel resin
- periostin was obtained by eluting the S2 recombinant periostin protein with an imidazole-containing buffer.
- the DNA sequence of the prepared plasmid was confirmed, and it was confirmed that the incorporated sequence was as intended.
- partial periostin consisting of R1 region, R2 region and R3 region of periostin Known literature (I. Takayama et al., J. Biochem., 146, 5, 713-723, published in 2009, etc.)
- partial periostin consisting of R1 region, R2 region and R3 region of periostin having 6 histidine residues bound to the C-terminus was prepared and obtained.
- partial periostin (R1, R2, R3 region) The DNA sequence of the prepared plasmid was confirmed, and it was confirmed that the incorporated sequence was as intended.
- periostin partial periostin
- partial periostin partial periostin
- partial periostin partial periostin
- partial periostin partial periostin
- partial periostin partial periostin
- partial periostin R1 * R2 * R3 area
- partial periostin R4 area
- partial periostin EMI area
- partial periostin C-terminal region
- Periostin, partial periostin (R1 ⁇ R2 region), partial periostin (R2 region), partial periostin (R1 ⁇ R2 ⁇ R3 region), and partial periostin (C-terminal region) prepared in Reference Example 1 were mixed with SDS-polyacrylamide. Confirmed by gel electrophoresis.
- SDS-1.5M Tris solution 18.2 g of tris (hydroxymethyl) aminomethane [Tris] and 0.4 g of sodium dodecyl sulfate [SDS] were added to pure water, mixed, and adjusted to pH 8.8 with hydrochloric acid. After that, an SDS-1.5M Tris solution [0.4% SDS-1.5M Tris-HCl buffer] was prepared as 100 mL.
- SDS-0.5M Tris solution 6.1 g of tris (hydroxymethyl) aminomethane [Tris] and 0.4 g of sodium dodecyl sulfate [SDS] were added to pure water, mixed, and adjusted to pH 6.8 with hydrochloric acid. After that, an SDS-0.5M Tris solution [0.4% SDS-0.5M Tris-HCl buffer] was prepared as 100 mL.
- Electrophoresis tank buffer 1.5 g of tris (hydroxymethyl) aminomethane [Tris], 0.5 g of sodium dodecyl sulfate [SDS], and 7.2 g of glycine were added to pure water, mixed, and then 500 mL.
- a tank buffer [0.1% SDS-192 mM glycine-25 mM Tris buffer] was prepared.
- SYPRO Ruby staining solution SYPRO Ruby protein gel stain of Molecular Probes (Eugene, Oregon, USA) was used as the SYPRO Ruby staining solution.
- SYPRO Ruby decoloring solution 10 mL of methanol and 7 mL of acetic acid were mixed with 83 mL of pure water to prepare a SYPRO Ruby decoloring solution.
- a separation gel solution having an acrylamide concentration of 13.5% was prepared using the reagent and pure water prepared in (1), (2), (4) and (5) of 1 above. After this separated gel solution was poured into the assembled glass plate, pure water was overlaid thereon, and the gelation reaction was allowed to proceed for 30 minutes.
- a concentrated gel solution having an acrylamide concentration of 1.3% was prepared using the reagent and pure water prepared in (1), (3), (4), and (5) of 1 above. Pure water was discarded from the glass plate of (1), and a small amount of this concentrated gel solution was injected and washed, and then the concentrated gel solution was injected. And sample comb was inserted here and gelatinization reaction was performed for 30 minutes.
- FIG. 2 shows the gel photographed in (3) above.
- periostin (lane denoted as “(a)”) and “partial periostin (R1 ⁇ R2) prepared in (1) to (4) and (7) of Reference Example 1 described above.
- (Region) ”(lane labeled“ (b) ”),“ partial periostin (R2 region) ”(lane labeled“ (c) ”),“ partial periostin (R1, R2, R3 region) ”(“ ( d) ”) and“ partial periostin (C-terminal region) ”(lane indicated as“ (e) ”) each have a band at the corresponding molecular weight.
- Electrophoresis tank buffer Preparation was carried out as described in (1) of (1) above to prepare an electrophoretic tank buffer (0.1% SDS-192 mM glycine-25 mM Tris buffer).
- sample treatment solution was prepared as described in (1) of (1) to prepare a sample treatment solution [4% SDS-12% 2-mercaptoethanol-20% glycerin-100 mM Tris buffer solution]. .
- A Partial periostin (EMI region)
- B Partial periostin (R4 region)
- C Periostin
- d Partial periostin ( ⁇ 17 ⁇ 18 ⁇ 21)
- E Molecular weight markers [Precision Plus Protein All Blue Standards markers; marker molecular weights 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa, and 250 KDa;
- Electrophoresis Using the reagent prepared in 1 above, each of the 2 samples was subjected to electrophoresis by SDS-polyacrylamide gel electrophoresis according to the following operation, and each of periostin, partial periostin and the like was analyzed for its molecular weight. A gel placed according to was obtained.
- a 9 cm ⁇ 9 cm polyvinyl difluoride membrane (BIO-RAD Laboratories, Hercules, Calif., USA) was layered on the gel, and 48 mM tris (hydroxymethyl) aminomethane [Tris], 39 mM glycine, Using a transfer buffer composed of 0.0375% (W / V) sodium dodecyl sulfate (SDS) and 20% (V / V) methanol, transfer was performed at a current of 200 mA for 2 hours, and the periostin or the partial periostin was added. The gel was transferred to the polyvinyl difluoride film.
- Tris hydroxymethyl aminomethane
- a blocking solution [50 mM Tris containing 0.5% casein, 100 mM sodium chloride, and 0.1% sodium azide (permural transfer of periostin or partial periostin) Blocking was performed by immersing in 20 mL of hydroxymethyl) aminomethane buffer [Tris buffer] (pH 8.0)] at 25 ° C. for 2 hours.
- Titer Max Gold (Funakoshi Co., Tokyo, Japan) as a chemical synthesis adjuvant was mixed with 1 volume of the periostin solution prepared in (1) of Reference Example 1 at a ratio of 1 volume.
- cell fusion was performed as follows.
- the mixed lymph node cells and myeloma cells (Sp2 / O cells) were centrifuged to remove the supernatant, suspended in 1 mL of polyethylene glycol (PEG 1500, Roche, Switzerland) at room temperature for 1 minute, and then at 37 ° C. Stir for 1 minute.
- 1 mL of serum-free medium was added over 1 minute, and then 10 mL of serum-free medium was added over 1 minute.
- the cells were washed several times, suspended in a medium containing hypoxanthine, aminopterin and thymidine, dispensed into a 96-well microtiter plate, and cultured at 37 ° C. in the presence of 5% CO 2 .
- fusion cell line As a method for selecting the grown monoclonal antibody-producing cell line (fusion cell line), 7 to 14 days after cell fusion, the above-described periostin used as an immunogen was solid-phased, and the fusion cell culture supernatant was used as a primary antibody. The ELISA method was used.
- this ELISA method was performed as follows. 1 ⁇ g / mL of the above periostin was dispensed into a 96-well microtiter plate and allowed to immobilize for several hours. After washing this solid phase solution, the fusion cell culture supernatant was added to each well and allowed to stand at room temperature for 1 hour.
- the fused cell culture supernatant was washed, and a peroxidase-labeled goat anti-rat IgG antibody (GE Healthcare, Little Charlotte, UK) was added as a secondary antibody and allowed to stand at room temperature for 1 hour.
- a peroxidase-labeled goat anti-rat IgG antibody GE Healthcare, Little Charlotte, UK
- ABTS peroxidase substrate KPL, Gaithersburg, MD, USA
- IgG was purified from the selected monoclonal antibody-producing cell line as follows. This monoclonal antibody-producing cell line was cultured at 37 ° C. in a CO 2 incubator using GIT medium (Nippon Pharmaceutical Co., Ltd., Tokyo, Japan).
- the IgG in the supernatant was bound to a protein G column (GE Healthcare, Little Charlotte, UK).
- the bound IgG was eluted with 50 mM aqueous citric acid solution (pH 2.6).
- Tris buffer 1M tris (hydroxymethyl) aminomethane buffer [Tris buffer] was added to 4 volumes of the eluate, and rat anti-periostin monoclonal antibody was obtained from the aforementioned monoclonal antibody-producing cell line as purified IgG.
- anti-periostin monoclonal antibody a rat anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS16A)”) was obtained from the SS16A monoclonal antibody-producing cell line.
- Example 2 Preparation of anti-periostin monoclonal antibody-second time
- the procedure described in Example 1 (1) to (4) was followed, and anti-periostin monoclonal antibody was prepared again. (Second time)
- anti-periostin monoclonal antibody (SS17B)”) was obtained from the SS17B monoclonal antibody-producing cell line.
- Example 3 Preparation of anti-periostin monoclonal antibody-third time
- anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS18A)”) was obtained from the SS18A monoclonal antibody-producing cell line.
- Example 4 Preparation of anti-periostin monoclonal antibody-fourth time
- Example 2 Preparation of anti-periostin monoclonal antibody-fourth time
- anti-periostin monoclonal antibody was prepared according to the following procedure. (4th)
- Titer Max Gold (Funakoshi Co., Tokyo, Japan) as a chemical synthesis adjuvant was mixed with 1 volume of the periostin solution prepared in (1) of Reference Example 1 at a ratio of 1 volume.
- spleen cells of immunized mice and myeloma cells are mixed in a ratio of 1: 1 to 10: 1, and polyethylene glycol is mixed by a general method. (PEG 1500, Roche, Switzerland) was added for cell fusion, and the grown hybridoma colonies were selected.
- cell fusion was performed as follows.
- the mixed lymph node cells and myeloma cells (Sp2 / O cells) were centrifuged to remove the supernatant, suspended in 1 mL of polyethylene glycol (PEG 1500, Roche, Switzerland) at room temperature for 1 minute, and then at 37 ° C. Stir for 1 minute.
- 1 mL of serum-free medium was added over 1 minute, and then 10 mL of serum-free medium was added over 1 minute.
- the cells were washed several times, suspended in a medium containing hypoxanthine, aminopterin and thymidine, dispensed into a 96-well microtiter plate, and cultured at 37 ° C. in the presence of 5% CO 2 .
- fusion cell line As a method for selecting the grown monoclonal antibody-producing cell line (fusion cell line), 7 to 14 days after cell fusion, the above-described periostin used as an immunogen was solid-phased, and the fusion cell culture supernatant was used as a primary antibody. The ELISA method was used.
- this ELISA method was performed as follows. 1 ⁇ g / mL of the above periostin was dispensed into a 96-well microtiter plate and allowed to immobilize for several hours.
- the fusion cell culture supernatant was added to each well and allowed to stand at room temperature for 1 hour.
- the fused cell culture supernatant was washed, and a peroxidase-labeled sheep anti-mouse IgG antibody (GE Healthcare, Little Charlotte, UK) was added as a secondary antibody and allowed to stand at room temperature for 1 hour.
- a peroxidase-labeled sheep anti-mouse IgG antibody GE Healthcare, Little Charlotte, UK
- ABTS peroxidase substrate KPL, Gaithersburg, MD, USA
- SS19A, SS19B, SS19C, and SS19D Four clones were established from the grown fused cell lines, and named SS19A, SS19B, SS19C, and SS19D, respectively.
- IgG was purified from the selected monoclonal antibody-producing cell line as follows. This monoclonal antibody-producing cell line was cultured at 37 ° C. in a CO 2 incubator using GIT medium (Nippon Pharmaceutical Co., Ltd., Tokyo, Japan). After culturing, the IgG in the supernatant was bound to a protein G column (GE Healthcare, Little Charlotte, UK). The bound IgG was eluted with 50 mM aqueous citric acid solution (pH 2.6).
- mice anti-periostin monoclonal antibody was obtained as purified IgG from the above monoclonal antibody-producing cell line.
- anti-periostin monoclonal antibody (SS19A)”) was obtained from the SS19A monoclonal antibody-producing cell line, and a mouse anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS19A)”).
- Anti-periostin monoclonal antibody (SS19B) from the SS19C monoclonal antibody-producing cell line, mouse anti-periostin monoclonal antibody (hereinafter referred to as“ anti-periostin monoclonal antibody (SS19C) ”), and SS19D strain monoclonal antibody-producing cell
- anti-periostin monoclonal antibody (SS19D) could be obtained from each strain.
- amino acid sequence of the heavy chain variable region of the anti-periostin monoclonal antibody is represented by SEQ ID NO: 16
- nucleotide sequence of the polynucleotide encoding this amino acid sequence is represented by SEQ ID NO: 15.
- amino acid sequence of the light chain variable region of the antibody is represented by SEQ ID NO: 18, and the base sequence of the polynucleotide encoding this amino acid sequence is represented by SEQ ID NO: 17.
- the antibody of the present invention comprises the amino acid sequence of the heavy chain variable region of the antibody comprising the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence of the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 18.
- Monoclonal antibodies are included.
- Example 5 (Preparation of anti-periostin monoclonal antibody-5th time) At a different time from Example 1, Example 2, Example 3 and Example 4, the procedure described in Example 4 (1) to (4) was followed to prepare an anti-periostin monoclonal antibody. went. (5th)
- anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS20A)”) could be obtained from the SS20A monoclonal antibody-producing cell line.
- Example 6 (Preparation of anti-periostin monoclonal antibody-sixth time)
- the anti-periostin monoclonal antibody was prepared as described in (1) to (4) of Example 1 at a different time from Example 1, Example 2, Example 3, Example 4 and Example 5.
- Antibody preparation was performed. (6th)
- anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS21A)”) was obtained from the SS21A monoclonal antibody-producing cell line.
- Example 7 (Confirmation of recognition site of anti-periostin monoclonal antibody) For each of the anti-periostin monoclonal antibodies obtained in Example 1, Example 2, Example 3, Example 4, Example 5 and Example 6, it was confirmed which region of periostin was recognized.
- periostin or partial periostin prepared in (1) to (7) of Reference Example 1 was adjusted to a concentration of 5 ⁇ g / mL with phosphate buffered saline. 50 ⁇ L of each of the prepared solutions or phosphate buffered saline as a control was injected into each well of a 96-well microtiter plate (Thermo Fisher Scientific Inc., Illinois, USA) and allowed to stand at 5 ° C. for 18 hours. The periostin or partial periostin was immobilized on the well of the microtiter plate.
- A "Periostin”
- B "Partial periostin (R1 / R2 region)”
- C “Partial periostin (R2 region)”
- D “Partial periostin (R1, R2, R3 region)”
- E “Partial periostin (R4 region)”
- F “Partial periostin (EMI region)”
- G “Partial periostin (C-terminal region)”
- the POD-labeled anti-IgG antibody is injected into a well of a microtiter plate.
- This POD-labeled anti-IgG antibody is referred to as “anti-periostin monoclonal antibody (SS16A)”, “anti-periostin monoclonal antibody (SS17B)”, “ In the case of “anti-periostin monoclonal antibody (SS18A)” or “anti-periostin monoclonal antibody (SS21A)”, the POD-labeled anti-rat IgG antibody was injected into the well of the microtiter plate.
- this POD-labeled anti-IgG antibody is an “anti-periostin monoclonal antibody (SS19A)”, “anti-periostin monoclonal antibody (SS19B)”, “anti-periostin monoclonal antibody (SS19C)”, “anti-periostin monoclonal antibody (SS19D)”, or
- anti-periostin monoclonal antibody (SS20A) the POD-labeled anti-mouse IgG antibody was injected into the well of the microtiter plate.
- the absorbance difference was 0.2 or more, it was determined that the anti-periostin monoclonal antibody recognized either the periostin or partial periostin region immobilized on the well of the microtiter plate. .
- the antiperiostin monoclonal antibody was measured by reacting with the periostin or partial periostin.
- the absorbance difference was 0.2 or more, that is, the anti-periostin monoclonal antibody was “ ⁇ ” was shown for combinations when it was determined that they were bound to periostin or partial periostin.
- the “anti-periostin monoclonal antibody (SS16A)” binds only to “periostin” and “partial periostin (R1, R2, R3 region)” of the above-mentioned periostin and partial periostin, Any of “partial periostin (R1 / R2 region)”, “partial periostin (R2 region)”, “partial periostin (R4 region)”, “partial periostin (EMI region)” and “partial periostin (C-terminal region)” You can see that they are not connected. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS16A)” recognizes the R3 region of periostin as an antigen recognition part (epitope).
- anti-periostin monoclonal antibody (SS17B) binds only to “periostin” and “partial periostin (R4 region)” among the above-mentioned periostin and partial periostin, (R1 • R2 region) ”,“ partial periostin (R2 region) ”,“ partial periostin (R1 • R2 • R3 region) ”,“ partial periostin (EMI region) ”and“ partial periostin (C-terminal region) ” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS17B)” recognizes the R4 region of periostin as an antigen recognition part (epitope).
- the “anti-periostin monoclonal antibody (SS18A)” is the “periostin”, “partial periostin (R1 and R2 region)” and “partial periostin (R1) among the above-mentioned periostin and partial periostin. R2 and R3 regions) ”only, and any of“ partial periostin (R2 region) ”,“ partial periostin (R4 region) ”,“ partial periostin (EMI region) ”and“ partial periostin (C-terminal region) ” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS18A)” recognizes the R1 region of periostin as an antigen recognition part (epitope).
- anti-periostin monoclonal antibody (SS19A) binds only to “periostin” and “partial periostin (R4 region)” among the above-mentioned periostin and partial periostin, (R1 • R2 region) ”,“ partial periostin (R2 region) ”,“ partial periostin (R1 • R2 • R3 region) ”,“ partial periostin (EMI region) ”and“ partial periostin (C-terminal region) ” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS19A)” recognizes the R4 region of periostin as an antigen recognition portion (epitope).
- anti-periostin monoclonal antibody (SS19B) binds only to “periostin” and “partial periostin (C-terminal region)” among the above-mentioned periostin and partial periostin, “Periostin (R1 • R2 region)”, “Partial periostin (R2 region)”, “Partial periostin (R1 • R2 • R3 region)”, “Partial periostin (R4 region)” and “Partial periostin (EMI region)” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS19B)” recognizes the C-terminal region of periostin as an antigen recognition portion (epitope).
- the “anti-periostin monoclonal antibody (SS19C)” is the “periostin”, “partial periostin (R1 and R2 region)”, “partial periostin (R2) among the above-mentioned periostin and partial periostin. "Region)” and “partial periostin (R1, R2, R3 region)” only, any of “partial periostin (R4 region)", “partial periostin (EMI region)” and “partial periostin (C-terminal region)” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS19C)” recognizes the R2 region of periostin as an antigen recognition part (epitope).
- the “anti-periostin monoclonal antibody (SS19D)” binds only to “periostin” and “partial periostin (R1, R2, R3 region)” of the above-described periostin and partial periostin.
- “Partial periostin (R1 / R2 region)”, “partial periostin (R2 region)”, “partial periostin (R4 region)”, “partial periostin (EMI region)” and “partial periostin (C-terminal region)” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS19D)” recognizes the R3 region of periostin as an antigen recognition part (epitope).
- anti-periostin monoclonal antibody (SS20A) binds only to “periostin” and “partial periostin (EMI region)” among the above-mentioned periostin and partial periostin.
- R1 • R2 region “ partial periostin (R2 region) ”,“ partial periostin (R1 • R2 • R3 region) ”,“ partial periostin (R4 region) ”and“ partial periostin (C-terminal region) ” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS20A)” recognizes the EMI region of periostin as an antigen recognition part (epitope).
- the “anti-periostin monoclonal antibody (SS21A)” binds only to “periostin” and “partial periostin (C-terminal region)” among the above-mentioned periostin and partial periostin. “Periostin (R1 • R2 region)”, “Partial periostin (R2 region)”, “Partial periostin (R1 • R2 • R3 region)”, “Partial periostin (R4 region)” and “Partial periostin (EMI region)” It can be seen that they are not combined. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS21A)” recognizes the C-terminal region of periostin as an antigen recognition part (epitope).
- Example 8 (Confirmation of reactivity of anti-periostin monoclonal antibody) For each of the anti-periostin monoclonal antibodies obtained in Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6, reaction with periostin monomer, multimer, and degradation product, respectively. The sex was confirmed.
- Anti-periostin monoclonal antibodies other than those derived from the SS19B strain “Anti-periostin monoclonal antibody (SS16A)”, “Anti-periostin monoclonal antibody (SS17B)”, “Anti-periostin monoclonal antibody (SS18A)”, “Anti-periostin monoclonal antibody (SS19A)” , “Anti-periostin monoclonal antibody (SS19C)”, “anti-periostin monoclonal antibody (SS19D)”, “anti-periostin monoclonal antibody (SS20A)” and “anti-periostin monoclonal antibody (SS21A)”
- the reactivity with each of the multimer and the decomposed product was confirmed as follows.
- Immunoprecipitation treatment (1) 5 ⁇ g of the anti-periostin monoclonal antibodies (a) to (h) below obtained in Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6 were used. Each one was individually placed in a 1.5 mL capacity tube.
- this tube was rotated at 5 ° C. for 1 hour, and each of the anti-periostin monoclonal antibodies was bound to the Protein G Sepharose.
- this tube was rotated at 5 ° C. for 2 hours to block the surface of Protein G Sepharose to which the anti-periostin monoclonal antibody was not bound.
- Protein G Sepharose-conjugated anti-periostin monoclonal antibody was included in the “mixture of periostin monomer, multimer and degradation product”. Each of periostin monomer, multimer and degradation product was contacted.
- the “anti-periostin monoclonal antibody” bound to Protein G Sepharose binds to any one or more of periostin monomer, multimer or degradation product recognized by this antibody (the antibody).
- the tube (4) was centrifuged. After removing the supernatant, by washing Protein G Sepharose in the tube with phosphate buffered saline, free periostin that did not bind to the “anti-periostin monoclonal antibody” bound to Protein G Sepharose. Monomers, multimers and degradation products (ie, periostin monomers, multimers or degradation products that were not recognized and bound by this “anti-periostin monoclonal antibody”) were all removed.
- molecular weight markers [Precision Plus Protein All Blue Standards markers; marker molecular weights 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa, and 250 KDa; BIO-RAc Labor State, California; It was.
- a protein G Sepharose “an anti-periostin monoclonal antibody” — “a periostin monomer, multimer or degradation product recognized and bound by the anti-periostin monoclonal antibody” in each of the above samples ” are mixed with a sample treatment solution to produce "Protein G Sepharose”, “anti-periostin monoclonal antibody”, and “periostin monomer, multimer or degradation product that is recognized and bound by the anti-periostin monoclonal antibody” Are dissociated respectively.
- the supernatant and the molecular weight marker injected into this gel are the following molecular weight markers (i) to (ix), and each ““ anti-periostin monoclonal antibody ”and “Supernatant containing periostin monomer, multimer or degradation product recognized and bound by the anti-periostin monoclonal antibody” was sequentially injected.
- the rightmost lane of this gel was used as a negative control lane in which no sample was injected.
- the periostin monomer, multimer or degradation product recognized and bound by this antibody (the antibody) is positioned in the gel according to its molecular weight. A gel was obtained.
