JP2012058048A - Method for improving accuracy of periostin measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for improving accuracy of measurement in the measurement using an antigen-antibody reaction of periostin contained in a sample.SOLUTION: An anti-periostin monoclonal antibody is used as the method for improving the accuracy of the measurement in the measurement using the antigen-antibody reaction of periostin contained in the sample.

Description

The present invention relates to a method for improving accuracy (sensitivity and specificity) of measurement using an antigen-antibody reaction of periostin (also referred to as osteoblast-specific factor 2 or OSF2) which can be a marker for allergic diseases and other diseases. is there.
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 (osteoblast-specific factor 2 or OSF2) is an extracellular matrix protein, but one of the inventors of the present invention is that the measurement of the expression level of this periostin gene is useful as a test method for allergic diseases. As a result, the inventors have completed the invention of the test method for allergic diseases (see Patent Document 1 and Non-Patent Document 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).

  In addition, 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). .)

  However, in the measurement of periostin useful for the examination of various diseases as described above, it has been desired to improve accuracy such as sensitivity and specificity in order to distinguish from normal subjects and those suffering from other diseases.

International Publication No. 02/052006 International Publication No. 09/148184 Japanese Patent Laid-Open No. 5-268882 Special Table 2002-502250 JP 2005-500059 gazette International Publication No. 07/077934

G. Takayama et. al. , J Allergy Clin Immunol, vol. 118, 98-104, 2006

In the above-described measurement using the antigen-antibody reaction of periostin contained in a sample, accuracy such as sensitivity and specificity is not sufficient.
For this reason, when it is going to utilize the measured value of periostin contained in a sample for the test | inspection of a disease, there existed a possibility of misdiagnosing this disease.
Therefore, in the measurement using the antigen-antibody reaction of periostin contained in the sample, further improvement in accuracy such as sensitivity and specificity has been desired in order to distinguish it from healthy subjects and those suffering from other diseases. .
On the other hand, an object of the present invention is to provide a method for improving the accuracy of measurement in measurement using an antigen-antibody reaction of periostin contained in a sample.

  The present invention is a method for improving the accuracy of measurement, characterized in that an anti-periostin monoclonal antibody is used in measurement using an antigen-antibody reaction of periostin contained in a sample.

The method for improving the accuracy of the measurement of the present invention is to use the anti-periostin monoclonal antibody in the measurement using the antigen-antibody reaction of periostin contained in the sample, thereby improving the sensitivity of the measurement [in the affected group in the measurement. Improve the accuracy of the measurement by improving the positive rate (true positive rate)] and specificity [negative rate in the unaffected group in the measurement (true negative rate; ie, 1-false positive rate)] It is something that can be done.
Thereby, it can prevent measuring substances other than periostin, and it becomes possible to measure only periostin correctly.
That is, the method for improving the accuracy of measurement of the present invention can obtain an accurate measurement value of periostin, improve discrimination from healthy subjects and patients with other diseases, and prevent misdiagnosis. It can be done.

It is the figure which showed the measurement result of the periostin density | concentration in each sample of the affected person of a cholangiocellular carcinoma, and a control | contrast person, when a polyclonal antibody is used as an anti- periostin antibody, and each when a monoclonal antibody is used. The ROC curve created based on the measurement results of the periostin concentration in each sample of a patient with cholangiocarcinoma and a control when a polyclonal antibody is used as an anti-periostin antibody and when a monoclonal antibody is used is shown. It is a figure. It is the figure which showed the measurement result of the periostin density | concentration in each sample of the affected person of a pulmonary fibrosis, and a healthy person in each when a polyclonal antibody is used as an anti- periostin antibody, and a monoclonal antibody is used. The ROC curve created based on the measurement results of the periostin concentration in each sample of patients with pulmonary fibrosis and healthy individuals in the case of using a polyclonal antibody as an anti-periostin antibody and in the case of using a monoclonal antibody is shown. It is a figure.

Hereinafter, the present invention will be described in detail.
1. Anti-periostin monoclonal antibody (1) antibody The anti-periostin monoclonal antibody in the present invention may be any monoclonal antibody as long as it can bind to periostin.

The anti-periostin monoclonal antibody in the present invention also includes a monoclonal antibody fragment (Fab, F (ab ′) _2, Fab ′ or the like) capable of binding to periostin.

  Although the method for obtaining the anti-periostin monoclonal antibody in the present invention is not particularly limited, the following methods and the like can be mentioned.

For example, a peptide or protein containing all or part of the amino acid sequence of periostin is prepared and obtained, and an animal is immunized with this as an immunogen, or a sequence encoding all or part of the periostin protein is obtained. Antibody-producing cells that produce monoclonal antibodies using periostin protein expressed in the animal as an immunogen by injecting the contained DNA or RNA directly into the animal or infecting the animal with a viral vector containing the same sequence, etc. To get.
Then, it is confirmed that the monoclonal antibody is a monoclonal antibody that binds (specifically) to periostin, or an antibody-producing cell that produces such a monoclonal antibody is selected, and the anti-periostin monoclonal antibody in the present invention is selected. get.

(2) Immunogen The immunogen for producing the anti-periostin monoclonal antibody in the present invention will be described below.
As described above, a peptide or protein containing all or part of the amino acid sequence of periostin can be used as an immunogen for producing the anti-periostin monoclonal antibody in the present invention.

By using a peptide or protein containing all or part of the amino acid sequence of periostin as an immunogen, the anti-periostin monoclonal antibody in the present invention can be obtained.
The immunogen for producing this anti-periostin monoclonal antibody has 1 to several (usually 1 to 8, preferably 1 to 6) amino acid residues 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 deletion, substitution, insertion, addition or modification of a group.

There is a report that an antibody can recognize an amino acid sequence consisting of three amino acids (F. Hudecz et al., J. Immunol. Methods, 147, 201-210, published in 1992).
Accordingly, the minimum unit of the amino acid sequence of the immunogen of the anti-periostin monoclonal antibody in the present invention is 1 to 1 in all or part of the amino acid sequence of periostin, or all or part of these amino acid sequences. From amino acid sequences obtained by performing deletion, substitution, insertion, addition or modification of several (usually 1 to 8, preferably 1 to 6) amino acid residues, from three consecutive amino acid residues The anti-periostin monoclonal antibody according to the present invention is a tripeptide consisting of an amino acid sequence consisting of these three consecutive amino acid residues, or a peptide added with other amino acids or peptides. Can be thought of as the smallest unit of immunogen.

  In addition, 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 as an immunogen for producing this anti-periostin monoclonal antibody A peptide or protein containing an amino acid sequence obtained by deletion, substitution, insertion, addition or modification of a residue binds to periostin when a monoclonal antibody is prepared using this peptide or protein as an immunogen. It must be possible to obtain a monoclonal antibody.

  Peptide or protein containing all or part of the amino acid sequence of periostin as the immunogen, or 1 to several (usually 1 to 8) of all or part of the amino acid sequence of periostin (Preferably 1 to 6) amino acid residues obtained by performing deletion, substitution, insertion, addition or modification of amino acid residues, such as peptides or proteins containing human or other animal body fluids, cells, tissues Alternatively, it can be obtained from an organ or the like by extraction, purification or the like by a known method or the like.

  In the present invention, 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.

For example, the following method etc. can be mentioned as a method of acquiring periostin.
(A) First, a recombinant periostin protein in which a V5 epitope / His tag is added to periostin (nucleotide sequence: nucleotide number of Accession Number. D13666 of nucleic acid database GenBank; amino acid sequence: amino acid sequence of nucleic acid database GenBank: Accession Number. BAA02837) Is expressed in S2 cells, which are insect cells, and purified.

(B) Specifically, a transformant of S2 cells is prepared as follows.
A cDNA encoding the above-mentioned part of periostin is inserted into pMT / Bip / V5-HisA plasmid (Invitrogen, Carlsbad, Calif., USA), and this is designated as pMT / Bip / V5-HisA-periostin.
A plasmid expressing pMT / Bip / V5-HisA-periodin and a hygromycin resistance gene, pCoHygro (Invitrogen, Carlsbad, Calif., USA) is co-introduced into S2 cells by a known method and transformed.
A transformant is selected with hygromycin to obtain a stable transformant.
And in the transformant of S2 cell, the periostin which the V5 epitope / His tag couple | bonded is expressed.

(C) 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 the S2 recombinant periostin protein to the resin.
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, for example, by the methods described in the following documents (I. Takayama et al., J. Biochem., 146, 5, 713-723, issued in 2009).

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 Dojin, 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.
In addition, 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.

Further, the immunogen may be prepared from DNA or RNA having a corresponding nucleobase sequence by using genetic engineering technology.
"Gene Research Method I", Tokyo Kagaku Doujin, 1986, edited by Japanese Biochemical Society
Genetic Research Method II ”, Tokyo Chemical Doujin, 1986, edited by Japanese Biochemical Society,“ Second Biochemistry Experiment Course 1 ”
It may be prepared with reference to “Gene Research Method III”, Tokyo Kagaku Dojin, 1987, and the like.

  By the way, 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.

  In addition, as a carrier when using a carrier for producing an antibody, mussel hemocyanin (KLH), bovine serum albumin (BSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, dipalmityl Any known carrier such as lysine, tetanus toxoid or polysaccharide can be used.

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- A known binding method such as a 3- (2-pyridyldithio) propionic acid method can be used.
Moreover, what made the immunogen adsorb | suck to carriers, such as a nitrocellulose particle | grain, polyvinylpyrrolidone, or a liposome, can also be used as an immunogen.

(3) Method for Obtaining Monoclonal Antibody The anti-periostin monoclonal antibody in the present invention can be obtained by the following operation.
The monoclonal antibody is an antibody-producing cell such as a hybridoma obtained by the cell fusion method of Keller et al. (G. Koehler et al., Nature, 256, 495-497, published in 1975) or a tumorigenic cell by a virus such as Epstan-Barr virus. Can be obtained.
Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.

  First, the immunogen or the conjugate of the immunogen and the carrier is used as a mammal (mouse, nude mouse, rat, rabbit, etc., for example, BALB / c of an inbred mouse) or birds (chicken, etc.), etc. To immunize.

  The immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like. Preferably, 0.1 μg to 5 mg of the immunogen or a combination of the immunogen and carrier is immunized per dose.

The immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing. Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.

  After the initial immunization, booster injections of the immunogen or a 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. Also in this case, the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.

  After the first immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the immunogen or the combined immunogen and carrier is added to physiological saline. A solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization. Three to five days after the final immunization, cells having the ability to produce antibodies such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.

  The cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). A cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferred. For example, a P3-X63-Ag8 strain (ATCC) which is a HGPRT-deficient cell line derived from BALB / c mice. TIB9), P3-X63-Ag8-U1 strain (Cancer Research Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8.653 strain (JCRB 0028) Or use SP2 / O-Ag-14 strain (JCRB 0029) etc. Rukoto can.

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.
When myeloma cells are of HGPRT-deficient strain or TK-deficient strain, fusion of cells capable of producing antibodies and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine Only cells (hybridomas) can be selectively cultured and propagated.

  The thus obtained hybridoma culture supernatant is obtained by using the above-mentioned immunogen, a conjugate of the immunogen and a carrier, or a human or other animal (if human periostin is measured, a human-derived one is used. A hybridoma that produces an antibody that binds (specifically) to periostin can be selected by measuring by an immunological assay such as ELISA or Western blot using a preferred periostin.