- a 9 cm ⁇ 9 cm polyvinyl difluoride membrane (BIO-RAD Laboratories, Hercules, Calif., USA) was layered on the gel, and 48 mM tris (hydroxymethyl) aminomethane [Tris], 39 mM glycine, Using a transfer buffer consisting of 0.0375% (W / V) sodium dodecyl sulfate (SDS) and 20% (V / V) methanol, transfer was carried out at a current of 200 mA for 2 hours, and positioned in the gel according to its molecular weight.
- the proteins such as “periostin monomer, multimer or degradation product recognized and bound by the anti-periostin monoclonal antibody” were transferred from the gel to the polyvinyl difluoride membrane.
- this polyvinyl difluoride membrane was washed by shaking in 20 mL of a cleaning solution (phosphate buffered saline containing 0.05% Tween 20) for 10 minutes. This operation was performed three times.
- a cleaning solution phosphate buffered saline containing 0.05% Tween 20
- anti-periostin monoclonal antibody (SS19C) obtained in Example 4 was subjected to biotin labeling using Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific Inc., Illinois, USA; product code number: 21335).
- Sulfo-NHS-LC-Biotin Thermo Fisher Scientific Inc., Illinois, USA; product code number: 21335.
- anti-periostin monoclonal antibody (SS19C) labeled with biotin.
- This “anti-periostin monoclonal antibody (SS19C)” is capable of recognizing and binding periostin monomers, multimers and degradation products, as will be described later.
- the polyvinyl difluoride membrane subjected to the washing treatment of (3) was immersed in this liquid mixture of biotin-labeled antibody at room temperature overnight for reaction.
- each of the above-mentioned “monomers, multimers or degradation products of periostin that is recognized and bound by the anti-periostin monoclonal antibody” transferred to the polyvinyl difluoride film, and the biotin-labeled “anti-periostin monoclonal antibody” (SS17B) ”and biotin-labeled“ anti-periostin monoclonal antibody (SS19C) ”were reacted.
- peroxidase-labeled streptavidin (Stereospecific Detection Technologies, Germany) was added to a labeled antibody diluent (50 mM tris (hydroxymethyl) amino containing 0.5% casein and 100 mM sodium chloride).
- the polyvinyl difluoride film of (5) above was immersed in a POD-labeled streptavidin solution diluted 15000 times with methane buffer [Tris buffer] (pH 8.0)] for 90 minutes at room temperature to react. .
- the molecular weight marker lane (denoted as “MM”), the periostin lane recognized by and bound to the “anti-periostin monoclonal antibody (SS16A)” (denoted as “16A”), the “anti-periostin” Periostin lane (designated “17B”) recognized and bound by “monoclonal antibody (SS17B)”, periostin lane (designated “18A”) recognized and bound by “anti-periostin monoclonal antibody (SS18A)”, “anti-periostin Periostin lane (represented as “19A”) recognized and bound by “monoclonal antibody (SS19A)”, periostin lane (represented as “19C”) recognized and bound by “anti-periostin monoclonal antibody (SS19C)”, “anti-periostin Monoclonal antibody (SS19 ) ”And recognized periostin lane (denoted“ 19D ”),“ periostin monoclonal antibody monoclonal antibody (SS19
- the thick band located between the 150 KDa and 250 KDa molecular weight markers represents the periostin multimer (presumed to be a periostin trimer from its molecular weight) and is near the 75 KDa molecular weight marker.
- the thick band seen from the high molecular weight side represents the monomer of periostin by its molecular weight
- the thick band near the 37 KDa molecular weight marker represents the degradation product of periostin by its molecular weight.
- Anti-periostin monoclonal antibody derived from SS19B strain With respect to “anti-periostin monoclonal antibody (SS19B)”, the reactivity with each of periostin monomer, multimer and degradation product was confirmed as follows.
- molecular weight markers [Precision Plus Protein All Blue Standards markers; marker molecular weights 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa, and 250 KDa; BIO-RAc Labor State, California; Used as.
- the monomer, multimer or degradation product of periostin was positioned in the gel according to its molecular weight, and this gel was obtained.
- the periostin monomer, multimer and degradation product located in the gel according to the molecular weight are transferred to the polyvinyl difluoride film from the gel obtained in (1) above, and this
- the transferred anti-periostin monomer, multimer and degradation product were brought into contact with the above-mentioned “anti-periostin monoclonal antibody (SS19B)” labeled with biotin, reacted, and the presence or absence of binding between them was confirmed.
- the molecular weight marker lane (indicated as “MM”) and then the periostin lane (indicated as “19B”) recognized and bound by the “anti-periostin monoclonal antibody (SS19B)” in order from the left lane.
- MM molecular weight marker
- periostin lane (indicated as “19B”) recognized and bound by the “anti-periostin monoclonal antibody (SS19B)” in order from the left lane.
- SS19B anti-periostin monoclonal antibody
- the thicker band on the higher molecular side of the molecular weight marker of 150 KDa represents a multimer of periostin than its molecular weight
- the thicker band on the higher molecular side of the molecular weight marker of 75 KDa represents a monomer of periostin from its molecular weight.
- Example 3 Summary For each anti-periostin monoclonal antibody obtained in Example 1, Example 2, Example 3, Example 4, Example 5 and Example 6, periostin monomers, multimers and degradation products A summary of the reactivity with each is shown in FIG.
- periostin monomers, multimers or degradation products that are recognized and bound are indicated by “ ⁇ ”.
- anti-periostin monoclonal antibody can recognize and bind to a degradation product of periostin.
- Anti-periostin monoclonal antibody (SS16A) ⁇ “Anti-periostin monoclonal antibody (SS18A)” ⁇ “Anti-periostin monoclonal antibody (SS19C)” ⁇ “Anti-periostin monoclonal antibody (SS19D)” ⁇ “Anti-periostin monoclonal antibody (SS20A)”
- the SS19D strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS19D)”, is the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology (Kazusa-Kamazu 2chome, Kisarazu-shi, Chiba, Japan 5). No. 8) is deposited as “Accession Number: NITE P-1068” on February 22, 2011.
- SS19D strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS19D)”
- SS19D anti-periostin monoclonal antibody
- the SS16A strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS16A)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Kazusa-Kamazu 2chome, Kisarazu City, Chiba Prefecture, Japan 5). No. 8) is deposited on March 16, 2012 as “Reception Number: NITE AP-1281”.
- SS16A anti-periostin monoclonal antibody
- the monoclonal antibody-producing cell line SS16A the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology (Kazusa-Kamazu 2-chome, Kisarazu-shi, Chiba, Japan) No. 8) was applied for transfer from domestic deposit to international deposit on July 19, 2012, and transferred to international deposit. [Transfer date: July 19, 2012] (Accession number: NITE BP-1281)
- the SS18A strain which is a monoclonal antibody-producing cell line of this “anti-periostin monoclonal antibody (SS18A)”, is the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology (Kazusa-Kamazu 2-chome, Kisarazu City, Chiba Prefecture, Japan 5). No. 8) is deposited as “Receipt Number: NITE AP-1282” on March 16, 2012.
- the SS19C strain which is a monoclonal antibody-producing cell line of this “anti-periostin monoclonal antibody (SS19C)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Kazusa-Kamazu 2chome, Kisarazu City, Chiba Prefecture, Japan 5). No. 8) is deposited on March 16, 2012 as “Reception Number: NITE AP-1283”.
- the SS20A strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS20A)”, is the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology (Kazusa-Kamazu 2-chome, Kisarazu City, Chiba Prefecture, Japan 5 No. 8) is deposited on March 16, 2012 as “Reception number: NITE AP-1284”.
- the SS20A strain which is a monoclonal antibody-producing cell line, is the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology (Kazusa-Kamazu 2-chome, Kisarazu City, Chiba Prefecture, Japan) No. 8) was applied for transfer from domestic deposit to international deposit on July 19, 2012, and transferred to international deposit. [Transfer date: July 19, 2012] (Accession number: NITE BP-1284)
- Example 9 (Confirmation of periostin in human serum) Periostin in human serum was confirmed by performing immunoprecipitation, electrophoresis and Western blotting.
- Immunoprecipitation treatment (1) In a 1.5 mL tube, 100 ⁇ L of NHS-activated Sepharose (GE Healthcare, Little Chalfont, UK) was placed in a bed volume, and the supernatant was removed by centrifugation.
- NHS-activated Sepharose GE Healthcare, Little Chalfont, UK
- this tube was kept warm at 25 ° C. for 1 hour with stirring, and the above-mentioned “anti-periostin monoclonal antibody (SS19C)” was bound to the above-mentioned NHS-activated Sepharose.
- SS19C anti-periostin monoclonal antibody
- the centrifugation operation was performed on the tube (7) (each having a serum amount of 25 ⁇ L, 50 ⁇ L, or 100 ⁇ L of a healthy person).
- the NHS-activated Sepharose in the tube was washed three times with a washing solution [10 mM Tris (hydroxymethyl) aminomethane [Tris] -150 mM sodium chloride-0.05% Tween 20 (pH 8.0)] All free periostin (monomer, multimer or degradation product) that did not bind to “an anti-periostin monoclonal antibody (SS19C) bound to NHS-activated Sepharose” was removed.
- SS19C anti-periostin monoclonal antibody
- the anti-periostin monoclonal antibody (SS19C) bound to NHS-activated Sepharose and “monomers, multimers or degradation products of periostin contained in the sample (serum of healthy subjects)” Of “NHS-activated Sepharose”-"Anti-periostin monoclonal antibody (SS19C)"-"Periostin monomer, multimer or degradation product contained in sample (serum of healthy subject)” A conjugate ” was prepared.
- (A) Acrylamide solution The acrylamide solution was prepared as described in 1 of (1) of Reference Example 2 to prepare an acrylamide solution [30% acrylamide storage solution].
- Electrophoresis buffer solution Prepared as described in (1) of (6) of Reference Example 2, and the electrophoretic buffer solution [0.1% SDS-192 mM glycine-25 mM Tris buffer] Prepared.
- molecular weight markers [Precision Plus Protein All Blue Standards markers; marker molecular weights 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa, and 250 KDa; Samples were prepared as follows.
- a separation gel solution having an acrylamide concentration of 7.5% was prepared using the reagent and pure water prepared in (a), (b), (d) and (e) of (1). After this separated gel solution was poured into the assembled glass plate, pure water was overlaid thereon, and the gelation reaction was allowed to proceed for 30 minutes.
- the periostin (monomer, multimer or degradation product) contained in the sample is positioned according to its molecular weight. This gel was obtained.
- the sample processing solution (h) in (1) is used in the absence of a reducing agent.
- the sample of the molecular weight marker was not injected into the comb hole of the gel, but only the supernatant was injected into the comb hole of the gel.
- the periostin (monomer, multimer or degradation product) contained in the sample (serum of healthy person) in the case where the reducing agent is not present in (c) was positioned according to its molecular weight and this gel was obtained.
- Blocking was performed by immersing in 50 mL of Tris-buffered physiological saline [10 mM Tris (hydroxymethyl) aminomethane [Tris] -150 mM sodium chloride (pH 8.0)] containing skim milk and 0.05% Tween 20 for 1 hour at room temperature. It was.
- Each polyvinyl difluoride membrane subjected to the washing treatment (3) was immersed in this antibody solution at 4 ° C. overnight to be reacted.
- periostin monomer, multimer or degradation product contained in the sample (serum of healthy subject) transferred to the polyvinyl difluoride film and the “anti-periostin in the antibody solution” Monoclonal antibody (SS19C) ” was reacted.
- Prestin monomers, multimers, or degradation products contained in the sample (serum of healthy subjects) based on the presence or absence of light emission on these polyvinyl difluoride films and their emission position (molecular weight) It was confirmed.
- the pattern on the left shows a polyvinyl difluoride film in the presence of a reducing agent using a sample processing solution (containing a reducing agent) during the electrophoresis treatment
- the pattern on the right shows the electrophoresis treatment described above.
- membrane when a reducing agent does not exist is sometimes shown using a sample processing liquid (it does not contain a reducing agent).
- the lane when the amount of serum of a healthy person added to the tube in (1) above (7) is 100 ⁇ L (indicated as “100 ⁇ L”), the above (7 )
- the lane when the amount of serum of a healthy person added to the tube in (1) above is 25 ⁇ L
- a thick band seen from the vicinity of the 150 KDa molecular weight marker to the high molecular weight side represents a periostin multimer (presumed to be a periostin dimer from the molecular weight), and a molecular weight of 75 KDa.
- the thick band near the marker represents the monomer of periostin by its molecular weight
- the thick band near the 37 KDa molecular weight marker represents the degradation product of periostin by its molecular weight.
- the lane indicated as “100 ⁇ L”) when the amount of the serum of the healthy person added to the tube in the above (7) is 100 ⁇ L, the above (7) )
- the lane when the amount of serum of a healthy person added to the tube in (1) above is 25 ⁇ L ( Lane expressed as “25 ⁇ L”) and the standard sample (mixture of periostin monomer, multimer, and degradation product) in (2) in (2) above (referred to as “standard sample”) ).
- the thick band seen from the vicinity of the 250 KDa molecular weight marker to the high molecular weight side represents a periostin multimer (presumed to be a periostin trimer from its molecular weight), and has a 75 KDa
- the thick band near the molecular weight marker represents the monomer of periostin by its molecular weight
- the thick band near the molecular weight marker of 37 KDa represents the degradation product of periostin by its molecular weight.
- the periostin (monomer, multimer, or degradation product) contained in the serum of these pulmonary fibrosis sufferers or healthy subjects was treated as follows: ““ The anti-periostin bound to NHS-activated Sepharose ” Monoclonal antibody (SS19C) "and” conjugate of periostin monomer, multimer or degradation product contained in sample (serum of pulmonary fibrosis sufferer or healthy person) ", ie ““ NHS-activated Sepharose ”—“ Anti-periostin monoclonal antibody (SS19C) ”—“ periostin monomer, multimer or degradation product contained in the sample (the serum of a pulmonary fibrosis-affected or healthy person) ” Each of the conjugates was prepared.
- molecular weight markers [Precision Plus Protein All Blue Standards markers; marker molecular weights 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa, and 250 KDa; Samples were prepared as follows.
- the electrophoresis was performed as described in (1) -2 (3) (a) to (g) except for the following items (a) and (b).
- Periostin monomer, multimer or degradation product contained in the sample (serum of pulmonary fibrosis sufferer or healthy person) in the presence of a reducing agent using (containing a reducing agent) has its molecular weight
- the gel was obtained by positioning according to
- a lane when a molecular weight marker is used as a sample (denoted as “MM”), a lane when a healthy person's serum is used as a sample (“healthy person”) Lane), lane when the serum of the first patient with pulmonary fibrosis is used as a sample (indicated as “1” with pulmonary fibrosis), lane when the serum of the second patient with pulmonary fibrosis is used as a sample (Indicated as “2” with pulmonary fibrosis), a lane (labeled as “3 with pulmonary fibrosis”) when using the serum of a third person with pulmonary fibrosis, and a standard sample (one periostin)
- the thick band near the 150 KDa molecular weight marker represents a periostin multimer (presumed to be a periostin dimer from its molecular weight), and the thick band near the 75 KDa molecular weight marker is The molecular weight represents a monomer of periostin, and the thick band near the 37 KDa molecular weight marker represents the degradation product of periostin by its molecular weight.
- each band in the sample of the pulmonary fibrosis patient is the value of the sample of the pulmonary fibrosis patient when the emission intensity of the corresponding band of the healthy sample is 1.0. It represents the relative value (relative ratio) of the emission intensity of each band.
- the relative value (relative ratio) in the serum samples of three patients with pulmonary fibrosis is 2.1 to 4.0 in the band of the degradation product, and the serum samples of pulmonary fibrosis patients
- the amount of the degradation product of periostin is more than twice that of the serum sample of a healthy subject, and it can be seen that the amount of the degradation product of periostin is significantly higher in those suffering from pulmonary fibrosis than in the healthy subject.
- Example 10 (Measurement of periostin in human serum-1) Periostin in human serum was measured, and the effects of the antibody of the present invention, measurement method, measurement reagent, accuracy improvement method and pulmonary fibrosis or interstitial pneumonia testing method were confirmed.
- periostin concentration was measured and determined as follows by an enzyme immunoassay (ELISA method) using an anti-periostin monoclonal antibody.
- Anti-periostin monoclonal antibody (SS18A) obtained in Example 3 (an antibody that recognizes and binds to the R1 region of periostin; recognizes and binds to any of periostin monomers, multimers, and degradation products)
- Antibody) phosphate buffered saline (PBS) [137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, and 8.04 mM disodium hydrogen phosphate. (PH 7.4)] was diluted to 2 ⁇ g / mL, and 100 ⁇ L was injected into each well of a 96-well microtiter plate (Thermo Fisher Scientific Inc., Illinois, USA), then 18 ° C. at 25 ° C. Let stand for a while, "Anti-periostin monoclonal antibody (SS18A)” was immobilized on each well of the microtiter plate.
- washing solution-2 phosphate buffered saline (PBS) containing 0.05% Tween 20.
- Anti-periostin monoclonal antibody (SS17B)” obtained in Example 2 (an antibody that recognizes and binds to the R4 region of periostin; although it can recognize and bind to the periostin monomer and multimer, periostin
- the POD-labeled anti-periostin monoclonal antibody labeled with peroxidase (POD) bound to an antibody that cannot recognize and bind to the degradation product of (4) is diluted to 50 ng / mL with the sample diluent of (4) above. 100 ⁇ L of the solution was injected into each well of the microtiter plate of (5) and allowed to stand at 25 ° C. for 90 minutes for reaction.
- anti-periostin monoclonal antibody (SS17B) labeled with POD was bound to periostin bound to the above-mentioned solid-phased “anti-periostin monoclonal antibody (SS18A)”.
- POD substrate solution [containing 0.8 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), 2.5 mM hydrogen peroxide, and 30 mM disodium hydrogen phosphate
- TMBZ 5,5′-tetramethylbenzidine
- disodium hydrogen phosphate 30 mM disodium hydrogen phosphate
- periostin in human serum was measured as follows. 1. Samples [1] (1) to (3) of human serum were used as samples.
- periostin in human serum was measured as follows. 1. Samples [1] (1) to (3) of human serum were used as samples.
- periostin concentration was measured and determined as follows by an enzyme immunoassay (ELISA method) using an anti-periostin monoclonal antibody.
- Anti-periostin monoclonal antibody (SS20A) obtained in Example 5 (an antibody that recognizes and binds to the EMI region of periostin; recognizes and binds to any monomer, multimer, and degradation product of periostin) Antibody) phosphate buffered saline (PBS) [137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, and 8.04 mM disodium hydrogen phosphate. (PH 7.4)] was diluted to 2 ⁇ g / mL, and 100 ⁇ L was injected into each well of a 96-well microtiter plate (Thermo Fisher Scientific Inc., Illinois, USA), then 18 ° C. at 25 ° C. Allowed to stand for "anti-periostin monoclonal antibody (SS20A) Was immobilized on each well of the microtiter plate.
- PBS phosphate buffered saline
- washing solution-2 phosphate buffered saline (PBS) containing 0.05% Tween 20.
- Anti-periostin monoclonal antibody (SS19D)” obtained in Example 4 an antibody that recognizes and binds to the R3 region of periostin; although it can recognize and bind to the periostin monomer and degradation product, periostin
- the biotin-labeled anti-periostin monoclonal antibody labeled with biotin bound to the antibody capable of recognizing and binding the multimers of the above is diluted to 50 ng / mL with the sample diluent of (4) above, and 100 ⁇ L thereof is diluted.
- the solution was poured into each well of the microtiter plate of (5) and allowed to stand at 25 ° C. for 90 minutes to carry out the reaction.
- biotin-labeled “anti-periostin monoclonal antibody (SS19D)” was bound to periostin bound to the solid-phased “anti-periostin monoclonal antibody (SS20A)”.
- peroxidase (POD) -labeled streptavidin (Stereospecific Detection Technologies, Germany) is diluted with a diluent (50 mM Tris (hydroxyl) containing 0.5% casein and 100 mM sodium chloride.
- Methyl) aminomethane buffer [Tris buffer] (pH 8.0)] was diluted 15,000 times, and 100 ⁇ L thereof was injected into each well of the microtiter plate of (7), and the mixture was incubated at 25 ° C. for 60 minutes. The reaction was allowed to stand.
- POD substrate solution [containing 0.8 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), 2.5 mM hydrogen peroxide, and 30 mM disodium hydrogen phosphate 100 ⁇ L of 20 mM citrate buffer solution (pH 3.9)] was injected into each well of the microtiter plate of (8) above, and allowed to stand at 25 ° C. for 10 minutes to cause the reaction to develop color.
- TMBZ 5,5′-tetramethylbenzidine
- disodium hydrogen phosphate 100 ⁇ L of 20 mM citrate buffer solution (pH 3.9)
- the horizontal axis represents the distinction of the measured sample, and the vertical axis represents the measured value (ng / mL) of the periostin concentration in the sample.
- Each “ ⁇ ” plotted in this figure represents a measured value of the concentration of periostin contained in each sample.
- the cut-off values were 82 ng / mL for “measurement by the prior art”, 136 ng / mL for “measurement by the present invention-A”, and “measurement by the present invention”.
- -B was set to 14 ng / mL.
- the line drawn parallel to the horizontal axis represents the cutoff value.
- anti-periostin monoclonal antibody (SS18A) that recognizes and binds to the R1 region of periostin is used as a solid-phase antibody
- anti-periostin monoclonal antibody that recognizes and binds to the R4 region of periostin The anti-periostin monoclonal antibody (SS18A) that recognizes and binds the R1 region of periostin to the measurement result of “measurement by the prior art” in [1] using “SS17B)” as a labeled antibody.
- EMI region of periostin R1 region
- R2 region and R3 region of periostin or a degradation product thereof EMI region of periostin, R1 region
- the sensitivity of the measurement can be improved and the accuracy of the measurement can be improved It was confirmed that.
- the sensitivity of the measurement can be improved and the accuracy of the measurement can be improved. It was confirmed that.
- the method for examining pulmonary fibrosis or interstitial pneumonia improves the sensitivity or specificity of the measurement, improves the accuracy of the measurement, and provides an accurate periostin measurement value. It can be obtained and can improve the discrimination between a person suffering from pulmonary fibrosis or interstitial pneumonia and a person suffering from a healthy person or another disease, and prevent misdiagnosis. It was confirmed.
- Anti-periostin monoclonal antibody (SS17B) that recognizes and binds to the R1 region of periostin is used as a solid phase antibody
- Anti-periostin monoclonal antibody (SS17B) recognizes and binds to the R4 region of periostin.
- the anti-periostin monoclonal antibody (SS20A) that recognizes and binds to the EMI region of periostin is used as a solid-phase antibody.
- an antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof and does not bind to a periostin multimer is used.
- the sensitivity of the measurement can be greatly improved, and It was confirmed that the specificity of the measurement can be improved and the accuracy of the measurement can be improved.
- periostin according to the present invention which can bind to a degradation product of periostin and does not bind to a multimer of periostin, the sensitivity of the measurement can be greatly improved, It was also confirmed that the specificity of the measurement can be improved and the accuracy of the measurement can be improved.