  By performing a combination of this hybridoma selection method and a known cloning method such as a limiting dilution method, the production cell line of the anti-periostin 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 anti-periostin monoclonal antibody of the present invention can be obtained from the culture supernatant. However, a serum-free medium or low-concentration serum medium can be used as the medium. In this case, it is preferable in that the antibody can be easily purified, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.
In addition, the monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and previously stimulated with pristane or the like, and after a certain period of time, the anti-periostin monoclonal antibody of the present invention is obtained from ascites collected in the abdominal cavity. It can also be obtained.

  The anti-periostin monoclonal antibody thus obtained can be purified by a salting-out method using ammonium sulfate, sodium sulfate, etc., an ion exchange chromatography, a gel filtration method or an affinity chromatography, or a combination of these methods. Anti-periostin monoclonal antibody obtained can be obtained.

2. Measurement using antigen-antibody reaction of periostin (1) General remarks The present invention uses an anti-periostin monoclonal antibody in measurement using the antigen-antibody reaction of periostin contained in a sample. It is an improvement method.

  That is, in measurement using the antigen-antibody reaction of periostin contained in a sample, the accuracy of measurement can be improved by using an anti-periostin monoclonal antibody.

  In the measurement using the antigen-antibody reaction of periostin contained in the sample, when two or more antibodies are used as antibodies that bind to periostin, any of the antibodies that bind to periostin contained in the sample in these measurements Must be an anti-periostin monoclonal antibody.

For example, in an ELISA sandwich method using an enzyme-labeled antibody and a solid-phased antibody, both the enzyme-labeled antibody and solid-phased antibody that bind to periostin contained in the sample are anti-periostin monoclonal antibodies. There is a need.
This anti-periostin monoclonal antibody is not limited to one type, and a plurality of types may be used simultaneously.
The details of this anti-periostin monoclonal antibody are as described in the above section “1. Anti-periostin monoclonal antibody”.

  The method for improving the accuracy of the measurement in the measurement using the antigen-antibody reaction of periostin contained in the sample of the present invention is suitable for the 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 measurement in the measurement using the antigen-antibody reaction of periostin contained in a sample according to the present invention is for examining whether or not cancer or lung disease is present or its degree (such as a medical condition). It is more suitable for measurement.

  Furthermore, in the measurement using the antigen-antibody reaction of periostin contained in a sample according to the present invention, the measurement accuracy improvement method is a test for the presence or absence of cholangiocellular carcinoma or pulmonary fibrosis (such as a medical condition). Particularly suitable for measurements for

Although periostin is elevated in individuals with cholangiocellular carcinoma, other diseases such as hepatocellular carcinoma, cirrhosis, sclerosing hepatocellular carcinoma, sarcomatous hepatocellular carcinoma, angiomyolipoma, or angiosarcoma It is low in those affected or healthy.
This is described by the inventors in the specification of the patent application (Japanese Patent Application No. 2009-274873) by the inventors, etc., and in the literature of Utispan et al. [Molecular Cancer, Vol. 9, no. 13, page 1-20, 2010].

(2) Measurement method using antigen-antibody reaction The method for improving the accuracy of measurement of the present invention is as long as it uses an anti-periostin monoclonal antibody in measurement using the antigen-antibody reaction of periostin contained in a sample. In particular, the measurement principle is not limited, and the desired effect is achieved.

  Examples of the measurement using the antigen-antibody reaction of periostin contained in this sample include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), and luminescence immunoassay ( LIA), enzyme antibody method, fluorescent antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination reaction measurement method, hemagglutination 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 specific binding substance, and specific to the measurement target substance (test substance) Measurement method using particles to which binding substance is immobilized, or ELSA method (Enzyme-linked Ligan shown by Dahlbeack et al. sorbent Assay) (Thromb.Haemost, 79, pp. 767-772, 1998 issue;. may be mentioned WO98 / 23963) and the like.

In the measurement using the antigen-antibody reaction, the measurement accuracy improving method of the present invention is applied to any method such as the sandwich method, the competitive method, or the homogeneous method (homogeneous method). Can do.
In addition, the measurement using the antigen-antibody reaction in the present invention may be performed by a method, or may be performed using an apparatus such as an analyzer.

(3) Samples Samples in the present invention include body fluids such as blood, serum, plasma, urine, semen, spinal fluid, saliva, ascites or amniotic fluid; or extracts such as blood vessels or liver, organs, tissues or cells, etc. Any biological sample that may contain periostin is the subject.

(4) Immunological measurement method using labeled antibody Immunological measurement method using labeled antibody such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay or luminescence immunoassay for the measurement in the present invention In other words, in the case of carrying out by a measuring method using an antigen-antibody reaction using a labeled antibody, it can be carried out by a sandwich method or a competition method, but when it is carried out by the sandwich method, it binds to periostin contained in the sample. It is necessary that both the immobilized antibody and the labeled antibody are anti-periostin monoclonal antibodies.

As a solid phase carrier used in the immunological measurement method using this labeled antibody, polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposome, gelatin, agarose, 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.
A solid-phase antibody can be prepared by adsorbing and binding an antibody such as the above-mentioned anti-periostin monoclonal antibody and a solid-phase carrier by a known method such as a physical adsorption method, a chemical binding method, or a combination thereof. .

In the case of the physical adsorption method, an anti-periostin monoclonal antibody or the like in which an antibody such as an anti-periostin monoclonal 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 the antibody in contact with a solid phase carrier.
When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with known methods described in ed. “Shinsei Kagaku Kenkyu Ken 1 Protein IV”, Tokyo Kagaku Dojin, published in 1991, etc., an antibody such as an anti-periostin monoclonal antibody and a solid phase carrier such as glutaraldehyde, carbodiimide, imidoester or maleimide It can be 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 monoclonal antibody and a solid phase carrier. .

  Further, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, the surface or inner wall surface of the solid phase carrier on which an antibody such as anti-periostin monoclonal antibody is immobilized is used. Treated with 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).

As the labeling substance, peroxidase (POD), alkaline phosphatase (ALP), β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase, or the like can be used in the case of enzyme immunoassay.
In the case of fluorescence immunoassay, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, or the like can be used.
In the case of a radioimmunoassay, tritium, iodine 125, iodine 131, or the like can be used.
In the luminescence immunoassay method, NADH-FMNH ₂ -luciferase system, luminol-hydrogen peroxide-POD system, acridinium ester system or dioxetane compound system can be used.

  The binding method between an antibody such as an anti-periostin monoclonal antibody and a labeling substance such as an enzyme is described in the “Clinical Pathology Special Issue 53: Immunoassay for Clinical Examination -Technology and Application” edited by the Japanese Society of Clinical Pathology. 1983; published by the Biochemical Society of Japan, “Shinsei Kagaku Kogaku Kenkyu 1 Protein IV”, Tokyo Kagaku Dojin, published in 1991, etc., in accordance with known methods such as anti-periostin monoclonal antibody and labeling substances such as glutaraldehyde and carbodiimide , Mixed with a bivalent cross-linking reagent such as imide ester or maleimide, brought into contact, and reacted with an amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, etc. of the antibody such as an anti-periostin monoclonal antibody and the labeling substance. Can be combined.

  The operation method of the measurement is a known method or the like (edited by the Japanese Society of Clinical Pathology “Special Issue on Extraordinary Clinical Pathology No. 53 Immunoassay for Clinical Examination -Technology and Application”, published by Clinical Pathology, 1983; edited by Ishikawa Yuji 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," published by Kyoritsu Publishing, 1987)

For example, a complex of “solid phase carrier = solid phase antibody = periostin = label antibody” by reacting the immobilized antibody with the sample and reacting the labeled antibody at the same time, or reacting the labeled antibody after washing. To form.
Then, the unbound labeled antibody was washed and separated and contained in the sample from the amount of labeled antibody bound to the solid phase carrier via “solid phase antibody = periostin” or the amount of unbound labeled antibody. Only the amount (concentration) of periostin can be measured.

Specifically, in the case of an enzyme immunoassay, a substrate is reacted with an enzyme labeled with an antibody under the optimum conditions, and the amount of the enzyme reaction product is measured by an optical method or the like.
In the case of the fluorescence immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of the radioimmunoassay, the radiation dose by the radioactive substance label is measured.
In the case of a luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.

(5) Immunological measurement method by the agglutination method The measurement in the present invention is carried out using an immunoturbidimetric method, latex turbidimetric method, latex agglutination method, erythrocyte agglutination method or particle agglutination method. In the case where the generation is carried out by measuring the transmitted light or scattered light by an optical method or by a visual measurement method, that is, by the measurement method for measuring the formation of a complex aggregate by an antigen-antibody reaction. In this case, a phosphate buffer, glycine buffer, Tris buffer, Good buffer, or the like can be used as a solvent, and a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor can be included. Good.

When an antibody such as an anti-periostin monoclonal antibody is used after sensitizing a solid support, polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride are used as the solid support. -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 heavy Particles made of a material such as coalescence, acrylic acid polymer, latex, gelatin, liposome, microcapsule, erythrocyte, silica, alumina, carbon black, metal compound, metal, ceramics or magnetic material can be used.
As a method for sensitizing an antibody such as an anti-periostin monoclonal antibody to a solid phase carrier, a known method such as a physical adsorption method, a chemical binding method, or a combination thereof can be used.

In the case of the physical adsorption method, according to a known method, an anti-periostin monoclonal antibody or the like mixed with an antibody such as an anti-periostin monoclonal antibody and a solid phase carrier in a solution such as a buffer solution or contacted, or dissolved in a buffer solution or the like It can be carried out by bringing the antibody in contact with a solid phase carrier.
When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with known methods described in ed. “Shinsei Kagaku Kenkyu Ken 1 Protein IV”, Tokyo Kagaku Dojin, published in 1991, etc., an antibody such as an anti-periostin monoclonal antibody and a solid phase carrier such as glutaraldehyde, carbodiimide, imidoester or maleimide It can be 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 monoclonal antibody and a solid phase carrier. .

  Further, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, the surface or inner wall surface of the solid phase carrier on which an antibody such as anti-periostin monoclonal antibody is immobilized is used. Treated with 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).

  When the latex turbidimetric method is used as the measurement principle, the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are bonded via a measurement target substance (periostin), and aggregates are formed. For reasons such as the degree of generation and the ease of measurement of the generated agglomerates, the latex particles preferably have an average particle size of 0.04 to 1 μm.

  In addition, when the latex turbidimetry is used as a measurement principle, the concentration of latex particles in which an antibody such as an anti-periostin monoclonal antibody is solid-phased is included in the concentration of periostin in a sample, the concentration of an antibody such as an anti-periostin monoclonal antibody. 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.

  However, in general, an antigen-antibody reaction between "antiperiostin monoclonal antibody and other antibodies" mixed with a sample and a latex particle and solidified in latex particles and "periostin" contained in the sample is performed. At the time of reaction, the concentration of latex particles on which “antibody such as anti-periostin monoclonal antibody” is immobilized is generally 0.005 to 1% (w / v) in the reaction mixture. In this case, the measurement reagent contains “latex particles on which an antibody such as an anti-periostin monoclonal antibody is immobilized” having such a concentration in the reaction mixture.

  In addition, when the indirect agglutination reaction method such as latex agglutination reaction method, erythrocyte agglutination reaction method or particle agglutination reaction method is used as the measurement principle, 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 μm, and more preferably in the range of 0.5 to 10 μm. And it is preferable that the specific gravity of these particle | grains exists in the range of 1-10, and it is more preferable that it exists in the range of 1-2.