- the method for examining pulmonary fibrosis or interstitial pneumonia which uses an antibody that can bind to a degradation product of periostin and does not bind to a multimer of periostin, Significantly improve specificity, improve measurement accuracy, and obtain accurate periostin measurement values, including those suffering from pulmonary fibrosis or interstitial pneumonia, healthy subjects, and others It was confirmed that it was possible to further improve the discrimination from those suffering from this disease and prevent misdiagnosis.
- Example 11 (ROC analysis of measurement results of periostin in human serum) Periostin in human serum was measured, and a ROC curve (receiver operating characteristic curve) as a result of the measurement was prepared and analyzed.
- Negative samples Serum of healthy subjects (66 cases in total) was used as a negative sample.
- periostin concentration was measured and determined as follows by an enzyme immunoassay (ELISA method) using an anti-periostin monoclonal antibody.
- biotin-labeled “anti-periostin monoclonal antibody (SS17B)” was bound to periostin bound to the solid-phased “anti-periostin monoclonal antibody (SS18A)”.
- POD substrate solution [containing 0.8 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), 2.5 mM hydrogen peroxide, and 30 mM disodium hydrogen phosphate 100 ⁇ L of 20 mM citrate buffer (pH 3.9)] was injected into each well of the microtiter plate of (h) and allowed to stand at 25 ° C. for 10 minutes to cause the reaction to develop color.
- TMBZ 5,5′-tetramethylbenzidine
- disodium hydrogen phosphate 100 ⁇ L of 20 mM citrate buffer (pH 3.9)
- Anti-periostin monoclonal antibody (SS20A) (an antibody that recognizes and binds to the EMI region of periostin; an antibody that recognizes and binds to any of periostin monomers, multimers, and degradation products), and (1
- the anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) is replaced with “anti-periostin monoclonal antibody (SS17B)” and “anti-periostin monoclonal antibody (SS19D)” (periostin) obtained in Example 4 above.
- the ROC curve was created by subtracting the measurement result in “Measurement by conventional technology-b” in (4) from the measurement result in “Measurement by the present invention-b” in (3).
- the measurement result of (3) above which can measure the monomer and degradation product of periostin
- the measurement result of (4) above which can measure only the monomer of periostin
- the measurement result of (4) above which can measure only the monomer of periostin
- Results (1) As described above, the measurement of the concentration of periostin contained in each of a positive sample composed of sera of patients suffering from pulmonary fibrosis and interstitial pneumonia (derived from collagen disease) and a negative sample composed of sera of healthy individuals FIG. 11 shows all the ROC curves created from the results.
- each ROC curve in this figure represents the value of “1-specificity” in this measurement, and the vertical axis represents “sensitivity” in this measurement.
- the value of this AUC represents the area under the ROC curve (Area Under the Curve), and the larger this AUC value, the more the measurement is performed.
- the sensitivity and specificity are high, that is, the measurement accuracy is high, which is preferable in the measurement.
- the ROC curve in “Measurement by conventional technology-b” in 2 (4) above that is, “Anti-periostin monoclonal antibody (SS19D)” that recognizes and binds the R3 region of periostin as a solid-phased antibody is used.
- the anti-periostin monoclonal antibody (SS17B) which recognizes and binds to the R4 region of periostin as a labeled antibody, detects the R3 and R4 regions of periostin, and only periostin monomer
- the calculated AUC value was 0.681.
- a region other than “periostin EMI region, R1 region, R2 region or R3 region” is detected, that is, R4 region and / or C-terminal region of periostin is detected, and a periostin degradation product is detected.
- AUC 0.850
- ROC curve of “measurement according to the present invention—a” in the above (2) is used (that is, the degradation product of periostin can be measured).
- UC 0.856
- ROC curve (AUC 0.972) of “Measurement according to the present invention—b” in 2 (3) above, and “Measurement according to the present invention—c” of 2 (5) above.
- an antibody that binds to a periostin degradation product and does not bind to a periostin multimer that is, although a periostin degradation product can be measured, a periostin multimer cannot be measured
- the increase in the AUC value is remarkable, and it can be seen that the sensitivity and specificity of the measurement, that is, the accuracy of the measurement is greatly improved.
- the periostin measurement method, the periostin measurement reagent, and the periostin measurement accuracy improvement method of the present invention improve the measurement sensitivity or specificity, and improve the measurement accuracy. It was confirmed that it was possible.
- the method for examining pulmonary fibrosis or interstitial pneumonia improves the sensitivity or specificity of the measurement, improves the accuracy of the measurement, and provides an accurate periostin measurement value. It can be obtained and can improve the discrimination between a person suffering from pulmonary fibrosis or interstitial pneumonia and a person suffering from a healthy person or another disease, and prevent misdiagnosis. It was confirmed.
- Example 12 (Measurement of periostin in human serum-2) Periostin in human serum was measured, and the effects of the antibody of the present invention, measurement method, measurement reagent, and accuracy improvement method were confirmed.
- Negative samples (54 cases in total) Serum of healthy subjects (a total of 54 cases) was used as a negative sample.
- periostin concentration was measured and determined as follows by an enzyme immunoassay (ELISA method) using an anti-periostin monoclonal antibody.
- biotin-labeled “anti-periostin monoclonal antibody (SS17B)” was bound to periostin bound to the solid-phased “anti-periostin monoclonal antibody (SS18A)”.
- POD substrate solution [containing 0.8 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), 2.5 mM hydrogen peroxide, and 30 mM disodium hydrogen phosphate 100 mM of 20 mM citrate buffer (pH 3.9)] was injected into each well of the microtiter plate of (h) and allowed to stand at 25 ° C. for 10 minutes to cause the reaction to develop color.
- TMBZ 5,5′-tetramethylbenzidine
- disodium hydrogen phosphate 100 mM of 20 mM citrate buffer (pH 3.9) was injected into each well of the microtiter plate of (h) and allowed to stand at 25 ° C. for 10 minutes to cause the reaction to develop color.
- Anti-periostin monoclonal antibody (SS20A) (an antibody that recognizes and binds to the EMI region of periostin; an antibody that recognizes and binds to any of periostin monomers, multimers, and degradation products), and (1
- the anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) is replaced with “anti-periostin monoclonal antibody (SS17B)” and “anti-periostin monoclonal antibody (SS19D)” (periostin) obtained in Example 4 above.
- Anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) of (1) was obtained in Example 4 instead of “anti-periostin monoclonal antibody (SS17B)”.
- Anti-periostin mono Ronal antibody (SS19A) (an antibody that recognizes and binds to the R4 region of periostin; an antibody that can recognize and bind to the periostin monomer and multimer, but cannot recognize and bind to a periostin degradation product) Except for the two cases described above, the measurement and treatment were performed as described in (a) to (m) of (1) above, and the concentration of periostin contained in each sample was determined.
- Anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) of (1) above was obtained in Example 6 instead of “anti-periostin monoclonal antibody (SS17B)”.
- Anti-periostin mono Ronal antibody (SS21A) (an antibody that recognizes and binds to the C-terminal region of periostin; an antibody that can recognize and bind to the periostin monomer and multimer, but cannot recognize and bind to a periostin degradation product)
- the measurement and treatment were performed as described in (a) to (m) of (1) above, and the concentration of periostin contained in each sample was determined.
- anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) of (1) was obtained in Example 5 in place of the “anti-periostin monoclonal antibody (SS17B)”.
- Anti-periostin mono except for two other things, it is referred to as “Ronal antibody (SS20A)” (an antibody that recognizes and binds to the EMI region of periostin; an antibody that recognizes and binds to any of periostin monomer, multimer and degradation product). Measurement and treatment were performed as described in (1) (a) to (m), and the concentration of periostin contained in each sample was determined.
- Anti-periostin monoclonal antibody (SS16A) (an antibody that recognizes and binds to the R3 region of periostin; an antibody that recognizes and binds to any of periostin monomers, multimers, and degradation products), and (1
- the anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) is replaced with “anti-periostin monoclonal antibody (SS17B)” and “anti-periostin monoclonal antibody (SS18A)” (periostin) obtained in Example 3 above.
- Anti-periostin monoclonal antibody (SS19C) (an antibody that recognizes and binds to the R2 region of periostin; an antibody that recognizes and binds to any of periostin monomers, multimers, and degradation products), and (1
- the anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) is replaced with “anti-periostin monoclonal antibody (SS17B)” and “anti-periostin monoclonal antibody (SS19D)” (periostin) obtained in Example 4 above.
- Anti-periostin monoclonal antibody (SS20A) (an antibody that recognizes and binds to the EMI region of periostin; an antibody that recognizes and binds to any of periostin monomers, multimers, and degradation products), and (1
- the anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) is replaced with “anti-periostin monoclonal antibody (SS17B)” and “anti-periostin monoclonal antibody (SS19C)” (periostin) obtained in Example 4 above.
- Measurement and treatment were performed as described in m), and the concentration of periostin contained in each sample was determined.
- Anti-periostin monoclonal antibody (SS20A) (an antibody that recognizes and binds to the EMI region of periostin; an antibody that recognizes and binds to any of periostin monomers, multimers, and degradation products), and (1
- the anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) is replaced with “anti-periostin monoclonal antibody (SS17B)” and “anti-periostin monoclonal antibody (SS21A)” (periostin) obtained in Example 6 above.
- FIG. 12 schematically shows which region of periostin is recognized in each of these measurements.
- the EMI region of periostin is denoted as “EMI”
- the R1 region is denoted as “R1”
- the R2 region is denoted as “R2”
- the R3 region is denoted as “R3”
- the R4 region is denoted as R4 region.
- R4 and the C-terminal region as “C-ter”.
- the region where the lower “Y” mark is bound indicates the region of periostin that is recognized and bound (ie, detected) by the immobilized antibody used in the measurement
- the region where the upper “Y” mark is bonded indicates the region of periostin recognized and bound (ie, detected) by the labeled antibody used in the measurement.
- the region surrounded by the bold line indicates the region recognized and bound (ie, detected) by the immobilized antibody and the labeled antibody, and the region sandwiched between these regions.
- the horizontal axis represents the distinction of the sample in which the measurement was performed, and the vertical axis represents the measured value (ng / mL) of the periostin concentration in the sample.
- Each “ ⁇ ” plotted in this figure represents the value of the periostin concentration contained in each sample.
- the cutoff value was set to a concentration at which the specificity exceeds 98% and the sensitivity is the highest.
- the line drawn parallel to the horizontal axis represents the level of the average value (ng / mL) of the measured value of periostin concentration contained in each sample.
- the immobilized antibody and labeled antibody used in the measurements of (2) to (13) above, the periostin region detected by the immobilized antibody, and the labeled antibody are detected.
- Periostin region whether the periostin that can be measured by the measurement is a monomer, multimer or degradation product, sensitivity of measurement, specificity of measurement, positive sample against the average value of negative sample
- the ratio of the average value of the measured values (“average value of the measured value of the positive sample” ⁇ “average value of the measured value of the negative sample”; that is, the degree of increase in the measured value of the positive sample relative to the measured value of the negative sample), “AUC value of the created ROC curve” is shown in FIG.
- EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof EMI region, R1 region, R2 region of periostin And R3 region is not detected, that is, when the degradation product of periostin cannot be measured [(1), (4), (5), (6), (9), (12) above In the case of measurement of (13) and (13)], “sensitivity of measurement” is 7.7% to 61.5% (average value: 35.7%), and “specificity of measurement” is 98.1.
- EMI region of periostin R1 region
- R2 region that is, when a degradation product of periostin can be measured [(2), (3), (7), (2) above 8), (10), and (11) in the measurement]
- the “sensitivity of measurement” is 73.1% to 96.2% (average value: 80.8%).
- “Degree” is 98.1%, “the ratio of the average value of the positive sample to the average value of the negative sample” (“the average value of the positive sample” ⁇ “the value of the negative sample Mean value; ie, measured value of negative sample The degree of increase in the measured value of the positive sample is 1.8 times to 7.7 times (average value: 4.8 times), and the “ROC curve AUC value” is 0.912 to 0.994 (average) Value: 0.943).
- an antibody other than “an antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof” Compared to the case of use, “sensitivity of measurement”, “ratio of average value of positive sample value to average value of negative sample value” (“average value of positive sample value” ⁇ “negative sample value” Both the “average value of the measured values”; that is, the degree of increase in the measured value of the positive sample relative to the measured value of the negative sample) and the “AUC value of the ROC curve” are both increased and improved. Ri, it is understood that accuracy of the measurement is improved.
- an antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region, and R3 region of periostin or a degradation product thereof and does not bind to a periostin multimer When at least one region selected from the group consisting of the EMI region, R1 region, R2 region and R3 region is detected, the periostin degradation product can be measured, and the periostin multimer is not measured [the above-mentioned In the case of the measurement of (3), (7) and (10) of 2], the “measurement sensitivity” is 73.1% to 96.2% (average value: 83.4%).
- the “specificity of the measurement” is 98.1%, and “the ratio of the average value of the positive sample to the average value of the negative sample” (“the average value of the positive sample” / “the negative sample”) Flatness of measured value Value ”; that is, the degree of increase in the measured value of the positive sample relative to the measured value of the negative sample) is 5.8 times to 7.7 times (average value: 7.1 times), and“ the AUC value of the ROC curve ” was 0.914 to 0.994 (average value: 0.950).
- the method for measuring periostin, the reagent for measuring periostin, and the method for improving the accuracy of periostin measurement according to the present invention improve the sensitivity of measurement in measuring periostin contained in a sample, thereby improving the accuracy of measurement. It was confirmed that it can be improved.
- the method for examining pulmonary fibrosis or interstitial pneumonia improves the sensitivity or specificity of the measurement, improves the accuracy of the measurement, and provides an accurate measured value of periostin. It can be obtained and can improve the discrimination between a person suffering from pulmonary fibrosis or interstitial pneumonia and a person suffering from a healthy person or another disease, and prevent misdiagnosis. It was confirmed.
- Example 13 (Measurement of periostin in a sample) Periostin was measured for a sample whose periostin concentration was known, and a standard curve (calibration curve) was prepared.
- Sample diluent not containing periostin 50 mM tris (hydroxymethyl) aminomethane buffer [Tris buffer] containing 0.5% casein, 100 mM sodium chloride, and 0.1% sodium azide (PH 8.0)] was used as a sample (standard sample) in which the periostin concentration in the following (1) was 0 ng / mL.
- Standard sample-1 (periostin concentration: 0 ng / mL) (2) Standard sample-2 (periostin concentration: 0.125 ng / mL) (3) Standard sample-3 (periostin concentration: 0.25 ng / mL) (4) Standard sample-4 (periostin concentration: 0.5 ng / mL) (5) Standard sample-5 (periostin concentration: 1.0 ng / mL) (6) Standard sample-6 (periostin concentration: 2.0 ng / mL)
- Measurement using conventional technology -7 (1) Measurement by conventional technology-1 (2) Measurement by the present invention-1 (3) Measurement-2 according to the present invention (4) Measurement by conventional technology-2 (5) Measurement by conventional technology-3 (6) Measurement by conventional technology-4 (7) Measurement according to the present invention-3 (8) Measurement according to the present invention-4 (9) Measurement by conventional technology-5 (10) Measurement by the present invention-5 (11) Measurement according to the present invention-6 (12) Measurement by conventional technology-6 (13) Measurement using conventional technology -7
- the horizontal axis represents the periostin concentration (ng / mL) in the sample
- the vertical axis represents the absorbance ( ⁇ OD) at 450 nm of each sample obtained by measurement.
- each standard curve (calibration curve) prepared in this example is the periostin concentration in the sample. It can be seen that the obtained absorbance increases as the value increases, and has quantitativeness.
- the method for measuring periostin and the reagent for measuring periostin of the present invention can accurately measure periostin in a sample.
- Example 14 (Preparation of anti-periostin monoclonal antibody-seventh time) At a different time from Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6, the operations were performed as described in (1) to (4) of Example 1. An anti-periostin monoclonal antibody was prepared. (7th)
- anti-periostin monoclonal antibody (SS25A)”) was obtained from the SS25A monoclonal antibody-producing cell line.
- Example 15 Preparation of anti-periostin monoclonal antibody-8th time
- Example 2 Example 3, Example 4, Example 5, Example 6, and Example 14.
- an anti-periostin monoclonal antibody was prepared.
- anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS27A)”) was obtained from the SS27A monoclonal antibody-producing cell line.
- Example 16 (Confirmation of recognition site of anti-periostin monoclonal antibody)
- Each of the anti-periostin monoclonal antibodies obtained in Example 14 and Example 15 was operated as described in 1 (1) to (8) of Example 7, and these anti-periostin monoclonal antibodies were converted to periostin. We confirmed which area of the worm was recognized.
- the “anti-periostin monoclonal antibody (SS25A)” obtained in Example 14 is the “periostin”, “partial periostin (R1 and R2 region)”, “part” of the above-described periostin and partial periostin. It binds only to “periostin (R2 region)” and “partial periostin (R1, R2, R3 region)”, and “partial periostin (R4 region)”, “partial periostin (EMI region)” and “partial periostin (C-terminal region)”. It was confirmed that they were not bound to any of the above. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS25A)” recognizes the R2 region of periostin as an antigen recognition part (epitope).
- the “anti-periostin monoclonal antibody (SS27A)” obtained in Example 15 is the “periostin”, “partial periostin (R1 and R2 region)” of the above periostin and partial periostin and It binds only to “partial periostin (R1, R2, R3 region)”, “partial periostin (R2 region)”, “partial periostin (R4 region)”, “partial periostin (EMI region)” and “partial periostin (C-terminal). It was confirmed that it was not bound to any of the “regions” ”. From this, it was confirmed that the “anti-periostin monoclonal antibody (SS27A)” recognizes the R1 region of periostin as an antigen recognition part (epitope).
- Example 17 (Confirmation of reactivity of anti-periostin monoclonal antibody) About each of the anti- periostin monoclonal antibody acquired in the said Example 14 and Example 15, the reactivity with the monomer of a periostin, a multimer, and each degradation product was confirmed.
- the molecular weight marker lane (indicated as “MM”), the periostin lane recognized by and bound to the “anti-periostin monoclonal antibody (SS18A)” (indicated as “18A”), and the “anti-periostin in order from the left lane.
- the thick band located between the 150 KDa and 250 KDa molecular weight markers represents the periostin multimer (presumed to be a periostin trimer from its molecular weight) and is near the 75 KDa molecular weight marker.
- the thick band seen from the high molecular weight side represents the monomer of periostin by its molecular weight
- the thick band near the 37 KDa molecular weight marker represents the degradation product of periostin by its molecular weight.
- the SS25A strain which is a monoclonal antibody-producing cell line of this “anti-periostin monoclonal antibody (SS25A)”, is the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology (Kazusa-Kamashita 2-chome, Kisarazu City, Chiba Prefecture, Japan 5 No. 8) is deposited on March 16, 2012 as “Reception Number: NITE AP-1285”.
- the SS27A strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS27A)”, is the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology (Kazusa-Kamazu 2-chome, Kisarazu City, Chiba Prefecture, Japan 5 No. 8) is deposited on March 16, 2012 as “Reception Number: NITE AP-1286”.
- anti-periostin monoclonal antibody (SS18A)” showed color development at any position showing a multimer, a monomer and a degradation product of periostin. It was confirmed that both the multimer, the monomer and the degradation product were recognized and bound.
- anti-periostin monoclonal antibody shows color development at positions showing periostin multimers and monomers, but no color development at positions showing periostin degradation products. More periostin multimers and monomers were recognized and bound, but periostin degradation products were not recognized and confirmed not to bind.
- Anti-periostin monoclonal antibody shows color development at the position showing the periostin monomer and degradation product, but no color development at the position showing the periostin multimer. More periostin monomers and degradation products were recognized and bound, but periostin multimers were not recognized and were confirmed not to bind.
- Example 18 (Measurement of periostin in human serum and ROC analysis of measurement results) Periostin in human serum was measured to confirm the effect of the present invention, and a ROC curve (receiver operating characteristic curve) as a measurement result was prepared and analyzed.
- Negative samples 64 cases in total Serum from healthy subjects (64 cases in total) was used as a negative sample.
- periostin concentration was measured and determined as follows by an enzyme immunoassay (ELISA method) using an anti-periostin monoclonal antibody.
- anti-periostin monoclonal antibody obtained in Example 4 above instead of “anti-periostin monoclonal antibody (SS17B)” instead of the anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) of (1) (SS19D) (An antibody that recognizes and binds to the R3 region of periostin; an antibody that can recognize and bind to a periostin monomer and degradation product, but cannot recognize and bind to a periostin multimer) Except for the above, the measurement and treatment were carried out as described in 2 (1) (a) to (n) of Example 11, the concentration of periostin contained in each sample was determined, and the ROC curve was obtained. Created.
- Anti-periostin monoclonal antibody (SS25A)” obtained in Example 14 an antibody that recognizes and binds to the R2 region of periostin; although it can recognize and bind to a periostin monomer and a degradation product
- an anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) of (1) above instead of “anti-periostin monoclonal antibody (SS17B)”, “5” obtained in Example 5
- peripheral antibody (SS20A) an antibody that recognizes and binds to the EMI region of periostin; an antibody that recognizes and binds to any of the monomer, multimer and degradation product of periostin
- Measurement and processing were performed as described in (1) (a) to (n) of Example 11-2 (1), the concentration of periostin contained in each sample was determined, and an ROC curve was prepared.
- Anti-periostin monoclonal antibody (SS27A)” obtained in Example 15 an antibody that recognizes and binds to the R1 region of periostin; although it can recognize and bind to the periostin monomer and degradation product
- an anti-periostin monoclonal antibody of the biotin-labeled anti-periostin monoclonal antibody in (f) of (1) above instead of “anti-periostin monoclonal antibody (SS17B)”, “5” obtained in Example 5
- peripheral antibody (SS20A) an antibody that recognizes and binds to the EMI region of periostin; an antibody that recognizes and binds to any of the monomer, multimer and degradation product of periostin
- Measurement and processing were performed as described in (1) (a) to (n) of Example 11-2 (1), the concentration of periostin contained in each sample was determined, and an ROC curve was prepared.
- each ROC curve in this figure represents the value of “1-specificity” in this measurement, and the vertical axis represents “sensitivity” in this measurement.
- the value of this AUC represents the area under the ROC curve (Area Under the Curve), and the larger this AUC value, the more the measurement is performed.
- the sensitivity and specificity are high, that is, the measurement accuracy is high, which is preferable in the measurement.
- the ROC curve in “Measurement according to the present invention— (i)” of ⁇ 2> above that is, “Anti-periostin monoclonal antibody (SS20A)” that recognizes and binds to the EMI region of periostin as a solid-phased antibody. , which detects and binds to the R3 region of periostin as a labeled antibody, detects the EMI region and R3 region of periostin, and is a monomer of periostin
- the calculated AUC value was 0.984.
- anti-periostin monoclonal antibody (SS20A) that recognizes and binds to the EMI region of periostin as a labeled antibody, and detects the R2 region and EMI region of periostin, and a monomer of periostin
- the calculated AUC value was 0.957.
- the ROC curve in “Measurement according to the present invention— (iii)” in ⁇ 4> of 2 above that is, “Anti-periostin monoclonal antibody (SS27A)” that recognizes and binds the R1 region of periostin as a solid-phased antibody.
- SS27A Anti-periostin monoclonal antibody
- the calculated AUC value was 0.993.
- a region other than “periostin EMI region, R1 region, R2 region or R3 region” is detected, that is, R4 region and / or C-terminal region of periostin is detected, and a degradation product of periostin is obtained.
- the periostin measurement method, the periostin measurement reagent, and the periostin measurement accuracy improvement method of the present invention improve the measurement sensitivity or specificity, and improve the measurement accuracy. It was confirmed that it was possible.
- the method for examining pulmonary fibrosis or interstitial pneumonia improves the sensitivity or specificity of the measurement, improves the accuracy of the measurement, and provides an accurate periostin measurement value. It can be obtained and can improve the discrimination between a person suffering from pulmonary fibrosis or interstitial pneumonia and a person suffering from a healthy person or another disease, and prevent misdiagnosis. It was confirmed.
- Measurement ⁇ 1> Measurement by Prior Art Measurement and treatment were performed as described in ⁇ 1> of ⁇ 1> above, and the concentration of periostin contained in each sample was determined.
- the “specificity of measurement” is 98.4%, “the ratio of the average value of the measurement value of the positive sample to the average value of the measurement value of the negative sample” (“average value of the measurement value of the positive sample” ⁇ “negative”
- the “average value of the measured values of the sample”; that is, the degree of increase in the measured value of the positive sample relative to the measured value of the negative sample) was 2.8 times, and the “AUC value of the ROC curve” was 0.872.
- an antibody that binds to at least one region selected from the group consisting of EMI region, R1 region, R2 region and R3 region of periostin or its degradation product and does not bind to a multimer of periostin is used.
- the “sensitivity of measurement” is 71.8% to 92.3% (average value: 81.2%) Yes
- the “specificity of measurement” is 98.4%
- the ratio of the average value of the positive sample to the average value of the negative sample (“the average value of the positive sample” ⁇ “ Negative sample measurement Mean value of the positive sample relative to the negative sample value) is 3.6 times to 16.1 times (mean value: 8.6 times). The value was 0.957 to 0.993 (average value: 0.978).
- the periostin measurement method, the periostin measurement reagent, and the periostin measurement accuracy improvement method of the present invention improve the measurement sensitivity and the like in the measurement of periostin contained in a sample, thereby improving the measurement accuracy. It was confirmed from the above that it was possible.
- the method for examining pulmonary fibrosis or interstitial pneumonia can improve the measurement sensitivity or specificity, improve the measurement accuracy, and obtain an accurate measurement value of periostin. It is possible to improve the discrimination between those suffering from pulmonary fibrosis or interstitial pneumonia and those suffering from healthy subjects and other diseases, and prevent misdiagnosis. It was confirmed from that.
- the SS19D strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS19D)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-8-8 Kazusa-Kamashita, Kisarazu, Chiba, Japan).
- NITE P-1068 As “Accession number: NITE P-1068” on February 22, 2011.
- the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology (2-5 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, Japan) No. 8) applied for transfer from domestic deposit to international deposit on February 7, 2012, and was transferred to international deposit.
- NITE BP-1068 accession in the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology
- the SS16A strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS16A)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-8-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, Japan). ) As “Receipt Number: NITE AP-1281” dated March 16, 2012. Regarding the anti-periostin monoclonal antibody (SS16A), the monoclonal antibody-producing cell line SS16A, the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology (Kazusa-Kamazu 2-chome, Kisarazu-shi, Chiba, Japan) No. 8) was applied for transfer from domestic deposit to international deposit on July 19, 2012, and transferred to international deposit. [Transfer date: July 19, 2012] (Accession number: NITE BP-1281)
- the SS18A strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS18A)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-8-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, Japan). ) As “Receipt Number: NITE AP-1282” dated March 16, 2012.
- the SS19C strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS19C)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-8-8 Kazusa-Kamashita, Kisarazu, Chiba, Japan). ) As “Receipt Number: NITE AP-1283” dated March 16, 2012.
- the SS20A strain a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS20A)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-8-8 Kazusa-Kamashita, Kisarazu, Chiba, Japan). ) As “Receipt Number: NITE AP-1284” dated March 16, 2012. Regarding the anti-periostin monoclonal antibody (SS20A), the SS20A strain, which is a monoclonal antibody-producing cell line, is the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology (Kazusa-Kamazu 2-chome, Kisarazu City, Chiba Prefecture, Japan) No. 8) was applied for transfer from domestic deposit to international deposit on July 19, 2012, and transferred to international deposit. [Transfer date: July 19, 2012] (Accession number: NITE BP-1284)
- the SS25A strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS25A)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-8-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, Japan). ) As “Receipt Number: NITE AP-1285” dated March 16, 2012.
- the SS27A strain which is a monoclonal antibody-producing cell line of the “anti-periostin monoclonal antibody (SS27A)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-8-8 Kazusa-Kamashita, Kisarazu, Chiba, Japan). ) As “Receipt Number: NITE AP-1286” on March 16, 2012. Regarding the SS27A strain, which is a monoclonal antibody-producing cell line of this “anti-periostin monoclonal antibody (SS27A)”, the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology (2-5 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, Japan) No. 8) was applied for transfer from domestic deposit to international deposit on July 19, 2012, and transferred to international deposit. [Transfer date: July 19, 2012] (Accession number: NITE BP-1286)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、臨床検査、臨床病理学、免疫学及び医学などの生命科学分野、並びに分析化学などの化学分野等において有用なものである。
また、出原は、このペリオスチン遺伝子の発現レベルの測定が特発性間質性肺炎の検査方法としても有用であることを見出した(特許文献2参照。)。
このため、試料に含まれるペリオスチンの測定値を疾患の検査に利用しようとする場合、この疾患の診断を誤らせる可能性があった。
従って、試料に含まれるペリオスチンの抗原抗体反応を利用した測定においては、健常者や他の疾患の罹患者との鑑別のため、更なる正確性の改善が望まれていた。
これに対して、本発明の課題は、試料に含まれるペリオスチンの抗原抗体反応等を利用した測定における、正確性が改善された測定方法及び測定試薬を提供すること、並びに測定の正確性の改善方法、並びに正確性が改善された肺線維症又は間質性肺炎の検査方法を提供することである。
(1)ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合する抗体。
(2)ペリオスチンの分解物に結合する抗体である、上記(1)に記載の抗体。
(3)ペリオスチンの多量体に結合しない抗体である、上記(1)又は(2)に記載の抗体。
(4)抗体がモノクローナル抗体である、上記(1)~(3)のいずれかに記載の抗体。
(5)抗体の重鎖可変領域のアミノ酸配列が、配列番号16で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号18で表されるアミノ酸配列を含むものである、上記(4)に記載の抗体。
(6)受託番号がNITE BP-1281であるハイブリドーマSS16A株、受託番号がNITE BP-1282であるハイブリドーマSS18A株、受託番号がNITE BP-1283であるハイブリドーマSS19C株、受託番号がNITE BP-1068であるハイブリドーマSS19D株、受託番号がNITE BP-1284であるハイブリドーマSS20A株、受託番号がNITE BP-1285であるハイブリドーマSS25A株及び受託番号がNITE BP-1286であるハイブリドーマSS27A株からなる群から選ばれるいずれかのハイブリドーマにより産生されるモノクローナル抗体。
(7)試料に含まれるペリオスチンの測定方法であって、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを特徴とする、前記方法。
(8)上記(1)~(6)のいずれかに記載の抗体を使用することを特徴とする、上記(7)に記載の方法。
(9)ペリオスチンがペリオスチン分解物である、上記(7)又は(8)に記載の方法。
(10)ペリオスチンが多量体ではない、上記(7)~(9)のいずれかに記載の方法。
(11)ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に特異的に結合する物質を含む、試料に含まれるペリオスチン又はその分解物の測定試薬。
(12)前記物質が上記(1)~(6)のいずれかに記載の抗体である、上記(11)に記載の試薬。
(13)ペリオスチンがペリオスチン分解物である、上記(11)又は(12)に記載の試薬。
(14)ペリオスチンが多量体ではない、上記(11)~(13)のいずれかに記載の試薬。
(15)試料に含まれるペリオスチンの量又は濃度の測定において、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを特徴とする、ペリオスチン測定の正確性の改善方法。
(16)上記(1)~(6)のいずれかに記載の抗体を使用することを特徴とする、上記(15)に記載の方法。
(17)ペリオスチンがペリオスチン分解物である、上記(15)又は(16)に記載の方法。
(18)ペリオスチンが多量体ではない、上記(15)~(17)のいずれかに記載の方法。
(19)次の工程を含む、肺線維症又は間質性肺炎の検査方法。
a) 被検者由来の試料におけるペリオスチンの量又は濃度を測定する工程であって、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを含む工程
b) 被検者由来の試料におけるペリオスチンの量又は濃度と、肺線維症又は間質性肺炎のいずれにも罹患していない生体由来の試料におけるペリオスチンの量又は濃度とを比較する工程
(20)上記(1)~(6)のいずれかに記載の抗体を使用することを特徴とする、上記(19)に記載の方法。
(21)ペリオスチンがペリオスチン分解物である、上記(19)又は(20)に記載の方法。
(22)ペリオスチンが多量体ではない、上記(19)~(21)のいずれかに記載の方法。
そして、この抗体は、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することに使用することができる抗体である。
また、この抗体は、ペリオスチンの分解物に結合することができる抗体である。
そして、この抗体は、ペリオスチンの分解物に結合することができ、かつペリオスチンの多量体には結合しない抗体である。
これにより、本発明のペリオスチンの測定方法、ペリオスチンの測定試薬、ペリオスチン測定の正確性の改善方法、及び肺線維症又は間質性肺炎の検査方法は、正確なペリオスチンの測定値を得ることができ、健常者や他の疾患の罹患者との鑑別を向上させ、診断を誤らせることを防ぐことができるものである。
本明細書において引用された全ての刊行物、例えば先行技術文献、公開公報、特許公報その他の特許文献は、参照として本明細書に組み込まれる。また、本明細書は、本願の優先権主張の基礎となる2011年9月6日に出願された日本国特許出願(特願2011-194323号)及び2012年3月29日に出願された日本国特許出願(特願2012-077774号)の明細書、特許請求の範囲及び図面に記載の内容を包含する。
1.抗体
本発明における抗体は、ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合する抗体(抗ペリオスチン特定領域抗体)である。
本発明において、ペリオスチン分解物とは、配列番号2で表されるアミノ酸配列を有するペリオスチンから、少なくとも配列番号14で表されるアミノ酸配列を有するC末端領域が欠損したポリペプチドを意味する。
また、本発明のペリオスチン分解物には、例えば、配列番号2で表されるアミノ酸配列を有するペリオスチンから、少なくとも配列番号14で表されるアミノ酸配列を有するC末端領域が欠損し、かつ配列番号12で表されるアミノ酸配列を有するR4領域の全部又は一部が欠損したポリペプチドが含まれる。
また、本発明のペリオスチン分解物には、例えば、配列番号2で表されるアミノ酸配列を有するペリオスチンから、少なくとも配列番号14で表されるアミノ酸配列を有するC末端領域が欠損し、かつ配列番号12で表されるアミノ酸配列を有するR4領域の全部が欠損し、かつ配列番号10で表されるアミノ酸配列を有するR3領域の一部が欠損したポリペプチドも含まれる。
本発明のペリオスチン分解物は、本発明者らが初めて見出したポリペプチドである。ペリオスチン分解物は、例えば、生体由来の試料に含まれるポリペプチドを本発明の抗体を用いて免疫学的手法により検出することにより、その存在を明確に確認することができる(図5、図9、図26等)。
ペリオスチン分解物としては、例えば、その分子量が約4万Da(約40kDa)のものなどを挙げることができるが、これに限定されない。また、ペリオスチン分解物は、生体由来の試料に含まれるものが好ましい。
すなわち、本発明における、ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合し、かつペリオスチンの多量体に結合しない抗ペリオスチン特定領域抗体は、測定の正確性の改善度が更に高いため好ましい。
本発明における抗ペリオスチン特定領域抗体を産生させるための免疫原について、以下説明を行う。
本発明における抗ペリオスチン特定領域抗体を産生させるための免疫原として、ペリオスチンの全部又は一部を用いることができる。
すなわち、ヒト等由来のペリオスチン、又は遺伝子組み換え操作により得たペリオスチン等のペリオスチンの全部又は一部を用いることができる。
なお、この抗ペリオスチン特定領域抗体を産生させるための免疫原は、ペリオスチンのアミノ酸配列の全部又は一部のアミノ酸配列に1ないし数個(通常1~8個、好ましくは1~6個)のアミノ酸残基の欠失、置換、挿入、付加、又は修飾を施すことにより得られるアミノ酸配列を含むペプチド又はタンパク質等であってもよい。
よって、本発明における抗ペリオスチン特定領域抗体の免疫原のアミノ酸配列の最小単位としては、ペリオスチンのアミノ酸配列の全部若しくは一部のアミノ酸配列、又はこれらのアミノ酸配列の全部若しくは一部のアミノ酸配列に1ないし数個(通常1~8個、好ましくは1~6個)のアミノ酸残基の欠失、置換、挿入、付加若しくは修飾を施すことにより得られるアミノ酸配列の内、連続する3つのアミノ酸残基よりなるアミノ酸配列を考えることができるので、これらの連続する3つのアミノ酸残基よりなるアミノ酸配列からなるトリペプチド、又はこれに他のアミノ酸若しくはペプチドが付加したもの等を、本発明における抗ペリオスチン特定領域抗体の免疫原の最小単位として考えることができる。
(a) まず、ペリオスチン(ポリヌクレオチドの塩基配列:核酸データベースGenBankのAccession NumberD13666(配列番号1);アミノ酸配列:核酸データベースGenBankのAccession NumberBAA02837(配列番号2))にV5/Hisタグを付加させたリコンビナントペリオスチンタンパク質を昆虫細胞であるS2細胞において発現させた上で精製する。
pMT/Bip/V5-HisAプラスミド(Invitrogen社、米国カリフォルニア州Carlsbad)にペリオスチンの上記部分をコードするcDNAを挿入して、これをpMT/Bip/periostin-V5-HisAとする。
S2細胞にpMT/Bip/periostin-V5-HisA及びハイグロマイシン耐性遺伝子を発現するプラスミドであるpAcHygro(Invitrogen社、米国カリフォルニア州Carlsbad)を公知の方法で共導入し、形質転換させる。
ハイグロマイシンにより形質転換体を選択し、安定形質転換体を得る。
そして、S2細胞の形質転換体では、カルボキシ末端にV5エピトープ/Hisタグの結合したペリオスチンを発現させる。
ペリオスチン遺伝子安定形質転換体S2細胞の培地に硫酸銅を加えることにより、S2リコンビナントペリオスチンタンパク質の発現を誘導する。
これにより、S2リコンビナントペリオスチンタンパク質は培養上清中に発現分泌される。
この培養上清をリン酸緩衝生理食塩水(PBS)に透析した後、ニッケルレジン(Ni-NTA Agarose、Qiagen社、ドイツ国Hilden)と混合して、S2リコンビナントペリオスチンタンパク質をレジンに結合させる。
レジンを洗浄して夾雑物を取り除き、イミダゾール含有緩衝液にてS2リコンビナントペリオスチンタンパク質を溶出させる。
溶出されたS2リコンビナントペリオスチンタンパク質をPBS等に透析し、精製されたペリオスチンタンパク質を取得する。
すなわち、ペリオスチンのcDNAを、GEX-KGベクター(“KL.Guanら,Anal.Biochem.,192巻,262~267頁,1991年発行”)に組み込んで、大腸菌BL21にトランスフェクションする。
これをアンピシリン入りLB培地にて培養し、菌体よりグルタチオンセファロース4B(GE Healthcare社、Little Chalfont、英国)により、グルタチオンSトランスフェラーゼ(GST)を付加したペリオスチンを精製する。
これにトロンビンにてGSTを切断し、GSTを付加しないペリオスチンを取得する。
これをブラッドフォード法にて定量して、その量(濃度)が明確となったペリオスチンを取得することができる。
また、非天然型アミノ酸の導入、各アミノ酸残基の化学修飾やシステイン残基を導入することにより分子内を環化させて構造を安定化させる等の修飾を施してもよい。
また、ニトロセルロース粒子、ポリビニルピロリドン又はリポソーム等の担体に免疫原を吸着させたものを免疫原とすることもできる。
ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合することができるポリクローナル抗体、すなわち、ポリクローナル抗体である抗ペリオスチン特定領域抗体は、以下の操作により調製することができる。
また、前記のノックアウト動物においては、その動物の体内でペリオスチンが生産されないため、免疫されたペリオスチンを異物と認識し易く、よって抗体の産生が高くなるためである。
アジュバントとしては、フロイント完全アジュバント、フロイント不完全アジュバント、水酸化アルミニウムアジュバント、化学合成アジュバント又は百日咳菌アジュバント等の公知のものを用いることができる。
免疫注射は、皮下、静脈内、腹腔内又は背部等の部位に行えばよい。
この追加免疫注射の回数としては、2~6回が一般的である。
この場合も、前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して追加免疫注射することが好ましい。
初回免疫の後、免疫動物の血清中の抗体価の測定をELISA法等により繰り返し行い、抗体価がプラトーに達したら全採血を行い、血清を分離して抗体を含む抗血清を得る。