  In addition, as 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, And 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 UV-type.

The measuring operation can be performed by a known method or the like. For example, in the case of measuring by an optical method, the sample and an antibody such as an anti-periostin monoclonal antibody, or the sample and the “solid phase carrier sensitized” An antibody such as an anti-periostin monoclonal antibody is reacted, and transmitted light or scattered light is measured by an endpoint method or a rate method.
In the case of visual measurement, in a container such as a plate or microplate, the sample is reacted with “an antibody such as an anti-periostin monoclonal antibody sensitized to a solid support” to visually check the state of aggregation. Judgment.
In addition, you may measure using apparatuses, such as a microplate reader, instead of measuring visually.

(6) Other components during measurement In measurement using the antigen-antibody reaction of periostin contained in a sample in the measurement accuracy improving method of the present invention, various aqueous solvents can be used as a solvent.
Examples of the aqueous solvent include purified water, physiological saline, and various buffer solutions such as Tris buffer, phosphate buffer, and phosphate buffered saline.
About the pH of this buffer solution, an appropriate pH may be appropriately selected and used. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3-12.

Further, in the measurement in the present invention, an antibody such as the above-mentioned anti-periostin monoclonal antibody, a solid phase carrier on which an antibody such as the above-mentioned anti-periostin monoclonal antibody was immobilized, and an antibody such as the above-mentioned anti-periostin monoclonal antibody were sensitized In addition to a solid phase carrier and / or a reagent component such as a labeled antibody in which an antibody such as the above-described anti-periostin monoclonal antibody and a labeling substance such as an enzyme are bound, bovine serum albumin (BSA), human serum albumin (HSA), Proteins such as casein or its salts; various salts; various sugars; skim milk powder; various animal sera such as normal rabbit serum; various preservatives such as sodium azide or antibiotics; activating substances; reaction accelerators; A sensitivity increasing substance; a nonspecific reaction inhibiting substance; or a nonionic surfactant, One or more may be appropriately used for various surfactants such as sexual surfactant or anionic surfactant.
And the density | concentration at the time of using these for a measurement is although it does not specifically limit, 0.001 to 10% (W / V) is preferable, and 0.01 to 5% (W / V) is especially preferable.

  Examples of the surfactant 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 acetic acid Anionic surfactants, such as and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by these Examples.

[Example 1] (Preparation of rabbit anti-periostin polyclonal antibody)
A rabbit anti-periostin polyclonal antibody was prepared by the following procedure.

  An insect cell is a recombinant periostin protein in which a V5 epitope / His tag is added to periostin (nucleotide sequence: nucleotide number of nucleic acid database GenBank Accession Number. D13666; amino acid sequence: amino acid database GenBank Accession Number. BAA02837 amino acid sequence). It was expressed in S2 cells and purified.

Specifically, S2 cell transformants were prepared as follows.
A cDNA encoding the above-mentioned portion of periostin was inserted into pMT / Bip / V5-HisA plasmid (Invitrogen, Carlsbad, Calif., USA), and this was designated as pMT / Bip / V5-HisA-periostin.
PCoHygro (Invitrogen, Carlsbad, Calif., USA), which is a plasmid expressing pMT / Bip / V5-HisA-periodin and a hygromycin resistance gene, was co-introduced into S2 cells by a known method and transformed.
A transformant was selected with hygromycin to obtain a stable transformant.

  And in the S2 cell transformant, periostin to which V5 epitope / His tag was bound was expressed.

Purification of S2 recombinant periostin protein was performed as follows.
The expression of S2 recombinant periostin protein was induced by adding copper sulfate to the medium of periostin gene stable transformant S2 cells.
As a result, the S2 recombinant periostin protein was expressed and secreted into the culture supernatant.

The culture supernatant was dialyzed against phosphate buffered saline (PBS) and then mixed with nickel resin (Ni-NTA Agarose, Qiagen, Hilden, Germany) to bind the S2 recombinant periostin protein to the resin.
The resin was washed to remove impurities, and S2 recombinant periostin protein was eluted with an imidazole-containing buffer.
The eluted S2 recombinant periostin protein was dialyzed against PBS and used as an animal immunogen.

  Next, these S2 recombinant periostin proteins are mixed with CFA (complete Freund's adjuvant) (Freund's Adjuvant, Complete, Sigma-Aldrich, St. Louis, Mo., USA), and then the rabbit is immunized, and then the serum is collected. did.

The specific procedure is as follows.
As a rabbit, New Zealand White rabbit (female, body weight 1.5-2 Kg) [KTB Oriental, Tosu, Saga, Japan] was used.

  These rabbits were immunized by injecting a mixture of S2 recombinant periostin protein, which is an immunogen, and CFA in several subcutaneous portions of the rabbit back.

  Five to seven weeks after immunization, blood was collected from the ear vein of the immunized rabbit using a needle and a syringe to obtain serum.

Finally, IgG was purified from the serum as follows.
That is, a double amount of 50 mM sodium acetate aqueous solution (pH 4.0) was added to the collected serum, and then the pH was adjusted to 4.0 with hydrochloric acid.
While stirring this serum, 1/15 volume of caprylic acid was added and stirred for 30 minutes, followed by centrifugation at 9,200 g for 10 minutes.
The supernatant was dialyzed against PBS to obtain rabbit anti-periostin polyclonal antibody as purified IgG.

[Example 2] (Preparation of rat anti-periostin monoclonal antibody)
A rat anti-periostin monoclonal antibody was prepared by the following procedure.

  A mixture of S2 recombinant periostin protein (20-50 μg / rat) and CFA (complete Freund's adjuvant) or Titer Max Gold (chemical synthesis adjuvant) [Funakoshi, Tokyo, Japan] Immunogen was used.