得られたポリクローナル抗体である抗ペリオスチン特定領域抗体は、ペリオスチンの分解物に結合することができる抗体である。
ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合することができるモノクローナル抗体、すなわち、モノクローナル抗体である抗ペリオスチン特定領域抗体は、以下の操作により調製することができる。
また、前記のノックアウト動物においては、その動物の体内でペリオスチンが生産されないため、免疫されたペリオスチンを異物と認識し易く、よって抗体の産生が高くなるためである。
アジュバントとしては、フロイント完全アジュバント、フロイント不完全アジュバント、水酸化アルミニウムアジュバント、化学合成アジュバント又は百日咳菌アジュバント等の公知なものを用いることができる。
免疫注射は、皮下、静脈内、腹腔内、足蹠又は背部等の部位に行えばよい。
この追加免疫注射の回数としては2~6回が一般的である。
この場合も前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して追加免疫注射することが好ましい。
ミエローマ細胞がHGPRT欠損株又はTK欠損株のものである場合には、ヒポキサンチン・アミノプテリン・チミジンを含む選別用培地(HAT培地)を用いることにより、抗体産生能を有する細胞とミエローマ細胞の融合細胞(ハイブリドーマ)のみを選択的に培養し、増殖させることができる。
また、このモノクローナル抗体産生細胞株を、これに適合性がありプリスタン等であらかじめ刺激した哺乳動物の腹腔内に注入し、一定期間の後、腹腔にたまった腹水より本発明における、モノクローナル抗体である抗ペリオスチン特定領域抗体を得ることもできる。
また、別の態様において、本発明におけるモノクローナル抗体としては、例えば、受託番号がNITE BP-1281であるハイブリドーマSS16A株、受託番号がNITE BP-1282であるハイブリドーマSS18A株、受託番号がNITE BP-1283であるハイブリドーマSS19C株、受託番号がNITE BP-1068であるハイブリドーマSS19D株、受託番号がNITE BP-1284であるハイブリドーマSS20A株、受託番号がNITE BP-1285であるハイブリドーマSS25A株及び受託番号がNITE BP-1286であるハイブリドーマSS27A株からなる群から選ばれるいずれかのハイブリドーマにより産生されるモノクローナル抗体が挙げられるが、これらに限定されるものではない。
1.総論
本発明のペリオスチンの測定方法は、試料に含まれるペリオスチンの測定方法であって、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを特徴とするものである。
(i) ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合する抗体、
(ii) ペリオスチンの分解物に結合する抗体、
(iii) ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合する抗体であって、ペリオスチンの多量体に結合しない抗体、
(iv) ペリオスチンの分解物に結合し、ペリオスチンの多量体に結合しない抗体、
(v) ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合するモノクローナル抗体、
(vi) ペリオスチンの分解物に結合するモノクローナル抗体、
(vii) ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合するモノクローナル抗体であって、ペリオスチンの多量体に結合しない前記抗体、
(viii) 抗体の重鎖可変領域のアミノ酸配列が配列番号16で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が配列番号18で表されるアミノ酸配列を含む、モノクローナル抗体、
(ix) 受託番号がNITE BP-1281であるハイブリドーマSS16A株、受託番号がNITE BP-1282であるハイブリドーマSS18A株、受託番号がNITE BP-1283であるハイブリドーマSS19C株、受託番号がNITE BP-1068であるハイブリドーマSS19D株、受託番号がNITE BP-1284であるハイブリドーマSS20A株、受託番号がNITE BP-1285であるハイブリドーマSS25A株及び受託番号がNITE BP-1286であるハイブリドーマSS27A株からなる群から選ばれるいずれかのハイブリドーマにより産生されるモノクローナル抗体。
例えば、酵素標識抗体と固相化抗体を用いるELISA法のサンドイッチ法においては、試料に含まれていたペリオスチンに結合させる酵素標識抗体及び固相化抗体のいずれもが、この抗ペリオスチン特定領域抗体である必要がある。
本発明のペリオスチンの測定方法としては、試料に含まれるペリオスチンを抗原抗体反応を利用して測定する測定方法であって、抗ペリオスチン特定領域抗体を使用するものが好ましいが、この抗ペリオスチン特定領域抗体を使用するものであれば、特にその測定原理に限定されるものではなく、所期の効果を奏するものである。
また、本発明のペリオスチンの測定方法における測定は、用手法により行ってもよく、又は分析装置等の装置を用いて行ってもよい。
本発明における試料としては、血液、血清、血漿、尿、精液、髄液、唾液、腹水若しくは羊水などの体液;あるいは血管若しくは肝臓などの臓器、組織又は細胞などの抽出液等、ペリオスチンが含まれる可能性のある生体試料等の試料であれば対象となる。
例えば、試料を抗ペリオスチン特定領域抗体と接触させ、結合させる前に、試料に希釈液を添加することにより希釈処理を行ってもよい。
この希釈液として、各種水系溶媒を用いることができる。
例えば、水、生理食塩水又はトリス(ヒドロキシメチル)アミノメタン緩衝液〔Tris緩衝液〕、リン酸緩衝液若しくはリン酸緩衝生理食塩水などの各種緩衝液等の水系溶媒を用いることができる。
なお、この緩衝液のpHについては、pH5~pH10の範囲にあることが好ましい。
本発明において、測定対象物質はペリオスチンである。
このペリオスチンには、ペリオスチンの一量体、ペリオスチンの二量体、三量体、四量体又はそれ以上のペリオスチンの多量体、又はペリオスチンの分解物(例えば、分子量約40KDaのペリオスチンの分解物など)等が含まれるが、いずれも本発明における測定対象物質となるものである。
すなわち、言い換えると、ペリオスチンの多量体以外のペリオスチン(本明細書において「多量体ではないペリオスチン」とも称する。)が測定対象物質として好適である。
すなわち、言い換えると、ペリオスチンの一量体及びペリオスチンの多量体以外のペリオスチンが測定対象物質として特に好適である。
本発明のペリオスチンの測定方法における測定を、酵素免疫測定法、蛍光免疫測定法、放射免疫測定法又は発光免疫測定法等の標識抗体を用いた免疫学的測定方法、すなわち標識抗体を用いる抗原抗体反応を利用した測定方法により実施する場合には、サンドイッチ法又は競合法等により行うことができるが、サンドイッチ法により実施する時には、試料に含まれていたペリオスチンに結合させる固相化抗体及び標識抗体のいずれの抗体もが抗ペリオスチン特定領域抗体である必要がある。
なお、前記の抗ペリオスチン特定領域抗体等の抗体と固相担体とを物理的吸着法、化学的結合法又はこれらの併用等の公知の方法により吸着、結合させて抗体を固相担体に固定化することができる。
また、化学的結合法により行う場合は、日本臨床病理学会編「臨床病理臨時増刊特集第53号 臨床検査のためのイムノアッセイ-技術と応用-」,臨床病理刊行会,1983年発行;日本生化学会編「新生化学実験講座1 タンパク質IV」,東京化学同人,1991年発行等に記載の公知の方法に従い、抗ペリオスチン特定領域抗体等の抗体と固相担体をグルタルアルデヒド、カルボジイミド、イミドエステル又はマレイミド等の二価性の架橋試薬と混合、接触させ、抗ペリオスチン特定領域抗体等の抗体と固相担体のそれぞれのアミノ基、カルボキシル基、チオール基、アルデヒド基又は水酸基等と反応させること等により行うことができる。
また、蛍光免疫測定法の場合には、フルオレセインイソチオシアネート、テトラメチルローダミンイソチオシアネート、置換ローダミンイソチオシアネート又はジクロロトリアジンイソチオシアネート等を用いることができる。
そして、放射免疫測定法の場合には、トリチウム、ヨウ素125又はヨウ素131等を用いることができる。
また、発光免疫測定法においては、NADH-FMNH2-ルシフェラーゼ系、ルミノール-過酸化水素-POD系、アクリジニウムエステル系又はジオキセタン化合物系等を用いることができる。
そして、未結合の標識抗体を洗浄分離して、「固相化抗体=ペリオスチン」を介して固相担体に結合した標識抗体の量又は未結合の標識抗体の量より試料に含まれていたペリオスチンの量(濃度)のみを測定することができる。
また、蛍光免疫測定法の場合には蛍光物質標識による蛍光強度等を、放射免疫測定法の場合には放射性物質標識による放射線量等を測定する。
そして、発光免疫測定法の場合は発光反応系による発光量等を測定する。
本発明のペリオスチンの測定方法における測定を、免疫比濁法、ラテックス比濁法、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等の免疫複合体凝集物の生成を、その透過光や散乱光を光学的方法により測るか、又は目視的に測る測定方法により実施する場合には、すなわち、抗原抗体反応による複合体の凝集物の生成を測る測定方法(凝集反応法)により実施する場合には、試料に含まれていたペリオスチンに結合させる抗体が抗ペリオスチン特定領域抗体である必要がある。
これらの容器の溶液収容部分(マイクロプレートのウェル等)の底面は、U型、V型又はUV型等の底面中央から周辺にかけて傾斜を持つ形状であることが好ましい。
また、目視的に測定する場合には、プレートやマイクロプレート等の前記容器中で、試料と「固相担体に固定化させた抗ペリオスチン特定領域抗体等の抗体」を反応させ、凝集の状態を目視的に測定する。
なお、この目視的に測定する代わりにマイクロプレートリーダー等の機器を用いて測定を行ってもよい。
例えば、まず、「抗ペリオスチン特定領域抗体を固定化した固相担体」を含有する測定試薬、又は「抗ペリオスチン特定領域抗体を固定化した固相担体」を含有する測定試薬及び「水系溶媒」を含有する測定試薬等を調製し、準備する。
なお、測定波長は、340nmから1,000nmの中から選ばれるのが一般的である。
測定装置は、汎用自動分析装置であっても、専用の測定装置(専用機)であってもよい。
第1試薬:
緩衝液(水系溶媒)
第2試薬:
「抗ペリオスチン特定領域抗体を固定化したラテックス粒子」を含有する緩衝液
なお、試料と第1試薬の混合比率(量比)は、適宜選択すればよい。
また、前記の静置時の温度は、室温(1℃~30℃)又は微温(30℃~40℃)の範囲内の一定温度であることが好ましい。(例えば、37℃等)
これにより、「抗ペリオスチン特定領域抗体を固定化したラテックス粒子」と試料とを接触させる。
なお、第2試薬の添加量は、適宜選択すればよい。
また、前記の静置時の温度は、室温(1℃~30℃)又は微温(30℃~40℃)の範囲内の一定温度であることが好ましい。(例えば、37℃等)
そして、前記の静置の時間は、1分以上、かつ10分以下の一定時間であることが好ましく、3分以上、かつ5分以下の一定時間であることがより好ましい。
本発明のペリオスチンの測定方法においては、溶媒として、各種の水系溶媒を用いることができる。
この水系溶媒としては、例えば、精製水、生理食塩水、又は、トリス(ヒドロキシメチル)アミノメタン緩衝液〔Tris緩衝液〕、リン酸緩衝液若しくはリン酸緩衝生理食塩水などの各種緩衝液等を挙げることができる。
この緩衝液のpHについては、適宜適当なpHを選択して用いればよく、特に制限はないものの、通常は、pH3~pH12の範囲内のpHを選択して用いることが一般的である。
そして、これらを測定に用いる際の濃度は特に限定されるものではないが、0.001~10%(W/V)が好ましく、特に0.01~5%(W/V)が好ましい。
1.総論
本発明のペリオスチン又はその分解物の測定試薬は、試料に含まれるペリオスチン又はその分解物の測定試薬であって、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを特徴とするものである。
各領域に特異的に結合する物質(特異的結合物質)の説明については上記の通りである。
例えば、このペリオスチンの測定が、酵素標識抗体と固相化抗体を用いるELISA法のサンドイッチ法による場合には、試料に含まれていたペリオスチンに結合させる酵素標識抗体及び固相化抗体のいずれもが、この抗ペリオスチン特定領域抗体である必要がある。
本発明における試料については、前記「〔2〕.ペリオスチンの測定方法」の「3.試料」に記載した通りである。
本発明における測定対象物質については、前記「〔2〕.ペリオスチンの測定方法」の「4.測定対象物質」に記載した通りである。
本発明のペリオスチン又はその分解物の測定試薬としては、試料に含まれるペリオスチン又はその分解物を抗原抗体反応を利用して測定する測定試薬であって、抗ペリオスチン特定領域抗体を含有するものが好ましいが、この抗ペリオスチン特定領域抗体を含有するものであれば、特にそのペリオスチン測定の測定原理に限定されるものではなく、所期の効果を奏するものである。
また、本発明のペリオスチン又はその分解物の測定試薬による測定は、用手法により行ってもよいし、又は分析装置等の装置を用いて行ってもよい。
この場合、抗ペリオスチン特定領域抗体は、その一つの測定試薬に含有される。
この場合、抗ペリオスチン特定領域抗体は、二つ以上の測定試薬の内の一つの測定試薬に含有されるものであってもよく、また、二つ以上の測定試薬に含有されるものであってもよい。
また、本発明のペリオスチン又はその分解物の測定試薬は、他の試薬と組み合わせて、販売し、又は試料に含まれるペリオスチン又はその分解物の測定に使用することもできる。
本発明のペリオスチン又はその分解物の測定試薬が、酵素免疫測定法、蛍光免疫測定法、放射免疫測定法又は発光免疫測定法等の標識抗体を用いた免疫学的測定方法、すなわち標識抗体を用いる抗原抗体反応を利用した測定方法を測定原理とする場合には、サンドイッチ法又は競合法等により行うことができるが、サンドイッチ法により実施する時には、試料に含まれていたペリオスチンに結合させる固相化抗体及び標識抗体のいずれの抗体もが抗ペリオスチン特定領域抗体である必要がある。
本発明のペリオスチン又はその分解物の測定試薬が、免疫比濁法、ラテックス比濁法、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等の免疫複合体凝集物の生成を、その透過光や散乱光を光学的方法により測るか、又は目視的に測る測定方法により実施する場合には、すなわち、抗原抗体反応による複合体の凝集物の生成を測る測定方法(凝集反応法)を測定原理とする場合には、試料に含まれていたペリオスチンに結合させる抗体が抗ペリオスチン特定領域抗体である必要がある。
これらの容器の溶液収容部分(マイクロプレートのウェル等)の底面は、U型、V型又はUV型等の底面中央から周辺にかけて傾斜を持つ形状であることが好ましい。
1.総論
本発明のペリオスチン測定の正確性の改善方法は、試料に含まれるペリオスチンの量又は濃度の測定において、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを特徴とするものである。
例えば、このペリオスチンの測定が、酵素標識抗体と固相化抗体を用いるELISA法のサンドイッチ法による場合には、試料に含まれていたペリオスチンに結合させる酵素標識抗体及び固相化抗体のいずれもが、この抗ペリオスチン特定領域抗体である必要がある。
本発明における試料については、前記「〔2〕.ペリオスチンの測定方法」の「3.試料」に記載した通りである。
本発明における測定対象物質については、前記「〔2〕.ペリオスチンの測定方法」の「4.測定対象物質」に記載した通りである。
本発明のペリオスチン測定の正確性の改善方法としては、試料に含まれるペリオスチンの抗原抗体反応を利用した測定において、抗ペリオスチン特定領域抗体を使用するものが好ましいが、この抗ペリオスチン特定領域抗体を使用するものであれば、特にその測定原理に限定されるものではなく、所期の効果を奏するものである。
また、本発明のペリオスチン測定の正確性の改善方法におけるペリオスチン測定は、用手法により行ってもよいし、又は分析装置等の装置を用いて行ってもよい。
この場合、抗ペリオスチン特定領域抗体は、その一つの測定試薬に含有される。
この場合、抗ペリオスチン特定領域抗体は、二つ以上の測定試薬の内の一つの測定試薬に含有されるものであってもよく、また、二つ以上の測定試薬に含有されるものであってもよい。
1.総論
本発明の肺線維症又は間質性肺炎の検査方法は、次の工程を含むものである。
a) 被検者由来の試料におけるペリオスチンの量又は濃度を測定する工程であって、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを含む工程
b) 被検者由来の試料におけるペリオスチンの量又は濃度と、肺線維症又は間質性肺炎のいずれにも罹患していない生体由来の試料におけるペリオスチンの量又は濃度とを比較する工程
これにより、本発明の肺線維症又は間質性肺炎の検査方法は、正確なペリオスチンの測定値を得ることができ、健常者や他の疾患の罹患者との鑑別を向上させ、診断を誤らせることを防ぐことができる検査方法である。
a) 被検者由来の試料におけるペリオスチン分解物の量又は濃度を測定する工程であって、ペリオスチン分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを含む工程
b) 被検者由来の試料におけるペリオスチン分解物の量又は濃度と、肺線維症又は間質性肺炎のいずれにも罹患していない生体由来の試料におけるペリオスチン分解物の量又は濃度とを比較する工程
a) 被検者由来の試料におけるペリオスチン又はペリオスチン分解物の量又は濃度を測定する工程であって、ペリオスチン又はペリオスチン分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出し、かつペリオスチンの多量体を検出しない工程
b) 被検者由来の試料におけるペリオスチン又はペリオスチン分解物の量又は濃度と、肺線維症又は間質性肺炎のいずれにも罹患していない生体由来の試料におけるペリオスチン又はペリオスチン分解物の量又は濃度とを比較する工程
本発明における試料については、前記「〔2〕.ペリオスチンの測定方法」の「3.試料」に記載した通りである。
本発明における測定対象物質については、前記「〔2〕.ペリオスチンの測定方法」の「4.測定対象物質」に記載した通りである。
本発明の肺線維症又は間質性肺炎の検査方法としては、試料に含まれるペリオスチンを抗原抗体反応を利用して測定することによる肺線維症又は間質性肺炎の検査方法であって、抗ペリオスチン特定領域抗体を使用するものが好ましいが、この抗ペリオスチン特定領域抗体を使用するものであれば、特にその測定原理に限定されるものではなく、所期の効果を奏するものである。
また、本発明の肺線維症又は間質性肺炎の検査方法におけるペリオスチン測定は、用手法により行ってもよいし、又は分析装置等の装置を用いて行ってもよい。
この場合、抗ペリオスチン特定領域抗体は、その一つの測定試薬に含有される。
この場合、抗ペリオスチン特定領域抗体は、二つ以上の測定試薬の内の一つの測定試薬に含有されるものであってもよく、また、二つ以上の測定試薬に含有されるものであってもよい。
本発明の肺線維症又は間質性肺炎の検査方法においては、試料に含まれるペリオスチンを、前記の通り、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することにより測定し、そして、被検者由来の試料におけるペリオスチンの量又は濃度と、肺線維症又は間質性肺炎のいずれにも罹患していない生体由来の試料におけるペリオスチンの量又は濃度とを比較する。
すなわち、本発明の肺線維症又は間質性肺炎の検査方法においては、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することにより測定を行った被検者由来の試料におけるペリオスチンの量又は濃度と、肺線維症又は間質性肺炎のいずれにも罹患していない生体由来の試料について、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することにより測定を行ったペリオスチンの量又は濃度とを、比較する。
なお、試料に含まれるペリオスチンを測定するに当たり、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することの詳細については、前記の通りである。
また、被検者由来の試料におけるペリオスチンの量又は濃度の上昇は、当該被検者の肺線維症又は間質性肺炎の病状が憎悪している可能性が高いことを示している。
そして、肺線維症又は間質性肺炎に罹患している可能性が高いと判断するための「所定タンパク質量」は、例えば、次のように求めることができる。先ず、2例以上の肺線維症又は間質性肺炎患者由来の試料におけるペリオスチンの量又は濃度を測定する。このとき、対象となる患者の例数は2例以上であり、例えば、5例以上、10例以上、50例以上、または100例以上である。一方、2例以上の対照試料におけるペリオスチンの量又は濃度も測定する。このとき、対象となる対照試料の例数は2例以上であり、例えば、5例以上、10例以上、50例以上、または100例以上である。そして、肺線維症又は間質性肺炎患者由来の試料群と対照試料群の両方を含む全体から、肺線維症又は間質性肺炎患者由来の試料群を抽出しうる、ペリオスチンの量又は濃度の至適閾値(cut-off値)を、統計処理により求める。統計処理としては、
例えば、Receiver-Operating-Characteristics(ROC)曲線を用いた解析等が挙げられる。
抗体のエピトープの特定等のため、次のペリオスチン及び部分ペリオスチン(ペリオスチンの部分よりなるもの)を調製した。
ペリオスチン(ヌクレオチド配列:核酸データベースGenBankのAccession NumberD13666のヌクレオチド配列、アミノ酸配列:核酸データベースGenBankのAccession NumberBAA02837のアミノ酸配列)にV5/Hisタグを付加させたリコンビナントペリオスチンタンパク質を昆虫細胞であるS2細胞において発現させた上で精製した。
pMT/Bip/V5-HisAプラスミド(Invitrogen社、米国カリフォルニア州Carlsbad)にペリオスチンの上記部分をコードするcDNAを挿入して、これをpMT/Bip/periostin-V5-HisAとした。
そして、S2細胞の形質転換体では、カルボキシ末端にV5/Hisタグの結合したペリオスチンを発現させた。
まず、ペリオスチン遺伝子安定形質転換体S2細胞の培地に硫酸銅を加えることにより、S2リコンビナントペリオスチンタンパク質の発現を誘導した。
これにより、S2リコンビナントペリオスチンタンパク質は培養上清中に発現分泌された。
次に、レジンを洗浄して夾雑物を取り除き、イミダゾール含有緩衝液にてS2リコンビナントペリオスチンタンパク質を溶出することにより、ペリオスチンを取得した。
なお、作成したプラスミドのDNAの配列を確認し、組み込まれた配列が目的通りのものであることを確認した。
公知の文献(I.Takayamaら,J.Biochem.,146巻,5号,713~723頁,2009年発行等)の記載に従い、C末端に6個のヒスチジン残基が結合した、ペリオスチンのR1領域及びR2領域よりなる部分ペリオスチンを、調製し、取得した。(以下、これを「部分ペリオスチン(R1・R2領域)」という)
なお、作成したプラスミドのDNAの配列を確認し、組み込まれた配列が目的通りのものであることを確認した。
公知の文献(I.Takayamaら,J.Biochem.,146巻,5号,713~723頁,2009年発行等)の記載に従い、C末端に6個のヒスチジン残基が結合した、ペリオスチンのR2領域よりなる部分ペリオスチンを、調製し、取得した。(以下、これを「部分ペリオスチン(R2領域)」という)
なお、作成したプラスミドのDNAの配列を確認し、組み込まれた配列が目的通りのものであることを確認した。
公知の文献(I.Takayamaら,J.Biochem.,146巻,5号,713~723頁,2009年発行等)の記載に従い、C末端に6個のヒスチジン残基が結合した、ペリオスチンのR1領域、R2領域及びR3領域よりなる部分ペリオスチンを、調製し、取得した。(以下、これを「部分ペリオスチン(R1・R2・R3領域)」という)
なお、作成したプラスミドのDNAの配列を確認し、組み込まれた配列が目的通りのものであることを確認した。
公知の文献(I.Takayamaら,J.Biochem.,146巻,5号,713~723頁,2009年発行等)の記載に従い、C末端に6個のヒスチジン残基が結合した、ペリオスチンのR4領域よりなる部分ペリオスチンを、調製し、取得した。(以下、これを「部分ペリオスチン(R4領域)」という)
なお、作成したプラスミドのDNAの配列を確認し、組み込まれた配列が目的通りのものであることを確認した。
公知の文献(I.Takayamaら,J.Biochem.,146巻,5号,713~723頁,2009年発行等)の記載に従い、C末端に6個のヒスチジン残基が結合した、ペリオスチンのEMI領域よりなる部分ペリオスチンを、調製し、取得した。(以下、これを「部分ペリオスチン(EMI領域)」という)
なお、作成したプラスミドのDNAの配列を確認し、組み込まれた配列が目的通りのものであることを確認した。
公知の文献(I.Takayamaら,J.Biochem.,146巻,5号,713~723頁,2009年発行等)の記載に従い、C末端に6個のヒスチジン残基が結合した、ペリオスチンのC末端領域よりなる部分ペリオスチンを、調製し、取得した。(以下、これを「部分ペリオスチン(C末端領域)」という)
なお、作成したプラスミドのDNAの配列を確認し、組み込まれた配列が目的通りのものであることを確認した。
I.ペリオスチン、部分ペリオスチン(R1・R2領域)、部分ペリオスチン(R2領域)、部分ペリオスチン(R1・R2・R3領域)、及び部分ペリオスチン(C末端領域)の確認
次の(1)~(11)の試薬をそれぞれ調製した。
アクリルアミド29.2g及びN,N’-メチレンビスアクリルアミド0.8gを純水に添加、混合した後100mLとして、アクリルアミド溶液〔30%アクリルアミド保存液〕を調製した。
トリス(ヒドロキシメチル)アミノメタン〔Tris〕18.2g及びドデシル硫酸ナトリウム〔SDS〕0.4gを純水に添加、混合し、塩酸でpH8.8に調整した後100mLとして、SDS-1.5Mトリス溶液〔0.4%SDS-1.5Mトリス塩酸緩衝液〕を調製した。
トリス(ヒドロキシメチル)アミノメタン〔Tris〕6.1g及びドデシル硫酸ナトリウム〔SDS〕0.4gを純水に添加、混合し、塩酸でpH6.8に調整した後100mLとして、SDS-0.5Mトリス溶液〔0.4%SDS-0.5Mトリス塩酸緩衝液〕を調製した。
過硫酸アンモニウム100mgを純水に添加、混合した後1mLとして、過硫酸アンモニウム溶液〔10%過硫酸アンモニウム水溶液〕を調製した。
N,N,N’,N’-テトラメチルエチレンジアミン(ナカライテスク社、日本国京都府)を使用した。
トリス(ヒドロキシメチル)アミノメタン〔Tris〕1.5g、ドデシル硫酸ナトリウム〔SDS〕0.5g、及びグリシン7.2gを純水に添加、混合した後500mLとして、泳動槽用緩衝液〔0.1%SDS-192mMグリシン-25mMトリス緩衝液〕を調製した。
メタノール10mL、及び酢酸7mLを、純水83mLと混合して、SYPRO Ruby固定液を調製した。
SYPRO Ruby染色液は、Molecular Probes社(Eugene、オレゴン州、米国)のSYPRO Ruby protein gel stainを使用した。
メタノール10mL、及び酢酸7mLを、純水83mLと混合して、SYPRO Ruby脱色液を調製した。
ドデシル硫酸ナトリウム〔SDS〕0.4g、2-メルカプトエタノール1.2mL、1Mのトリス(ヒドロキシメチル)アミノメタン〔Tris〕-塩酸緩衝液(pH6.8)の1mL、及びグリセリン2mLを純水に添加、混合した後10mLとして、試料処理液〔4%SDS-12%2-メルカプトエタノール-20%グリセリン-100mMトリス緩衝液〕を調製した。
前記参考例1の(1)~(4)及び(7)で調製した、次のペリオスチン及び部分ペリオスチンをそれぞれ試料とした。また、次の分子量マーカーも試料とした。
(b)部分ペリオスチン(R1・R2領域)
(c)部分ペリオスチン(R2領域)
(d)部分ペリオスチン(R1・R2・R3領域)
(e)部分ペリオスチン(C末端領域)
(f)分子量マーカー〔Precision Plus Protein All Blue Standardsマーカー;マーカー分子量 10KDa、15KDa、20KDa、25KDa、37KDa、50KDa、75KDa、100KDa、150KDa及び250KDa;BIO-RAD Laboratories社、Hercules、カリフォルニア州、米国〕