The specific procedure is as follows.
S2 recombinant periostin protein was prepared as follows.
First, the expression of S2 recombinant periostin protein was induced by adding copper sulfate to the medium of periostin gene stable transformant S2 cells.
As a result, the S2 recombinant periostin protein was expressed and secreted into the culture supernatant.

The culture supernatant was dialyzed against PBS and then mixed with nickel resin (Ni-NTA Agarose, Qiagen, Hilden, Germany) to bind the S2 recombinant periostin protein to the resin.
The resin was washed to remove impurities, and S2 recombinant periostin protein was eluted with an imidazole-containing buffer.

Freund's Adjuvant, Complete (Sigma-Aldrich, St. Louis, MO, USA) was used as CFA (complete Freund's adjuvant).
Moreover, Titer Max Gold (Funakoshi Co., Tokyo, Japan) was used as a chemical synthesis adjuvant.
Then, CFA or Titer Max Gold was mixed at a ratio of 1 volume to 1 volume of the S2 recombinant periostin protein solution.

Next, 20-50 μg of S2 recombinant periostin protein solution and a mixture of CFA or Titer Max Gold are injected subcutaneously into the footpads of female rats, and after 10 days to 2 weeks, again in the footpads of the rats. As an immunogen, a mixture of 20-50 μg of S2 recombinant periostin protein solution and CFA or Titer Max Gold was injected subcutaneously.
Here, Wistar rats (female, 6 to 8 weeks old) [Charles River Japan, Yokohama, Kanagawa, Japan] were used as the rats.

  Three to four days after the final immunization, cells in the popliteal, inguinal and iliac lymph nodes of immunized rats and myeloma cells (Sp2 / O cells) were mixed at a ratio of 1: 1 to 10: 1. In this way, polyethylene glycol (PEG 1500, Roche, Switzerland) was added to cause cell fusion, and the grown hybridoma colonies were selected.

Specifically, 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 .

As a method for selecting the grown monoclonal antibody-producing cell line (fused cell line), an ELISA method in which the protein used for the immunogen is immobilized on a solid phase after 7 to 14 days from cell fusion and the fused cell culture supernatant is used as a primary antibody. I went in the system.
Specifically, this ELISA method was performed as follows.
The protein used for 1 μg / mL of the immunogen 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 supernatant of the fused cell culture was washed, a peroxidase-labeled goat anti-rat IgG antibody (GE Healthcare, Little Chalfont, UK) was added as a secondary antibody, and the mixture was allowed to stand at room temperature for 1 hour.
After washing this secondary antibody, ABTS peroxidase substrate (KPL, Gaithersburg, MD, USA) was added to develop color, and the absorbance at 405 nm was measured.

Two clones were established from the grown fusion cell lines, and named SS17B strain and SS18A strain, respectively.
Then, IgG was purified from these selected monoclonal antibody-producing cell lines 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).
One volume of 1M tris (hydroxymethyl) aminomethane buffer was added to 4 volumes of the eluate, and rat anti-periostin monoclonal antibody was obtained from each monoclonal antibody-producing cell line as purified IgG.
That is, rat anti-periostin monoclonal antibodies could be obtained from the respective monoclonal antibody-producing cell lines of the SS17B strain and the SS18A strain.

[Example 3] (Confirmation of effect of the present invention-1)
The effect of the measurement accuracy improving method of the present invention was confirmed.

1. Sample As a sample, the serum of a patient with cholangiocellular carcinoma or the serum of a control person was used as follows.
(1) Sera of patients with cholangiocellular carcinoma (total 8 cases) (2) Sera of controls (total 71 cases) Breakdown of controls:
・ Patients with hepatocellular carcinoma (total 24 cases)
・ Patients with mixed hepatocellular carcinoma (total 7 cases)
・ Patients with cirrhosis (26 cases in total)
・ Patients with sclerosing hepatocellular carcinoma (total 3 cases)
・ Patients with sarcomatous hepatocellular carcinoma (total 2 cases)
・ Patients with angiomyolipoma (1 case in total)
・ Patients with angiosarcoma (1 patient in total)
・ Healthy people (total 7 cases)

2. Measurement of periostin concentration in a sample (1) Measurement using a polyclonal antibody as an anti-periostin antibody The concentration of periostin contained in each sample of (1) and (2) above is determined by fluorescence immunization using an anti-periostin polyclonal antibody. It measured by the measuring method.

(A) First, in each well of a 96-well microtiter plate, phosphate buffered saline (PBS) [137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, and 8. Inject 50 μL of the rabbit anti-periostin polyclonal antibody of Example 1 diluted to 10 μg / mL with an aqueous solution containing 04 mM disodium hydrogen phosphate (pH 7.4)], and allow to stand overnight at 4 ° C. Rabbit anti-periostin polyclonal antibody was immobilized on each well of the microtiter plate.

(B) Next, after removing the liquid in the wells of this microtiter plate, 180 μL of blocking liquid-A [PBS containing 3% BSA] was injected into each well and allowed to stand at room temperature for 1 hour.

(C) Next, each well was washed three times with a washing solution-A [TBS containing 0.05% Tween-20].

(D) Next, for each of the samples of (1) and (2) above, Sample Diluent-A [Tris (hydroxymethyl) aminomethane buffered saline containing 0.05% Tween-20 After 10-fold dilution with [TBS], 50 μL of the solution was injected into the wells and allowed to stand at room temperature for 1 hour for reaction.
Thus, periostin contained in the sample was bound to the rabbit anti-periostin polyclonal antibody immobilized on the well.

(E) Next, each well was washed three times with the washing solution-A.

(F) A biotin-labeled anti-periostin polyclonal antibody labeled with biotin bound to the rabbit anti-periostin polyclonal antibody of Example 1 was diluted with the sample diluent-A to 1 μg / mL, and 50 μL thereof was diluted. The solution was poured into the well and allowed to stand at room temperature for 1 hour for reaction.
As a result, the biotin-labeled anti-periostin polyclonal antibody was bound to periostin bound to the immobilized rabbit anti-periostin polyclonal antibody.

(G) Next, each well was washed 3 times with the washing solution-A.

(H) Next, 50 μL of 1,000-fold diluted europium (Eu) -labeled streptavidin (DELFIA, PerkinElmer, Mass., USA) was injected into the wells and allowed to stand at 25 ° C. for 60 minutes. The reaction was carried out.
Thus, streptavidin of “Europium-labeled streptavidin” is bound to biotin of “well of 96-well microtiter plate”-“rabbit anti-periostin polyclonal antibody”-“periostin”-“biotin-labeled anti-periostin polyclonal antibody”. I let you.

(I) Next, each well was washed five times with the above-described washing solution dedicated to europium-labeled streptavidin (DELFIIA, PerkinElmer, Massachusetts, USA).

(J) Next, 50 μL of an enhancement solution (Enhancement Solution, DELFIA, PerkinElmer, Massachusetts, USA) was injected into the well and allowed to stand at room temperature for 10 to 15 minutes to perform the reaction. The fluorescence intensity was measured for each well using a decomposition fluorometer.

(K) It should be noted that the purified periostin protein was diluted with the sample diluent to prepare a diluted series of purified periostin protein, and the measurement was performed for each of these samples according to the description in (a) to (j) above. The standard curve of “periostin concentration-fluorescence intensity” in this fluorescence immunoassay was prepared.

(L) The fluorescence intensity of each well measured in the above (j) is applied to the standard curve of “periostin concentration−fluorescence intensity” prepared in the above (k), and each sample of (1) and (2) in 1 above Each periostin concentration was determined.

(2) Measurement using monoclonal antibody as anti-periostin antibody The concentration of periostin contained in each sample of (1) and (2) above is determined by enzyme immunoassay (ELISA method) using anti-periostin monoclonal antibody. It was measured.

(A) First, in each well of a 96-well microtiter plate, phosphate buffered saline (PBS) [137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, and 8. 100 μL of the rat anti-periostin monoclonal antibody (monoclonal antibody-producing cell line: SS18A strain) of Example 2 diluted to 2 μg / mL with an aqueous solution containing 04 mM disodium hydrogen phosphate (pH 7.4)], The rat anti-periostin monoclonal antibody (SS18A strain) was allowed to stand at 25 ° C. for 18 hours, and was immobilized on each well of the microtiter plate.

(B) Next, after removing the solution in the well of this microtiter plate, blocking solution-B [50 mM containing 0.5% casein, 100 mM sodium chloride, and 0.1% sodium azide. Of Tris (hydroxylmethyl) aminomethane buffer [Tris buffer] (pH 8.0)] was injected into each well and allowed to stand at 4 ° C. for 18 hours.

(C) Next, each well was washed 3 times with a washing solution-B [phosphate buffered saline (PBS) containing 0.05% Tween-20].

(D) Next, for each sample of (1) and (2) above, sample diluent-B [0.5% casein, 100 mM sodium chloride, and 0.1% sodium azide was added. After diluting 200-fold with 50 mM Tris (hydroxylmethyl) aminomethane buffer [Tris buffer] (pH 8.0)], 100 μL of the diluted solution was injected into the well and allowed to stand at 25 ° C. for 18 hours. The reaction was carried out.
This allowed periostin contained in the sample to bind to the rat anti-periostin monoclonal antibody (SS18A strain) immobilized on the well.

(E) Next, each well was washed 5 times with the washing solution-B.

(F) POD-labeled anti-periostin monoclonal antibody obtained by binding peroxidase (POD) to the rat anti-periostin monoclonal antibody (monoclonal antibody-producing cell line: SS17B strain) of Example 2 as described above was used as the sample diluent-B. Was diluted to 50 ng / mL, and 100 μL thereof was injected into the well and allowed to stand at 25 ° C. for 90 minutes to carry out the reaction.
As a result, the POD-labeled anti-periostin monoclonal antibody (SS17B strain) was bound to periostin bound to the solid-phased rat anti-periostin monoclonal antibody (SS18A strain).

(G) Next, each well was washed 5 times with the washing solution-B.

(H) Next, a substrate solution of POD [containing 0.8 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), 2.5 mM hydrogen peroxide, and 30 mM disodium hydrogen phosphate The 20 mM citrate buffer solution] was injected into the wells and allowed to stand at 25 ° C. for 10 minutes to cause the reaction to develop color.
Thereafter, 0.7N sulfuric acid was injected into each well to stop the reaction.

(I) Next, the absorbance at 450 nm was measured for each well using a spectrophotometer.

(J) The purified periostin protein is diluted with the sample diluent to prepare a diluted series of purified periostin protein, and each of these samples is measured according to the description in (a) to (i) above. The standard curve of “periostin concentration-absorbance” in this enzyme immunoassay was prepared.

(K) The absorbance of each well measured in (i) above is applied to the standard curve of “periostin concentration-absorbance” prepared in (j) above, and each of the samples in (1) and (2) in 1 above is applied. Periostin concentration was determined.

3. Measurement Result (1) Measurement Result of Periostin Concentration in Sample In each sample of the affected individuals (total 8 cases) and control persons (total 71 cases) of cholangiocellular carcinoma in each of the above 2 (1) and (2) The measurement results of the periostin concentration are shown in FIG.

The measurement result in the measurement using the polyclonal antibody as the anti-periostin antibody in (2) above is shown on the left side of FIG.
Moreover, the measurement result in the measurement using a monoclonal antibody as the anti-periostin antibody of the above (2) is shown on the right side of FIG.

  And in either of the left side and the right side of FIG. 1, the horizontal axis represents whether the sample is the serum of a patient suffering from cholangiocellular carcinoma or the serum of a control person, and the vertical axis represents each measured It represents the concentration (ng / mL) of periostin contained in the sample.