前記1で調製した試薬を使用し、前記2の試料のそれぞれについて、次の操作によりSDS-ポリアクリルアミドゲル電気泳動法での電気泳動を行った。
この分離ゲル溶液を、組み立てたガラスプレートに注入した後、純水を上に重層し、そしてゲル化反応を30分間行わせた。
前記(1)のガラスプレートより純水を捨て、ここにこの濃縮ゲル溶液を少量注入し洗浄した後、濃縮ゲル溶液を注入した。そして、ここにサンプルコウムを挿入し、そしてゲル化反応を30分間行わせた。
なお、このゲルに注入した前記(3)の処理を行った試料及び前記2の(f)の分子量マーカーの試料であるが、レーン(a)に前記の「ペリオスチン」を、その右側のレーン(b)に前記の「部分ペリオスチン(R1・R2領域)」を、その右側のレーン(c)に前記の「部分ペリオスチン(R2領域)」を、その右側のレーン(d)に前記の「部分ペリオスチン(R1・R2・R3領域)」を、その右側のレーン(e)に前記の「部分ペリオスチン(C末端領域)」を、そしてレーン(a)の左側のレーンに前記の「分子量マーカー」を注入した。
前記3の(10)で撮影したゲルを図2に示した。
すなわち、「ペリオスチン」、「部分ペリオスチン(R1・R2領域)」、「部分ペリオスチン(R2領域)」、「部分ペリオスチン(R1・R2・R3領域)」及び「部分ペリオスチン(C末端領域)」をそれぞれ調製できていることが確認できた。
前記参考例1で調製した部分ペリオスチン(R4領域)、及び部分ペリオスチン(EMI領域)を、SDS-ポリアクリルアミドゲル電気泳動法及びウエスタンブロッティング法で確認した。
1.試薬
次の(1)~(4)の試薬をそれぞれ調製した。
Funakoshi Easy-Gel(III) プリキャスト・ゲル(15%)(フナコシ社、日本国東京都)を使用した。
前記Iの1の(6)の記載の通りに調製を行い、泳動槽用緩衝液〔0.1%SDS-192mMグリシン-25mMトリス緩衝液〕を調製した。
前記Iの1の(10)の記載の通りに調製を行い、試料処理液〔4%SDS-12%2-メルカプトエタノール-20%グリセリン-100mMトリス緩衝液〕を調製した。
前記参考例1の(1)、(5)及び(6)で調製した、次のペリオスチン及び部分ペリオスチンをそれぞれ試料とした。
(b)部分ペリオスチン(R4領域)
(c)ペリオスチン
(d)部分ペリオスチン(Δ17・18・21)
(e)分子量マーカー〔Precision Plus Protein All Blue Standardsマーカー;マーカー分子量 10KDa、15KDa、20KDa、25KDa、37KDa、50KDa、75KDa、100KDa、150KDa及び250KDa;BIO-RAD Laboratories社、Hercules、カリフォルニア州、米国〕
前記1で調製した試薬を使用し、前記2の試料のそれぞれについて、次の操作によりSDS-ポリアクリルアミドゲル電気泳動法での電気泳動を行って、ペリオスチン及び部分ペリオスチン等の各々をその分子量に従って位置させたゲルを取得した。
なお、このゲルに注入した前記(1)の処理を行った試料及び前記2の(e)の分子量マーカーの試料であるが、レーン(a)に前記の「部分ペリオスチン(EMI領域)」を、その右側のレーン(b)に前記の「部分ペリオスチン(R4領域)」を、その右側のレーン(c)に前記の「ペリオスチン」を、その右側のレーン(d)に前記の「部分ペリオスチン(Δ17・18・21)」を、そしてレーン(a)の左側のレーン(e)に前記の「分子量マーカー」を注入した。
1.ウエスタンブロッティング
(1) 前記〔1〕の3で取得したゲルの転写を、トランスブロットSDセル(BIO-RAD Laboratories社、Hercules、カリフォルニア州、米国)を用いて、その使用説明書に従い、セミドライ方式で行った。
まず、前記〔1〕の3で取得したゲルを転写用装置上に置いた。
前記1の(9)で撮影したポリビニルジフルオライド膜を図3に示した。
すなわち、「ペリオスチン」、「部分ペリオスチン(EMI領域)」、「部分ペリオスチン(R4領域)」及び「部分ペリオスチン(Δ17・18・21)」をそれぞれ調製できていることが確認できた。
以下の手順により、抗ペリオスチンモノクローナル抗体の調製を行った。(1回目)
なお、ここで、ラットは、Wistarラット〔雌、6~8週齢〕(日本チャールズリバー社、日本国神奈川県横浜市)を用いた。
混合した前記リンパ節細胞とミエローマ細胞(Sp2/O細胞)を遠心して上清を除き、室温でポリエチレングリコール(PEG1500、Roche社、スイス国)1mLに1分間かけて懸濁した後、37℃で1分間撹拌した。
血清不含培地1mLを1分間かけて加え、その後、血清不含培地10mLを1分間かけて加えた。
細胞を数回洗浄した後、ヒポキサンチン、アミノプテリン、チミジン含有培地に懸濁して96穴マイクロタイタープレートに分注して、37℃において5%CO2存在下で培養した。
1μg/mLの前記のペリオスチンを96穴マイクロタイタープレートに分注し、数時間固相化させた。
この固相化溶液を洗浄した後、融合細胞培養上清を各ウェルに加え、1時間室温にて静置した。
このモノクローナル抗体産生細胞株を、GIT培地(日本製薬社、日本国東京都)を用いてCO2インキュベータ内37℃で培養した。
結合させたIgGを、50mMクエン酸水溶液(pH2.6)で溶出した。
前記実施例1とは別の時に、前記実施例1の(1)~(4)の記載の通りに操作を行い、再度、抗ペリオスチンモノクローナル抗体の調製を行った。(2回目)
前記実施例1及び実施例2とは別の時に、前記実施例1の(1)~(4)の記載の通りに操作を行い、三たび、抗ペリオスチンモノクローナル抗体の調製を行った。(3回目)
前記実施例1、実施例2及び実施例3とは別の時に、以下の手順により、抗ペリオスチンモノクローナル抗体の調製を行った。(4回目)
なお、ここで、マウスは、Riosらの文献(H.Riosら,Molecular and Cellular Biology,25巻,24号,11131~11144頁,2005年発行)の記載に従って得たペリオスチンノックアウトマウス(BALB/c)を用いた。
混合した前記リンパ節細胞とミエローマ細胞(Sp2/O細胞)を遠心して上清を除き、室温でポリエチレングリコール(PEG1500、Roche社、スイス国)1mLに1分間かけて懸濁した後、37℃で1分間撹拌した。
血清不含培地1mLを1分間かけて加え、その後、血清不含培地10mLを1分間かけて加えた。
1μg/mLの前記のペリオスチンを96穴マイクロタイタープレートに分注し、数時間固相化させた。
このモノクローナル抗体産生細胞株を、GIT培地(日本製薬社、日本国東京都)を用いてCO2インキュベータ内37℃で培養した。
培養後、上清中のIgGをプロテインGカラム(GE Healthcare社、Little Chalfont、英国)に結合させた。
結合させたIgGを、50mMクエン酸水溶液(pH2.6)で溶出した。
すなわち、本発明の抗体には、抗体の重鎖可変領域のアミノ酸配列が配列番号16で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が配列番号18で表されるアミノ酸配列を含む、モノクローナル抗体が含まれる。
前記実施例1、実施例2、実施例3及び実施例4とは別の時に、前記実施例4の(1)~(4)の記載の通りに操作を行い、抗ペリオスチンモノクローナル抗体の調製を行った。(5回目)
前記実施例1、実施例2、実施例3、実施例4及び実施例5とは別の時に、前記実施例1の(1)~(4)の記載の通りに操作を行い、抗ペリオスチンモノクローナル抗体の調製を行った。(6回目)
前記実施例1、実施例2、実施例3、実施例4、実施例5及び実施例6において取得した抗ペリオスチンモノクローナル抗体のそれぞれについて、ペリオスチンのどの領域を認識しているかの確認を行った。
(1) 前記参考例1の(1)~(7)で調製したペリオスチン又は部分ペリオスチン〔下記の(a)~(g)〕のそれぞれをリン酸緩衝生理食塩水により5μg/mLの濃度となるように調製したもの、又は対照としてのリン酸緩衝生理食塩水を、各々96穴マイクロタイタープレート(Thermo Fisher Scientific Inc社、イリノイ州、米国)のウェルに50μL注入し、5℃で18時間静置し、前記のペリオスチン又は部分ペリオスチンを前記マイクロタイタープレートのウェルに固相化した。
(b) 「部分ペリオスチン(R1・R2領域)」
(c) 「部分ペリオスチン(R2領域)」
(d) 「部分ペリオスチン(R1・R2・R3領域)」
(e) 「部分ペリオスチン(R4領域)」
(f) 「部分ペリオスチン(EMI領域)」
(g) 「部分ペリオスチン(C末端領域)」
(ii) 「抗ペリオスチンモノクローナル抗体(SS17B)」
(iii) 「抗ペリオスチンモノクローナル抗体(SS18A)」
(iv) 「抗ペリオスチンモノクローナル抗体(SS19A)」
(v) 「抗ペリオスチンモノクローナル抗体(SS19B)」
(vi) 「抗ペリオスチンモノクローナル抗体(SS19C)」
(vii) 「抗ペリオスチンモノクローナル抗体(SS19D)」
(viii) 「抗ペリオスチンモノクローナル抗体(SS20A)」
(ix) 「抗ペリオスチンモノクローナル抗体(SS21A)」
また、このPOD標識抗IgG抗体が「抗ペリオスチンモノクローナル抗体(SS19A)」、「抗ペリオスチンモノクローナル抗体(SS19B)」、「抗ペリオスチンモノクローナル抗体(SS19C)」、「抗ペリオスチンモノクローナル抗体(SS19D)」、又は「抗ペリオスチンモノクローナル抗体(SS20A)」である場合には、前記のマイクロタイタープレートのウェルには前記POD標識抗マウスIgG抗体を注入した。
(1) 前記1における測定の結果を図4に示した。
このことより、「抗ペリオスチンモノクローナル抗体(SS16A)」は、ペリオスチンのR3領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS17B)」は、ペリオスチンのR4領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS18A)」は、ペリオスチンのR1領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS19A)」は、ペリオスチンのR4領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS19B)」は、ペリオスチンのC末端領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS19C)」は、ペリオスチンのR2領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS19D)」は、ペリオスチンのR3領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS20A)」は、ペリオスチンのEMI領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS21A)」は、ペリオスチンのC末端領域を抗原認識部(エピトープ)として認識していることが確かめられた。
前記実施例1、実施例2、実施例3、実施例4、実施例5及び実施例6において取得した抗ペリオスチンモノクローナル抗体の各々について、ペリオスチンの一量体、多量体及び分解物それぞれとの反応性を確認した。
「抗ペリオスチンモノクローナル抗体(SS16A)」、「抗ペリオスチンモノクローナル抗体(SS17B)」、「抗ペリオスチンモノクローナル抗体(SS18A)」、「抗ペリオスチンモノクローナル抗体(SS19A)」、「抗ペリオスチンモノクローナル抗体(SS19C)」、「抗ペリオスチンモノクローナル抗体(SS19D)」、「抗ペリオスチンモノクローナル抗体(SS20A)」及び「抗ペリオスチンモノクローナル抗体(SS21A)」の各々について、ペリオスチンの一量体、多量体及び分解物それぞれとの反応性を以下の通り確かめた。
(1) 前記実施例1、実施例2、実施例3、実施例4、実施例5及び実施例6において取得した下の(a)~(h)の抗ペリオスチンモノクローナル抗体の5μgを、1種類ずつ個別に1.5mL容量のチューブに入れた。
(b) 「抗ペリオスチンモノクローナル抗体(SS17B)」
(c) 「抗ペリオスチンモノクローナル抗体(SS18A)」
(d) 「抗ペリオスチンモノクローナル抗体(SS19A)」
(e) 「抗ペリオスチンモノクローナル抗体(SS19C)」
(f) 「抗ペリオスチンモノクローナル抗体(SS19D)」
(g) 「抗ペリオスチンモノクローナル抗体(SS20A)」
(h) 「抗ペリオスチンモノクローナル抗体(SS21A)」
上清を除いた後、このチューブの中の「抗ペリオスチンモノクローナル抗体が結合したProtein G Sepharose」の20μLに、1mLのブロッキング液〔0.5%のカゼイン、100mMの塩化ナトリウム、及び0.1%のアジ化ナトリウムを含有する50mMのトリス(ヒドロキシメチル)アミノメタン緩衝液[Tris緩衝液](pH8.0)〕を加えた。
上清を除いた後、このチューブの中の「抗ペリオスチンモノクローナル抗体が結合したProtein G Sepharose」の20μLに、前記のブロッキング液により100ng/mLとなるように希釈した前記参考例2のIIの〔1〕の2の「部分ペリオスチン(Δ17・18・21)」の1mLを加えた。
なお、この「部分ペリオスチン(Δ17・18・21)」は、別途確認した結果、ペリオスチンの一量体、多量体及び分解物をそれぞれ含むことが分かっている。(以下、この「部分ペリオスチン(Δ17・18・21)」を、「ペリオスチンの一量体、多量体及び分解物の混合物」ということがある。)
ここで、このProtein G Sepharoseに結合した「抗ペリオスチンモノクローナル抗体」は、この抗体(当該抗体)が認識するペリオスチンの一量体、多量体又は分解物のいずれか一つ以上と結合する。
上清を除いた後、リン酸緩衝生理食塩水によりチューブの中のProtein G Sepharoseを洗浄することにより、Protein G Sepharoseに結合している「抗ペリオスチンモノクローナル抗体」に結合しなかった遊離のペリオスチンの一量体、多量体及び分解物(すなわち、この「抗ペリオスチンモノクローナル抗体」が認識せず結合しなかったペリオスチンの一量体、多量体又は分解物)を全て除去した。
(1)試薬
次の(a)~(c)の試薬をそれぞれ調製した。
Funakoshi Easy-Gel(III) プリキャスト・ゲル(10%)(フナコシ社、日本国東京都)を使用した。
前記参考例2のIの1の(6)の記載の通りに調製を行い、泳動槽用緩衝液〔0.1%SDS-192mMグリシン-25mMトリス緩衝液〕を調製した。
ドデシル硫酸ナトリウム〔SDS〕0.1g、2-メルカプトエタノール0.1mL、1Mのトリス(ヒドロキシメチル)アミノメタン〔Tris〕-塩酸緩衝液(pH6.8)の0.5mL、及びグリセリン2mLを純水に添加、混合した後10mLとして、試料処理液〔1%SDS-1%2-メルカプトエタノール-20%グリセリン-50mMトリス緩衝液〕を調製した。
前記1の(5)の処理を行った後の各々のチューブの中の、『「Protein G Sepharose」-「抗ペリオスチンモノクローナル抗体」-「当該抗ペリオスチンモノクローナル抗体が認識し結合するペリオスチンの一量体、多量体又は分解物」の結合物』を、それぞれ試料とした。
前記(1)で調製した試薬を使用し、前記(2)の試料のそれぞれについて、次の操作によりSDS-ポリアクリルアミドゲル電気泳動法での電気泳動を行った。
すなわち、この添加された試料処理液との混合により、「Protein G Sepharose」と、「抗ペリオスチンモノクローナル抗体」と、「当該抗ペリオスチンモノクローナル抗体が認識し結合するペリオスチンの一量体、多量体又は分解物」は、それぞれ遊離状態のものとなった。
また、前記(2)の分子量マーカーの試料の5μLを前記(b)のゲルのコウム穴に注入した。
(ii) 『「抗ペリオスチンモノクローナル抗体(SS16A)」、及び「当該抗ペリオスチンモノクローナル抗体(SS16A)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(iii) 『「抗ペリオスチンモノクローナル抗体(SS17B)」、及び「当該抗ペリオスチンモノクローナル抗体(SS17B)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(iv) 『「抗ペリオスチンモノクローナル抗体(SS18A)」、及び「当該抗ペリオスチンモノクローナル抗体(SS18A)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(v) 『「抗ペリオスチンモノクローナル抗体(SS19A)」、及び「当該抗ペリオスチンモノクローナル抗体(SS19A)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(vi) 『「抗ペリオスチンモノクローナル抗体(SS19C)」、及び「当該抗ペリオスチンモノクローナル抗体(SS19C)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(vii) 『「抗ペリオスチンモノクローナル抗体(SS19D)」、及び「当該抗ペリオスチンモノクローナル抗体(SS19D)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(viii) 『「抗ペリオスチンモノクローナル抗体(SS20A)」、及び「当該抗ペリオスチンモノクローナル抗体(SS20A)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(ix) 『「抗ペリオスチンモノクローナル抗体(SS21A)」、及び「当該抗ペリオスチンモノクローナル抗体(SS21A)が認識し結合するペリオスチンの一量体、多量体又は分解物」を含む上清』
(1) 前記2の(3)の(e)で取得したゲルの転写を、トランスブロットSDセル(BIO-RAD Laboratories社、Hercules、カリフォルニア州、米国)を用いて、その使用説明書に従い、セミドライ方式で行った。
まず、前記2の(3)の(e)で取得したゲルを転写用装置上に置いた。
(1) 前記3の(9)で撮影したポリビニルジフルオライド膜を図5に示した。
「抗ペリオスチンモノクローナル抗体(SS19B)」について、ペリオスチンの一量体、多量体及び分解物それぞれとの反応性を以下の通り確かめた。
(1)試薬
次の(a)~(c)の試薬をそれぞれ調製した。
Funakoshi Easy-Gel(III) プリキャスト・ゲル(10%)(フナコシ社、日本国東京都)を使用した。
前記参考例2のIの1の(6)の記載の通りに調製を行い、泳動槽用緩衝液〔0.1%SDS-192mMグリシン-25mMトリス緩衝液〕を調製した。
前記〔1〕の2の(1)の(c)の記載の通りに調製を行い、試料処理液〔1%SDS-1%2-メルカプトエタノール-20%グリセリン-50mMトリス緩衝液〕を調製した。
前記参考例2のIIの〔1〕の2の「部分ペリオスチン(Δ17・18・21)」、すなわち「ペリオスチンの一量体、多量体及び分解物の混合物」の50ngを標準試料として用いた。
前記の通り、この「ペリオスチンの一量体、多量体及び分解物の混合物」(「部分ペリオスチン(Δ17・18・21)」)は、ペリオスチンの一量体、多量体及び分解物をそれぞれ含むことが分かっている。
前記(1)で調製した試薬を使用し、前記(2)の試料のそれぞれについて、次の操作によりSDS-ポリアクリルアミドゲル電気泳動法での電気泳動を行った。
また、前記(2)の分子量マーカーの試料の5μLを前記(b)のゲルのコウム穴に注入した。
なお、このゲルに注入した前記の上清及び分子量マーカーであるが、向かって左側のレーンより、次の(i)~(ii)の分子量マーカー、及び『「ペリオスチンの一量体、多量体及び分解物」を含む上清』を順に注入した。
(ii) 『「ペリオスチンの一量体、多量体及び分解物」を含む上清』
(1) 実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19B)」をSulfo-NHS-LC-Biotin(Thermo Fisher Scientific Inc社、イリノイ州、米国;商品コード番号:21335)を用いてビオチン標識処理を行い、得られたビオチンで標識した「抗ペリオスチンモノクローナル抗体(SS19B)」を前記の標識抗体希釈液(アジ化ナトリウム含有)により5μg/mLとなるように希釈し、ビオチンで標識した「抗ペリオスチンモノクローナル抗体(SS19B)」の溶解液を調製した。
(1) 前記2で得たポリビニルジフルオライド膜を図6に示した。
しかし、前記のペリオスチンの一量体を表すバンドよりも低分子量側には発色(バンド)は認められない。すなわち、ペリオスチンの分解物を示す位置においては発色は認められない。
よって、「抗ペリオスチンモノクローナル抗体(SS19B)」はペリオスチンの多量体及び一量体を認識し結合するが、ペリオスチンの分解物は認識せず、結合しないことが確かめられた。
前記実施例1、実施例2、実施例3、実施例4、実施例5及び実施例6において取得した各々の抗ペリオスチンモノクローナル抗体について、ペリオスチンの一量体、多量体及び分解物それぞれとの反応性をまとめたものを図7に示す。
・ 「抗ペリオスチンモノクローナル抗体(SS17B)」
・ 「抗ペリオスチンモノクローナル抗体(SS19A)」
・ 「抗ペリオスチンモノクローナル抗体(SS19B)」
・ 「抗ペリオスチンモノクローナル抗体(SS21A)」
・ 「抗ペリオスチンモノクローナル抗体(SS16A)」
・ 「抗ペリオスチンモノクローナル抗体(SS18A)」
・ 「抗ペリオスチンモノクローナル抗体(SS19C)」
・ 「抗ペリオスチンモノクローナル抗体(SS19D)」
・ 「抗ペリオスチンモノクローナル抗体(SS20A)」
・ 「抗ペリオスチンモノクローナル抗体(SS19D)」
ヒト血清中のペリオスチンを、免疫沈降、電気泳動及びウエスタンブロッティングの処理を行うことによって確認した。
健常者の血清のペリオスチンについて、以下の通り確かめた。
(1) 1.5mL容量のチューブに、ベッド容量で100μLのNHS-activated Sepharose(GE Healthcare社、Little Chalfont、英国)を入れ、遠心分離操作を行い上清を除去した。
上清を除いた後、このチューブの中の前記「抗ペリオスチンモノクローナル抗体(SS19C)」が結合したNHS-activated Sepharoseに、1mLのブロッキング液〔0.5M 2-アミノエタノール-0.5M塩化ナトリウム(pH8.3)〕を加えた。
上清を除いた後、このチューブに1mLの20%エタノールを入れて、4℃に18時間保存した。
上清を除いた後、洗浄液〔10mMトリス(ヒドロキシメチル)アミノメタン[Tris]-150mM塩化ナトリウム-0.05%Tween20(pH8.0)〕によりチューブ中のNHS-activated Sepharoseを3回洗浄し、『NHS-activated Sepharoseに結合した「抗ペリオスチンモノクローナル抗体(SS19C)」』に結合しなかった遊離のペリオスチン(一量体、多量体又は分解物)を全て除去した。
(1)試薬
次の(a)~(h)の試薬をそれぞれ調製した。
前記参考例2のIの1の(1)の記載の通りに調製を行い、アクリルアミド溶液〔30%アクリルアミド保存液〕を調製した。
前記参考例2のIの1の(2)の記載の通りに調製を行い、SDS-1.5Mトリス溶液〔0.4%SDS-1.5Mトリス塩酸緩衝液〕を調製した。
前記参考例2のIの1の(3)の記載の通りに調製を行い、SDS-0.5Mトリス溶液〔0.4%SDS-0.5Mトリス塩酸緩衝液〕を調製した。
前記参考例2のIの1の(4)の記載の通りに調製を行い、過硫酸アンモニウム溶液〔10%過硫酸アンモニウム水溶液〕を調製した。
前記参考例2のIの1の(5)の記載の通り、N,N,N’,N’-テトラメチルエチレンジアミン(ナカライテスク社、日本国京都府)を使用した。
前記参考例2のIの1の(6)の記載の通りに調製を行い、泳動槽用緩衝液〔0.1%SDS-192mMグリシン-25mMトリス緩衝液〕を調製した。
ドデシル硫酸ナトリウム〔SDS〕0.4g、2-メルカプトエタノール(還元剤)1.2mL、1Mのトリス(ヒドロキシメチル)アミノメタン〔Tris〕-塩酸緩衝液(pH6.8)の1mL、及びグリセリン2mLを純水に添加、混合した後10mLとして、試料処理液(還元剤含有)〔4%SDS-12%2-メルカプトエタノール-20%グリセリン-100mMトリス緩衝液〕を調製した。
ドデシル硫酸ナトリウム〔SDS〕0.4g、1Mのトリス(ヒドロキシメチル)アミノメタン〔Tris〕-塩酸緩衝液(pH6.8)の1mL、及びグリセリン2mLを純水に添加、混合した後10mLとして、試料処理液(還元剤不含有)〔1%SDS-20%グリセリン-100mMトリス緩衝液〕を調製した。
前記1の(8)の処理を行った後のそれぞれのチューブ(健常者の血清量が25μL、50μL又は100μLのものの各々)の中の『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』を次の通りそれぞれ試料とした。
前記の通り、この「ペリオスチンの一量体、多量体及び分解物の混合物」(「部分ペリオスチン(Δ17・18・21)」)は、ペリオスチンの一量体、多量体及び分解物をそれぞれ含むことが分かっている。
(b) 『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』 (健常者の血清量:50μL)
(c) 『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』 (健常者の血清量:25μL)
(d) 標準試料(ペリオスチンの一量体、多量体及び分解物の混合物)
(e) 分子量マーカー〔マーカー分子量 10KDa、15KDa、20KDa、25KDa、37KDa、50KDa、75KDa、100KDa、150KDa及び250KDa〕
前記(1)で調製した試薬を使用し、前記(2)の試料のそれぞれについて、次の操作によりSDS-ポリアクリルアミドゲル電気泳動法での電気泳動を行った。
この分離ゲル溶液を、組み立てたガラスプレートに注入した後、純水を上に重層し、そしてゲル化反応を30分間行わせた。
前記(a)のガラスプレートより純水を捨て、ここにこの濃縮ゲル溶液を少量注入し洗浄した後、濃縮ゲル溶液を注入した。そして、ここにサンプルコウムを挿入し、そしてゲル化反応を30分間行わせた。
すなわち、この添加された試料処理液との混合により、「NHS-activated Sepharose」と、「抗ペリオスチンモノクローナル抗体(SS19C)」と、「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」は、それぞれ遊離状態のものとなった。
また、前記(2)の(e)の分子量マーカーの試料の5μLを前記(d)のゲルのコウム穴に注入した。
なお、このゲルに注入した前記の上清及び分子量マーカーであるが、向かって左側のレーンより、次の(i)~(v)の各「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の上清、標準試料(ペリオスチンの一量体、多量体及び分解物の混合物)の上清、及び分子量マーカーを順に注入した。
(ii) 「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」(健常者の血清量:50μL)の上清
(iii) 「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」(健常者の血清量:25μL)の上清
(iv) 標準試料(ペリオスチンの一量体、多量体及び分解物の混合物)の上清
(v) 分子量マーカー
(1) 前記2の(3)の(g)及び(h)でそれぞれ取得したゲルについて、その転写をMini Protean II Cellキット(BIO-RAD Laboratories社、Hercules、カリフォルニア州、米国)を用いて、その使用説明書に従い、ウェット方式で行った。
まず、前記2の(3)の(g)及び(h)でそれぞれで取得したゲルを転写用装置上に置いた。
そして、このパーオキシダーゼ標識抗マウスIgG抗体溶液に、前記(4)のそれぞれのポリビニルジフルオライド膜を室温で45分間浸漬して反応させた。
(1) 前記3の(9)で得たそれぞれのポリビニルジフルオライド膜の写真を図8に示した。
肺線維症罹患者の血清のペリオスチンについて、以下の通り確かめた。
前記〔1〕の1の(7)における「一人の健常者の血清の25μL、50μL又は100μLを個別にそれぞれのチューブに加えた」ことを、「三人の肺線維症罹患者それぞれの血清及び一人の健常者の血清の1mLを個々にそれぞれのチューブ(1.5mL容量)に加えた」ことに代えること以外は、前記〔1〕の1の(1)~(8)の記載の通りに操作を行い、これらの肺線維症罹患者又は健常者の血清に含まれているペリオスチン(一量体、多量体又は分解物)に関し、『「NHS-activated Sepharoseに結合した前記抗ペリオスチンモノクローナル抗体(SS19C)」と「試料(肺線維症罹患者又は健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」との結合物』、すなわち『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(肺線維症罹患者又は健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』をそれぞれ調製した。
(1)試薬
前記〔1〕の2の(1)における(a)~(h)の記載の通りに各試薬をそれぞれ調製した。
前記1において調製した『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(肺線維症罹患者又は健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』を次の通りそれぞれ試料とした。
前記の通り、この「ペリオスチンの一量体、多量体及び分解物の混合物」(「部分ペリオスチン(Δ17・18・21)」)は、ペリオスチンの一量体、多量体及び分解物をそれぞれ含むことが分かっている。
(b) 『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(一人目の肺線維症罹患者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』
(c) 『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(二人目の肺線維症罹患者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』
(d) 『「NHS-activated Sepharose」-「抗ペリオスチンモノクローナル抗体(SS19C)」-「試料(三人目の肺線維症罹患者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の結合物』
(e) 標準試料(ペリオスチンの一量体、多量体及び分解物の混合物)
(f) 分子量マーカー〔マーカー分子量 10KDa、15KDa、20KDa、25KDa、37KDa、50KDa、75KDa、100KDa、150KDa及び250KDa〕
前記(1)で調製した試薬を使用し、前記(2)の試料のそれぞれについて、SDS-ポリアクリルアミドゲル電気泳動法での電気泳動を行った。
(b) 前記〔1〕の2の(3)の(e)において(i)~(iii)の各「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の上清、(iv)の標準試料(ペリオスチンの一量体、多量体及び分解物の混合物)の上清、並びに(v)の分子量マーカーの順に、向かって左側のレーンよりゲルのコウム穴に注入したことに代えて、分子量マーカー、「試料(健常者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の上清、「試料(一人目の肺線維症罹患者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の上清、「試料(二人目の肺線維症罹患者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の上清、「試料(三人目の肺線維症罹患者の血清)に含まれていたペリオスチンの一量体、多量体又は分解物」の上清、並びに標準試料(ペリオスチンの一量体、多量体及び分解物の混合物)の上清の順に、向かって左側のレーンよりゲルのコウム穴に注入したこと。
前記〔1〕の3の(1)~(9)の記載の通りに操作を行い、前記2でSDS-ポリアクリルアミドゲル電気泳動法により電気泳動を行い取得したゲルについて、ポリビニルジフルオライド膜に転写を行い、ペリオスチン(一量体、多量体又は分解物)の分子量に応じた位置に発光を生じさせ、このポリビニルジフルオライド膜上の発光の有無及びその発光した位置(分子量)より、前記の肺線維症罹患者及び健常者の血清に含まれるペリオスチン(一量体、多量体又は分解物)の存在を確認した。
(1) 前記3で得たポリビニルジフルオライド膜を図9に示した。
ヒト血清中のペリオスチンの測定を行い、本発明の抗体、測定方法、測定試薬、正確性の改善方法及び肺線維症又は間質性肺炎の検査方法の効果を確かめた。
従来の技術により、ヒト血清中のペリオスチンの測定を以下の通り行なった。
1.試料
次の(1)~(3)のヒト血清をそれぞれ試料とした。
(2)間質性肺炎〔膠原病由来〕罹患者(計40例)の血清
(3)健常者(計66例)の血清
各試料に含まれるペリオスチンの濃度を、抗ペリオスチンモノクローナル抗体を使用した酵素免疫測定法(ELISA法)により、以下の通り測定し、求めた。
これにより、試料に含まれるペリオスチンを、前記のウェルに固相化した「抗ペリオスチンモノクローナル抗体(SS18A)」に結合させた。
本発明により、ヒト血清中のペリオスチンの測定を以下の通り行なった。
1.試料
前記〔1〕の1の(1)~(3)のヒト血清をそれぞれ試料とした。
前記〔1〕の2の(6)におけるPOD標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19C)」(ペリオスチンのR2領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とする以外は、前記〔1〕の2の(1)~(12)の記載の通りに測定を行い、各試料に含まれていたペリオスチンの濃度を求めた。
本発明により、ヒト血清中のペリオスチンの測定を以下の通り行なった。
1.試料
前記〔1〕の1の(1)~(3)のヒト血清をそれぞれ試料とした。
各試料に含まれるペリオスチンの濃度を、抗ペリオスチンモノクローナル抗体を使用した酵素免疫測定法(ELISA法)により、以下の通り測定し、求めた。
これにより、試料に含まれるペリオスチンを、前記のウェルに固相化した抗ペリオスチンモノクローナル抗体に結合させた。
(1) 前記の〔1〕における「従来技術による測定」の測定結果、〔2〕における「本発明による測定-A」の測定結果、及び〔3〕における「本発明による測定-B」の測定結果を、それぞれ図10に示した。
ヒト血清中のペリオスチンの測定を行い、その測定結果のROC曲線(受信者動作特性曲線;Receiver Operating Characteristic Curve)を作成し、解析を行なった。
次の(1)及び(2)のヒト血清をそれぞれ試料とした。
肺線維症罹患者(計37例)の血清、及び間質性肺炎〔膠原病由来〕罹患者(計40例)の血清を、陽性試料として用いた。
健常者(計66例)の血清を、陰性試料として用いた。
各試料に含まれるペリオスチンの濃度を、抗ペリオスチンモノクローナル抗体を使用した酵素免疫測定法(ELISA法)により、以下の通り測定し、求めた。
(a) 前記実施例3で取得した「抗ペリオスチンモノクローナル抗体(SS18A)」(ペリオスチンのR1領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)を、リン酸緩衝生理食塩水(PBS)〔137mMの塩化ナトリウム、2.68mMの塩化カリウム、1.47mMのリン酸二水素カリウム、及び8.04mMのリン酸水素二ナトリウムを含有する水溶液(pH7.4)〕により2μg/mLとなるように希釈し、これを96穴マイクロタイタープレート(Thermo Fisher Scientific Inc社、イリノイ州、米国)の各ウェルに100μLずつ注入した後、25℃で18時間静置し、「抗ペリオスチンモノクローナル抗体(SS18A)」をこのマイクロタイタープレートの各ウェルに固相化した。
これにより、試料に含まれるペリオスチンを、前記のウェルに固相化した「抗ペリオスチンモノクローナル抗体(SS18A)」に結合させた。
前記1の(1)の陽性試料及び前記1の(2)の陰性試料それぞれについての前記(m)におけるペリオスチン濃度の測定値より、ROC曲線を作成した。
前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19C)」(ペリオスチンのR2領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること以外は、前記(1)の(a)~(n)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求め、そしてROC曲線を作成した。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすることの二つのこと以外は、前記(1)の(a)~(n)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求め、そしてROC曲線を作成した。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすること以外は、前記(1)の(a)~(n)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求め、そしてROC曲線を作成した。
ペリオスチンの分解物のみを測定することができる測定系のROC曲線を計算により求め、作成した。
(1) 前記の通り、肺線維症罹患者及び間質性肺炎〔膠原病由来〕罹患者の血清からなる陽性試料、及び健常者の血清からなる陰性試料それぞれに含まれるペリオスチンの濃度の測定結果等より作成したROC曲線の全てを図11に示した。
ヒト血清中のペリオスチンの測定を行い、本発明の抗体、測定方法、測定試薬、及び正確性の改善方法の効果を確かめた。
次の(1)及び(2)のヒト血清をそれぞれ試料とした。
肺線維症罹患者(計16例)の血清、及び間質性肺炎〔膠原病由来〕罹患者(計10例)の血清を、陽性試料として用いた。
健常者(計54例)の血清を、陰性試料として用いた。
各試料に含まれるペリオスチンの濃度を、抗ペリオスチンモノクローナル抗体を使用した酵素免疫測定法(ELISA法)により、以下の通り測定し、求めた。
(a) 前記実施例3で取得した「抗ペリオスチンモノクローナル抗体(SS18A)」(ペリオスチンのR1領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)を、リン酸緩衝生理食塩水(PBS)〔137mMの塩化ナトリウム、2.68mMの塩化カリウム、1.47mMのリン酸二水素カリウム、及び8.04mMのリン酸水素二ナトリウムを含有する水溶液(pH7.4)〕により2μg/mLとなるように希釈し、これを96穴マイクロタイタープレート(Thermo Fisher Scientific Inc社、イリノイ州、米国)の各ウェルに100μLずつ注入した後、25℃で18時間静置し、「抗ペリオスチンモノクローナル抗体(SS18A)」をこのマイクロタイタープレートの各ウェルに固相化した。
これにより、試料に含まれるペリオスチンを、前記のウェルに固相化した「抗ペリオスチンモノクローナル抗体(SS18A)」に結合させた。
前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19C)」(ペリオスチンのR2領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすること以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19A)」(ペリオスチンのR4領域を認識し結合する抗体;ペリオスチンの一量体及び多量体を認識し結合することができるものの、ペリオスチンの分解物を認識し結合することはできない抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例6で取得した「抗ペリオスチンモノクローナル抗体(SS21A)」(ペリオスチンのC末端領域を認識し結合する抗体;ペリオスチンの一量体及び多量体を認識し結合することができるものの、ペリオスチンの分解物を認識し結合することはできない抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例1で取得した「抗ペリオスチンモノクローナル抗体(SS16A)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例3で取得した「抗ペリオスチンモノクローナル抗体(SS18A)」(ペリオスチンのR1領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19B)」(ペリオスチンのC末端領域を認識し結合する抗体;ペリオスチンの一量体及び多量体を認識し結合することができるものの、ペリオスチンの分解物を認識し結合することはできない抗体)とすること以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19C)」(ペリオスチンのR2領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19C)」(ペリオスチンのR2領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例6で取得した「抗ペリオスチンモノクローナル抗体(SS21A)」(ペリオスチンのC末端領域を認識し結合する抗体;ペリオスチンの一量体及び多量体を認識し結合することができるものの、ペリオスチンの分解物を認識し結合することはできない抗体)とすることの二つのこと以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例6で取得した「抗ペリオスチンモノクローナル抗体(SS21A)」(ペリオスチンのC末端領域を認識し結合する抗体;ペリオスチンの一量体及び多量体を認識し結合することができるものの、ペリオスチンの分解物を認識し結合することはできない抗体)とすること以外は、前記(1)の(a)~(m)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
(1) 前記2の(1)~(3)における測定結果を図13に、前記2の(4)~(6)における測定結果を図14に、前記2の(7)~(9)における測定結果を図15に、前記2の(10)~(12)における測定結果を図16に、そして前記2の(13)における測定結果を図17に示した。
ペリオスチン濃度が分かっている試料についてペリオスチンの測定を行い、標準曲線(検量線)を作成した。
前記参考例2のIIの〔1〕の2の「部分ペリオスチン(Δ17・18・21)」(すなわち、「ペリオスチンの一量体、多量体及び分解物の混合物」であって、ペリオスチンの一量体、多量体及び分解物をそれぞれ含むことが分かっている。)を、試料希釈液〔0.5%のカゼイン、100mMの塩化ナトリウム、及び0.1%のアジ化ナトリウムを含有する50mMのトリス(ヒドロキシメチル)アミノメタン緩衝液[Tris緩衝液](pH8.0)〕で希釈して、次の(2)~(6)のペリオスチン濃度が分かっている標準試料をそれぞれ調製し、試料とした。
また、ペリオスチンを含有しない試料希釈液〔0.5%のカゼイン、100mMの塩化ナトリウム、及び0.1%のアジ化ナトリウムを含有する50mMのトリス(ヒドロキシメチル)アミノメタン緩衝液[Tris緩衝液](pH8.0)〕を、次の(1)のペリオスチン濃度が0ng/mLの試料(標準試料)とした。
(2)標準試料-2 (ペリオスチン濃度:0.125ng/mL)
(3)標準試料-3 (ペリオスチン濃度:0.25ng/mL)
(4)標準試料-4 (ペリオスチン濃度:0.5ng/mL)
(5)標準試料-5 (ペリオスチン濃度:1.0ng/mL)
(6)標準試料-6 (ペリオスチン濃度:2.0ng/mL)
各試料に含まれるペリオスチンの濃度を、抗ペリオスチンモノクローナル抗体を使用した酵素免疫測定法(ELISA法)により測定し、求めた。
(b) 前記実施例12の2の(1)の(l)及び(m)における操作に代えて、前記実施例12の2の(1)の(k)において測定した各標準試料の吸光度より「ペリオスチン濃度-吸光度」の標準曲線(検量線)を作成したこと
(2)本発明による測定-1
(3)本発明による測定-2
(4)従来技術による測定-2
(5)従来技術による測定-3
(6)従来技術による測定-4
(7)本発明による測定-3
(8)本発明による測定-4
(9)従来技術による測定-5
(10)本発明による測定-5
(11)本発明による測定-6
(12)従来技術による測定-6
(13)従来技術による測定-7
(1) 前記2における測定結果を図19、図20、図21、図22、図23、図24及び図25に分けて示した。
前記実施例1、実施例2、実施例3、実施例4、実施例5及び実施例6とは別の時に、前記実施例1の(1)~(4)の記載の通りに操作を行い、抗ペリオスチンモノクローナル抗体の調製を行った。(7回目)
前記実施例1、実施例2、実施例3、実施例4、実施例5、実施例6及び実施例14とは別の時に、前記実施例1の(1)~(4)の記載の通りに操作を行い、抗ペリオスチンモノクローナル抗体の調製を行った。(8回目)
前記実施例14及び実施例15において取得した抗ペリオスチンモノクローナル抗体のそれぞれについて、前記実施例7の1の(1)~(8)の記載の通りに操作を行い、これらの抗ペリオスチンモノクローナル抗体がペリオスチンのどの領域を認識しているかの確認を行った。
このことより、「抗ペリオスチンモノクローナル抗体(SS25A)」は、ペリオスチンのR2領域を抗原認識部(エピトープ)として認識していることが確かめられた。
このことより、「抗ペリオスチンモノクローナル抗体(SS27A)」は、ペリオスチンのR1領域を抗原認識部(エピトープ)として認識していることが確かめられた。
前記実施例14及び実施例15において取得した抗ペリオスチンモノクローナル抗体の各々について、ペリオスチンの一量体、多量体及び分解物それぞれとの反応性を確認した。
(1) 前記実施例14において取得した「抗ペリオスチンモノクローナル抗体(SS25A)」、及び前記実施例15において取得した「抗ペリオスチンモノクローナル抗体(SS27A)」のそれぞれについて、前記実施例8の〔1〕の1~3の記載の通りに、免疫沈降処理、SDS-ポリアクリルアミドゲル電気泳動法、及びウエスタンブロッティング法の各々の操作を行った。
そして、この発色させたポリビニルジフルオライド膜を撮影した。
このポリビニルジフルオライド膜上の発色の有無及びその発色した位置(分子量)より、前記の各々の抗ペリオスチンモノクローナル抗体について、ペリオスチンの一量体、多量体及び分解物のそれぞれとの反応性を確認した。
(1) 前記1の(2)において撮影したポリビニルジフルオライド膜を図26に示した。
・ 「抗ペリオスチンモノクローナル抗体(SS25A)」
・ 「抗ペリオスチンモノクローナル抗体(SS27A)」
ヒト血清中のペリオスチンの測定を行い、本発明の効果を確かめ、そして、測定結果のROC曲線(受信者動作特性曲線;Receiver Operating Characteristic Curve)を作成し、解析を行なった。
1.試料
次の(1)及び(2)のヒト血清をそれぞれ試料とした。
肺線維症罹患者(計20例)の血清、及び間質性肺炎〔膠原病由来〕罹患者(計19例)の血清を、陽性試料として用いた。
健常者(計64例)の血清を、陰性試料として用いた。
各試料に含まれるペリオスチンの濃度を、抗ペリオスチンモノクローナル抗体を使用した酵素免疫測定法(ELISA法)により、以下の通り測定し、求めた。
前記実施例11の2の(1)の(a)~(n)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求め、そしてROC曲線を作成した。
前記実施例11の2の(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例4で取得した「抗ペリオスチンモノクローナル抗体(SS19D)」(ペリオスチンのR3領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすることの二つのこと以外は、前記実施例11の2の(1)の(a)~(n)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求め、そしてROC曲線を作成した。
前記実施例11の2の(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例14で取得した「抗ペリオスチンモノクローナル抗体(SS25A)」(ペリオスチンのR2領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすることの二つのこと以外は、前記実施例11の2の(1)の(a)~(n)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求め、そしてROC曲線を作成した。
前記実施例11の2の(1)の(a)におけるマイクロタイタープレートのウェルに固相化した抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS18A)」に代えて、前記実施例15で取得した「抗ペリオスチンモノクローナル抗体(SS27A)」(ペリオスチンのR1領域を認識し結合する抗体;ペリオスチンの一量体及び分解物を認識し結合することができるものの、ペリオスチンの多量体を認識し結合することはできない抗体)とすること、及び前記(1)の(f)におけるビオチン標識抗ペリオスチンモノクローナル抗体の抗ペリオスチンモノクローナル抗体を、「抗ペリオスチンモノクローナル抗体(SS17B)」に代えて、前記実施例5で取得した「抗ペリオスチンモノクローナル抗体(SS20A)」(ペリオスチンのEMI領域を認識し結合する抗体;ペリオスチンの一量体、多量体及び分解物のいずれについても認識し結合する抗体)とすることの二つのこと以外は、前記実施例11の2の(1)の(a)~(n)の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求め、そしてROC曲線を作成した。
(1) 前記の通り、肺線維症罹患者及び間質性肺炎〔膠原病由来〕罹患者の血清からなる陽性試料、及び健常者の血清からなる陰性試料それぞれに含まれるペリオスチンの濃度の測定結果等より作成したROC曲線の全てを図27に示した。
1.試料
次の(1)及び(2)のヒト血清をそれぞれ試料とした。
前記〔1〕の1の(1)と同じ肺線維症罹患者(計20例)の血清、及び間質性肺炎〔膠原病由来〕罹患者(計19例)の血清を、陽性試料として用いた。
前記〔1〕の1の(2)と同じ健常者(計64例)の血清を、陰性試料として用いた。
<1>従来技術による測定
前記〔1〕の2の<1>の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記〔1〕の2の<2>の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記〔1〕の2の<3>の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
前記〔1〕の2の<4>の記載の通りに測定及び処理を行い、各試料に含まれていたペリオスチンの濃度を求めた。
(1) 前記2の<1>~<4>の各々の測定における、使用した固相化抗体及び標識抗体、前記固相化抗体により検出されるペリオスチンの領域、前記標識抗体により検出されるペリオスチンの領域、当該測定により測定することができるペリオスチンが一量体、多量体又は分解物のいずれであるか、測定の感度、測定の特異度、陰性試料の測定値の平均値に対する陽性試料の測定値の平均値の比率(「陽性試料の測定値の平均値」÷「陰性試料の測定値の平均値」;すなわち、陰性試料の測定値に対する陽性試料の測定値の上昇度)、並びに「作成したROC曲線のAUCの値」を、図28に示した。
Claims (22)
- ペリオスチン又はその分解物のEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に結合する抗体。
- ペリオスチンの分解物に結合する抗体である、請求項1に記載の抗体。
- ペリオスチンの多量体に結合しない抗体である、請求項1又は2に記載の抗体。
- 抗体がモノクローナル抗体である、請求項1~3のいずれか1項に記載の抗体。
- 抗体の重鎖可変領域のアミノ酸配列が、配列番号16で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号18で表されるアミノ酸配列を含むものである、請求項4に記載の抗体。
- 受託番号がNITE BP-1281であるハイブリドーマSS16A株、受託番号がNITE BP-1282であるハイブリドーマSS18A株、受託番号がNITE BP-1283であるハイブリドーマSS19C株、受託番号がNITE BP-1068であるハイブリドーマSS19D株、受託番号がNITE BP-1284であるハイブリドーマSS20A株、受託番号がNITE BP-1285であるハイブリドーマSS25A株及び受託番号がNITE BP-1286であるハイブリドーマSS27A株からなる群から選ばれるいずれかのハイブリドーマにより産生されるモノクローナル抗体。
- 試料に含まれるペリオスチンの測定方法であって、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを特徴とする、前記方法。
- 請求項1~6のいずれか1項に記載の抗体を使用することを特徴とする、請求項7に記載の方法。
- ペリオスチンがペリオスチン分解物である、請求項7又は8に記載の方法。
- ペリオスチンが多量体ではない、請求項7~9のいずれか1項に記載の方法。
- ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域に特異的に結合する物質を含む、試料に含まれるペリオスチン又はその分解物の測定試薬。
- 前記物質が請求項1~6のいずれか1項に記載の抗体である、請求項11に記載の試薬。
- ペリオスチンがペリオスチン分解物である、請求項11又は12に記載の試薬。
- ペリオスチンが多量体ではない、請求項11~13のいずれか1項に記載の試薬。
- 試料に含まれるペリオスチンの量又は濃度の測定において、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを特徴とする、ペリオスチン測定の正確性の改善方法。
- 請求項1~6のいずれか1項に記載の抗体を使用することを特徴とする、請求項15に記載の方法。
- ペリオスチンがペリオスチン分解物である、請求項15又は16に記載の方法。
- ペリオスチンが多量体ではない、請求項15~17のいずれか1項に記載の方法。
- 次の工程を含む、肺線維症又は間質性肺炎の検査方法。
a) 被検者由来の試料におけるペリオスチンの量又は濃度を測定する工程であって、ペリオスチンのEMI領域、R1領域、R2領域及びR3領域からなる群から選ばれる少なくとも一つの領域を検出することを含む工程
b) 被検者由来の試料におけるペリオスチンの量又は濃度と、肺線維症又は間質性肺炎のいずれにも罹患していない生体由来の試料におけるペリオスチンの量又は濃度とを比較する工程 - 請求項1~6のいずれか1項に記載の抗体を使用することを特徴とする、請求項19に記載の方法。
- ペリオスチンがペリオスチン分解物である、請求項19又は20に記載の方法。
- ペリオスチンが多量体ではない、請求項19~21のいずれか1項に記載の方法。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/342,996 US9347954B2 (en) | 2011-09-06 | 2012-09-06 | Antibody capable of binding to specific region of periostin, and method of measuring periostin using the same |
EP12829373.5A EP2754672B1 (en) | 2011-09-06 | 2012-09-06 | Antibody capable of binding to specific region of periostin, and method of measuring periostin using the same |
JP2013532647A JP6183809B2 (ja) | 2011-09-06 | 2012-09-06 | ペリオスチンの特定領域に結合する抗体及びこれを用いたペリオスチンの測定方法 |
US15/143,196 US20160313351A1 (en) | 2011-09-06 | 2016-04-29 | Antibody capable of binding to specific region of periostin, and method for measuring periostin using same |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2011-194323 | 2011-09-06 | ||
JP2011194323 | 2011-09-06 | ||
JP2012077774 | 2012-03-29 | ||
JP2012-077774 | 2012-03-29 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/342,996 A-371-Of-International US9347954B2 (en) | 2011-09-06 | 2012-09-06 | Antibody capable of binding to specific region of periostin, and method of measuring periostin using the same |
US15/143,196 Continuation US20160313351A1 (en) | 2011-09-06 | 2016-04-29 | Antibody capable of binding to specific region of periostin, and method for measuring periostin using same |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013035799A1 true WO2013035799A1 (ja) | 2013-03-14 |
Family
ID=47832239
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2012/072774 WO2013035799A1 (ja) | 2011-09-06 | 2012-09-06 | ペリオスチンの特定領域に結合する抗体及びこれを用いたペリオスチンの測定方法 |
Country Status (4)
Country | Link |
---|---|
US (2) | US9347954B2 (ja) |
EP (1) | EP2754672B1 (ja) |
JP (1) | JP6183809B2 (ja) |
WO (1) | WO2013035799A1 (ja) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015120185A1 (en) * | 2014-02-07 | 2015-08-13 | Medimmune, Llc | Novel assay to detect human periostin |
US9684000B2 (en) | 2010-12-16 | 2017-06-20 | Genentech, Inc. | Diagnosis and treatments relating to TH2 inhibition |
WO2018030456A1 (ja) * | 2016-08-10 | 2018-02-15 | 株式会社シノテスト | 試料に含まれるペリオスチンの測定試薬、ペリオスチン測定用前処理剤、ペリオスチン測定方法及びペリオスチン測定の感度の改善方法 |
WO2018070397A1 (ja) * | 2016-10-12 | 2018-04-19 | 株式会社シノテスト | ペリオスチンの吸着防止剤及び吸着防止方法 |
JP2020186175A (ja) * | 2019-05-10 | 2020-11-19 | 株式会社シノテスト | 免疫グロブリンaに結合しているペリオスチン並びに免疫グロブリンaに結合しているペリオスチンに結合する抗体、ペリオスチンの測定方法、ペリオスチンの測定試薬及びペリオスチン測定の正確性の改善方法 |
CN113687168A (zh) * | 2021-08-30 | 2021-11-23 | 北京航空航天大学 | 一种10kHz-18GHz电场辐射发射的系统电磁兼容性指标分解方法 |
WO2023027186A1 (ja) * | 2021-08-26 | 2023-03-02 | 国立大学法人大阪大学 | 肺線維化疾患バイオマーカー |
WO2023036305A1 (zh) * | 2021-09-13 | 2023-03-16 | 沈阳眼产业技术研究院有限公司 | 抗periostin人源化单克隆抗体及其制备方法与应用 |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05268982A (ja) | 1992-03-27 | 1993-10-19 | Hoechst Japan Ltd | 骨形成作用を有する新規な蛋白質およびその製造法 |
JPH09229936A (ja) | 1996-02-26 | 1997-09-05 | Shinotesuto:Kk | 磁性粒子を用いる被検物質の測定方法並びに該方法に使用する担体及び測定器具 |
JPH10132819A (ja) | 1996-10-25 | 1998-05-22 | Shinotesuto:Kk | 粒子を用いる被検物質の測定方法及び該方法に使用する測定器具 |
WO1998023963A1 (en) | 1996-11-27 | 1998-06-04 | Thrombosis And Coagulation Aktiebolag (T.A.C. Ab) | Methods and reagents for determining protein s |
JP2002502250A (ja) | 1997-05-29 | 2002-01-22 | ボード・オヴ・リージェンツ,ザ・ユニヴァーシティ・オヴ・テキサス・システム | Osf2/cbfa1組成物および使用の方法 |
WO2002052006A1 (en) | 2000-12-26 | 2002-07-04 | Genox Research, Inc. | Method of examining allergic disease |
JP2005500059A (ja) | 2001-08-13 | 2005-01-06 | ダナ−ファーバー キャンサー インスティテュート インク. | ペリオスチンに基づく診断アッセイ法 |
WO2007077934A1 (ja) | 2005-12-28 | 2007-07-12 | Asubio Pharma Co., Ltd. | 抗ペリオスチン抗体およびそれを含有するペリオスチンが関与する疾患の予防または治療用医薬組成物 |
WO2009148184A1 (ja) | 2008-06-05 | 2009-12-10 | 国立大学法人佐賀大学 | 特発性間質性肺炎の検出方法 |
WO2010139768A1 (fr) * | 2009-06-03 | 2010-12-09 | Synarc Sas | Sequences, anticorps, procedes et necessaires pour la detection et le dosage in vitro de la periostine, en vue du diagnostic du suivi ou du pronostic de pathologies ou de phenomenes biologiques impliquant la periostine |
JP2012058048A (ja) * | 2010-09-08 | 2012-03-22 | Shino Test Corp | ペリオスチン測定の正確性の改善方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09229935A (ja) | 1996-02-20 | 1997-09-05 | Toshiba Corp | 免疫型情報処理装置及び情報処理方法 |
JP5885243B2 (ja) * | 2009-12-02 | 2016-03-15 | 国立大学法人佐賀大学 | 胆管細胞癌の検出方法および予防・治療剤のスクリーニング方法 |
SG11201405830VA (en) | 2012-03-27 | 2014-10-30 | Genentech Inc | Methods of prognosing, diagnosing and treating idiopathic pulmonary fibrosis |
-
2012
- 2012-09-06 EP EP12829373.5A patent/EP2754672B1/en active Active
- 2012-09-06 WO PCT/JP2012/072774 patent/WO2013035799A1/ja active Application Filing
- 2012-09-06 JP JP2013532647A patent/JP6183809B2/ja active Active
- 2012-09-06 US US14/342,996 patent/US9347954B2/en active Active
-
2016
- 2016-04-29 US US15/143,196 patent/US20160313351A1/en not_active Abandoned
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05268982A (ja) | 1992-03-27 | 1993-10-19 | Hoechst Japan Ltd | 骨形成作用を有する新規な蛋白質およびその製造法 |
JPH09229936A (ja) | 1996-02-26 | 1997-09-05 | Shinotesuto:Kk | 磁性粒子を用いる被検物質の測定方法並びに該方法に使用する担体及び測定器具 |
JPH10132819A (ja) | 1996-10-25 | 1998-05-22 | Shinotesuto:Kk | 粒子を用いる被検物質の測定方法及び該方法に使用する測定器具 |
WO1998023963A1 (en) | 1996-11-27 | 1998-06-04 | Thrombosis And Coagulation Aktiebolag (T.A.C. Ab) | Methods and reagents for determining protein s |
JP2002502250A (ja) | 1997-05-29 | 2002-01-22 | ボード・オヴ・リージェンツ,ザ・ユニヴァーシティ・オヴ・テキサス・システム | Osf2/cbfa1組成物および使用の方法 |
WO2002052006A1 (en) | 2000-12-26 | 2002-07-04 | Genox Research, Inc. | Method of examining allergic disease |
JP2005500059A (ja) | 2001-08-13 | 2005-01-06 | ダナ−ファーバー キャンサー インスティテュート インク. | ペリオスチンに基づく診断アッセイ法 |
WO2007077934A1 (ja) | 2005-12-28 | 2007-07-12 | Asubio Pharma Co., Ltd. | 抗ペリオスチン抗体およびそれを含有するペリオスチンが関与する疾患の予防または治療用医薬組成物 |
WO2009148184A1 (ja) | 2008-06-05 | 2009-12-10 | 国立大学法人佐賀大学 | 特発性間質性肺炎の検出方法 |
WO2010139768A1 (fr) * | 2009-06-03 | 2010-12-09 | Synarc Sas | Sequences, anticorps, procedes et necessaires pour la detection et le dosage in vitro de la periostine, en vue du diagnostic du suivi ou du pronostic de pathologies ou de phenomenes biologiques impliquant la periostine |
JP2012058048A (ja) * | 2010-09-08 | 2012-03-22 | Shino Test Corp | ペリオスチン測定の正確性の改善方法 |
Non-Patent Citations (30)
Title |
---|
"Koso Men-eki Sokutei-ho", 1987, IGAKU-SHOIN LTD. |
"RINSHO BYORI", 1983, RINSHO BYORI KANKO KAI, article "Rinsho Kensa notameno Imunoassei - Gijutsu to Oyo" |
"Shin Seikagaku Jikken Koza", 1991, TOKYO KAGAKU DOJIN, article "Tanpakushitsu" |
"Shin Seikagaku Jikken Koza", vol. 1, 1991, TOKYO KAGAKU DOJIN, article "Tanpakushitsu" |
"Shin Seikagaku Jikken Koza", vol. 1, 1991, TOKYO KAGAKU DOJIN, article "Tanpakusitsu" |
"Tanpakushitsu Kakusan Koso", vol. 31, 1987, KYORITSU SHUPPAN CO., LTD., article "Koso Men-eki Sokutei-ho" |
"Zoku-Seikagaku Jikken Koza", vol. 1, 1986, TOKYO KAGAKU DOJIN |
"Zoku-Seikagaku Jikken Koza", vol. 1, 1986, TOKYO KAGAKU DOJIN, article "Idenshi Kenkyu-ho" |
"Zoku-Seikagaku Jikken Koza", vol. 1, 1987, TOKYO KAGAKU DOJIN, article "Idenshi Kenkyu-ho" |
"Zoku-Seikagaku Jikken Koza", vol. 2, 1987, TOKYO KAGAKU DOJIN, article "Tanpakushitsu no Kagaku Ge-kan" |
1. KII ET AL., J. BIOL. CHEM., vol. 285, no. 3, 2010, pages 2028 - 2039 |
1. TAKAYAMA ET AL., J. BIOCHEM., vol. 146, no. 5, 2009, pages 713 - 723 |
BLANCHARD, C. ET AL.: "Periostin facilitates eosinophil tissue infiltration in allergic lung and esophageal responses", MUCOSAL. IMMUNOL., vol. 1, no. 4, 2008, pages 289 - 296, XP009164763 * |
DAHLBEACK ET AL., THROMB. HAEMOST, vol. 79, 1998, pages 767 - 772 |
DAHLBEACK ET AL., THROMB. HAEMOST., vol. 79, 1998, pages 767 - 772 |
F. HUDECZ ET AL., ET AL., J. IMMUNOL. METHODS, vol. 147, 1992, pages 201 - 210 |
G TAKAYAMA ET AL., J. ALLERGY CLIN. IMMUNOL, vol. 118, 2006, pages 98 - 104 |
G. KOEHLER ET AL., NATURE, vol. 256, 1975, pages 495 - 497 |
G. TAKAYAMA ET AL., J. ALLERGY CLIN. IMMUNOL., vol. 118, no. 1, 2006, pages 713 - 723 |
H. RIOS ET AL., MOL. CELL. BIOL., vol. 25, no. 24, 2005, pages 11131 - 11144 |
H. RIOS ET AL., MOLECULAR AND CELLULAR BIOLOGY, vol. 25, no. 24, 2005, pages 11131 - 11144 |
I. TAKAYAMA ET AL., J. BIOCHEM, vol. 146, no. 5, 2009, pages 713 - 723 |
I. TAKAYAMA ET AL., J. BIOCHEM., vol. 146, no. 5, 2009, pages 713 - 723 |
IZUMIYA ET AL.: "Pepuchido Gosei no Kiso to Jikken", 1985, MARUZEN |
K. L. GUAN ET AL., ANAL. BIOCHEM., vol. 192, 1991, pages 262 - 267 |
KIYAMA ET AL., THE PHARMACEUTICAL SOCIETY OF JAPAN, THE 112TH ANNUAL MEETING LECTURE SUMMARIES 3, 1992, pages 122 |
M. MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 554 |
ORECCHIA, PAOLA ET AL.: "Identification of a novel cell binding site of periostin involved in tumour growth", EUR. J. CANCER, vol. 47, no. 14, 2011, pages 2221 - 2229, XP028286215 * |
T. MARUHASHI ET AL., J. BIOL. CHEM., vol. 285, no. 17, 2010, pages 13294 - 13303 |
THE JAPANESE BIOCHEMICAL SOCIETY: "Seikagaku Jikken Koza", vol. 1, 1975, TOKYO KAGAKU DOJIN, article "Protein Chemistry" |
Cited By (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9684000B2 (en) | 2010-12-16 | 2017-06-20 | Genentech, Inc. | Diagnosis and treatments relating to TH2 inhibition |
US11226341B2 (en) | 2010-12-16 | 2022-01-18 | Genentech, Inc. | Method of treating asthma using an IL-13 antibody |
US9995755B2 (en) | 2010-12-16 | 2018-06-12 | Genentech, Inc. | Diagnosis and treatments relating to TH2 inhibition |
WO2015120185A1 (en) * | 2014-02-07 | 2015-08-13 | Medimmune, Llc | Novel assay to detect human periostin |
WO2015120171A1 (en) * | 2014-02-07 | 2015-08-13 | Medimmune, Llc | Novel assay to detect human periostin |
JP2017506064A (ja) * | 2014-02-07 | 2017-03-02 | メディミューン,エルエルシー | ヒトペリオスチンを検出する新規アッセイ |
JP2017507322A (ja) * | 2014-02-07 | 2017-03-16 | メディミューン,エルエルシー | ヒトペリオスチンを検出する新規アッセイ |
US9862762B2 (en) | 2014-02-07 | 2018-01-09 | Medimmune, Llc | Monoclonal antibodies which bind human periostin |
US10775388B2 (en) | 2014-02-07 | 2020-09-15 | Medimmune, Llc | Methods of using antibodies to determine periostin levels in a sample |
JP2020000236A (ja) * | 2014-02-07 | 2020-01-09 | メディミューン,エルエルシー | ヒトペリオスチンを検出する新規アッセイ |
JPWO2018030456A1 (ja) * | 2016-08-10 | 2019-09-12 | 株式会社シノテスト | 試料に含まれるペリオスチンの測定試薬、ペリオスチン測定用前処理剤、ペリオスチン測定方法及びペリオスチン測定の感度の改善方法 |
JP2021073435A (ja) * | 2016-08-10 | 2021-05-13 | 株式会社シノテスト | 試料に含まれるペリオスチン測定の感度の改善方法 |
WO2018030456A1 (ja) * | 2016-08-10 | 2018-02-15 | 株式会社シノテスト | 試料に含まれるペリオスチンの測定試薬、ペリオスチン測定用前処理剤、ペリオスチン測定方法及びペリオスチン測定の感度の改善方法 |
JP7006599B2 (ja) | 2016-08-10 | 2022-02-10 | 株式会社シノテスト | 試料に含まれるペリオスチンの測定試薬、ペリオスチン測定用前処理剤、ペリオスチン測定方法及びペリオスチン測定の感度の改善方法 |
JP7066142B2 (ja) | 2016-08-10 | 2022-05-13 | 株式会社シノテスト | 試料に含まれるペリオスチン測定の感度の改善方法 |
JPWO2018070397A1 (ja) * | 2016-10-12 | 2019-08-29 | 株式会社シノテスト | ペリオスチンの吸着防止剤及び吸着防止方法 |
WO2018070397A1 (ja) * | 2016-10-12 | 2018-04-19 | 株式会社シノテスト | ペリオスチンの吸着防止剤及び吸着防止方法 |
JP2020186175A (ja) * | 2019-05-10 | 2020-11-19 | 株式会社シノテスト | 免疫グロブリンaに結合しているペリオスチン並びに免疫グロブリンaに結合しているペリオスチンに結合する抗体、ペリオスチンの測定方法、ペリオスチンの測定試薬及びペリオスチン測定の正確性の改善方法 |
JP7475584B2 (ja) | 2019-05-10 | 2024-04-30 | 株式会社シノテスト | 免疫グロブリンaに結合しているペリオスチン並びに免疫グロブリンaに結合しているペリオスチンに結合する抗体、ペリオスチンの測定方法、ペリオスチンの測定試薬及びペリオスチン測定の正確性の改善方法 |
WO2023027186A1 (ja) * | 2021-08-26 | 2023-03-02 | 国立大学法人大阪大学 | 肺線維化疾患バイオマーカー |
CN113687168A (zh) * | 2021-08-30 | 2021-11-23 | 北京航空航天大学 | 一种10kHz-18GHz电场辐射发射的系统电磁兼容性指标分解方法 |
CN113687168B (zh) * | 2021-08-30 | 2022-02-22 | 北京航空航天大学 | 一种10kHz-18GHz电场辐射发射的系统电磁兼容性指标分解方法 |
WO2023036305A1 (zh) * | 2021-09-13 | 2023-03-16 | 沈阳眼产业技术研究院有限公司 | 抗periostin人源化单克隆抗体及其制备方法与应用 |
Also Published As
Publication number | Publication date |
---|---|
EP2754672A4 (en) | 2015-09-02 |
JPWO2013035799A1 (ja) | 2015-03-23 |
EP2754672B1 (en) | 2019-02-27 |
US20140308685A1 (en) | 2014-10-16 |
JP6183809B2 (ja) | 2017-08-23 |
EP2754672A1 (en) | 2014-07-16 |
US9347954B2 (en) | 2016-05-24 |
US20160313351A1 (en) | 2016-10-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6183809B2 (ja) | ペリオスチンの特定領域に結合する抗体及びこれを用いたペリオスチンの測定方法 | |
WO1994004563A1 (en) | PEPTIDES CONTAINING RESPECTIVE AMINO ACID SEQUENCES SELECTED FROM AMONG THOSE OF LIPOPROTEIN(a) AND APOLIPOPROTEIN(a), ANTIBODIES RESPECTIVELY RECOGNIZING THESE AMINO ACID SEQUENCES, AND METHOD OF ASSAYING WITH THESE ANTIBODIES | |
US20130244259A1 (en) | Novel testing method and testing reagent for angiitis | |
CN111316099A (zh) | 用于1型糖尿病诊断的基于脂蛋白体的znt8自身抗原 | |
WO2016104439A1 (ja) | 抗活性型gip抗体 | |
US20230032690A1 (en) | New tau species | |
JP2019536006A (ja) | 診断アッセイに使用するためのタンパク質抗原としてのrepタンパク質 | |
JP5002001B2 (ja) | インスリンレセプターαサブユニットの測定方法 | |
JP2012058048A (ja) | ペリオスチン測定の正確性の改善方法 | |
JP5137015B2 (ja) | Ptx3高感度測定法 | |
RU2607588C2 (ru) | Способ получения агента, связывающегося с препро-вазопрессином или с его фрагментами | |
CN107110848B (zh) | 以脱氧羟腐胺缩赖氨酸合酶基因作为指标使用的动脉硬化及癌的检测方法 | |
CN108026522B (zh) | 特异性纯化的抗普莱晒谱星抗体 | |
JP2009050269A (ja) | ヒトhmg−1に特異的に結合する抗体及びその製造方法 | |
JP7066142B2 (ja) | 試料に含まれるペリオスチン測定の感度の改善方法 | |
JP7475584B2 (ja) | 免疫グロブリンaに結合しているペリオスチン並びに免疫グロブリンaに結合しているペリオスチンに結合する抗体、ペリオスチンの測定方法、ペリオスチンの測定試薬及びペリオスチン測定の正確性の改善方法 | |
JP5750646B2 (ja) | Scca2濃度測定によるアレルギー疾患の検査方法 | |
WO2021187173A1 (ja) | 消化管間質腫瘍を検出する方法および検出試薬 | |
JP5750645B2 (ja) | アレルギー疾患の検査方法 | |
JP5626681B2 (ja) | 癌の検出方法 | |
WO2018034332A1 (ja) | EphA2 N末端フラグメント抗体 | |
JP2000069963A (ja) | アポリポプロテインe4特異モノクローナル抗体 | |
JP2021063020A (ja) | Hmgb1の分解産物に結合する抗体、hmgb1分解産物の測定方法及びhmgb1分解産物の測定試薬 | |
CN115746134A (zh) | 半乳糖凝集素-3的免疫测定 | |
JP2005314397A (ja) | 抗コンドロモジュリン−1特異的抗体及びその利用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12829373 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2013532647 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012829373 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14342996 Country of ref document: US |