(2) ROC curve based on measurement results Periostin concentration in each sample of affected individuals (total 8 cases) and control persons (total 71 cases) of cholangiocellular carcinoma in each of the above (1) and (2) Based on the measurement results, an ROC curve (Receiver Operating Characteristic Curve) was created.
This ROC curve is shown in FIG.
In FIG. 2, the horizontal axis represents “1-specificity” and the vertical axis represents “sensitivity”.

  In FIG. 2, the ROC curve of the measurement result in the measurement using the polyclonal antibody as the anti-periostin antibody in (2) above is represented by a broken line, and the monoclonal antibody is used as the anti-periostin antibody in (2) above The ROC curve of the measurement result in the measured measurement is represented by a solid line.

In addition, the area under the curve (Area Under the Curve; AUC) of the ROC curve of the measurement result in the measurement using the polyclonal antibody was 0.791.
Moreover, the area under the curve (AUC) of the ROC curve of the measurement result in the measurement using the monoclonal antibody was 0.852.

4). Discussion (1) About the measurement result of the periostin concentration in the sample (a) From the above measurement result when using the polyclonal antibody on the left side of FIG. It can be seen that the periostin concentration in the serum is widely distributed from a low value to a high value.

By the way, as described above, periostin is high in those suffering from cholangiocellular carcinoma, but other diseases such as hepatocellular carcinoma, cirrhosis, sclerosing hepatocellular carcinoma, sarcomatous hepatocellular carcinoma, vascular muscle It is low in patients with lipoma or angiosarcoma, or in healthy individuals.
Therefore, in the measurement results when using the polyclonal antibody on the left side of FIG. 1, the periostin concentration in the serum in the control person is moderate to high because of false positives.
Therefore, in this measurement of the periostin concentration, it is desirable to clearly discriminate positive in those suffering from cholangiocellular carcinoma and clearly discriminate negative in the subject, that is, non-affected.

In the measurement result when using a polyclonal antibody on the left side of FIG. 1, the cut-off value for distinguishing between affected (positive) and unaffected (negative) is high in specificity and sensitivity. The cut-off value was set so that the balance between the two would be improved, and the periostin concentration was set to 200 ng / mL.
At this time, the sensitivity of the measurement using this polyclonal antibody [positive rate in the affected group in this measurement (true positive rate)] is 62.5%, and the specificity [non-affected group in this measurement] The negative rate (true negative rate; that is, 1-false positive rate)] was 74.6%.

(B) From the above measurement result when using a monoclonal antibody on the right side of FIG. 1, it can be seen that the distribution of the periostin concentration in the serum of the control person is gathered in a lower value range.
In the measurement result when using a monoclonal antibody on the right side of FIG. 1, the cutoff value for distinguishing between affected (positive) and unaffected (negative) has high specificity and higher sensitivity. The cut-off value was set so as to improve the balance between the two so that it could be taken and the periostin concentration was set to 154 ng / mL.
At this time, the sensitivity of the measurement using this monoclonal antibody was 75.0%, and the specificity was 94.4%.

(C) In other words, as a result of this, when a monoclonal antibody is used as an anti-periostin antibody used for measurement of periostin, it is distinguished between affected (positive) and unaffected (negative) when using a monoclonal antibody. It can be seen that the cut-off value for reducing the periostin concentration by 23% from the periostin concentration of 200 ng / mL to 154 ng / mL.

  It can be seen that the sensitivity of the measurement can be increased from 62.5% to 75.0% when the monoclonal antibody is used as compared with the case where the polyclonal antibody is used as the anti-periostin antibody.

  Furthermore, similarly, it can be seen that the specificity of measurement could be increased from 74.6% to 94.4% by using a monoclonal antibody.

  From these, in the measurement using the antigen-antibody reaction of periostin contained in the sample, the sensitivity of measurement and the specificity of measurement can be improved by using a monoclonal antibody as an anti-periostin antibody, that is, It was confirmed that the accuracy of the measurement can be improved.

(2) About ROC curve based on measurement result From FIG. 2, compared with the ROC curve (broken line) of the measurement result when a polyclonal antibody is used as an anti-periostin antibody, the measurement result when a monoclonal antibody is used It can be seen that the ROC curve (solid line) is improved in both sensitivity and specificity.
And compared with the case where a polyclonal antibody is used as an anti-periostin antibody (dashed line), the area under the curve (AUC) is increased (improved) from 0.791 to 0.852 when a monoclonal antibody is used (solid line). It can be seen that the accuracy of the measurement is improved.

  From the results of this ROC curve, it is possible to improve the measurement sensitivity and the measurement specificity by using a monoclonal antibody as the anti-periostin antibody in the measurement using the antigen-antibody reaction of periostin contained in the sample. It has been determined that what can be done, that is, the accuracy of the measurement can be improved.

[Example 4] (Confirmation of effect of the present invention -2)
The effect of the measurement accuracy improving method of the present invention was confirmed.

1. Sample As a sample, the serum of a patient suffering from pulmonary fibrosis or the serum of a healthy person was used as follows.
(1) Serum of persons with pulmonary fibrosis (total 37 cases) (2) Serum of healthy persons (total 66 cases)

2. Measurement of periostin concentration in a sample (1) Measurement using a polyclonal antibody as an anti-periostin antibody The concentration of periostin contained in each sample of (1) and (2) above is determined by fluorescence immunization using an anti-periostin polyclonal antibody. It measured by the measuring method.

  This measurement operation was performed in accordance with the procedure of (a) to (l) in 2 (1) of Example 3.

(2) Measurement using monoclonal antibody as anti-periostin antibody The concentration of periostin contained in each sample of (1) and (2) above is determined by enzyme immunoassay (ELISA method) using anti-periostin monoclonal antibody. It was measured.

  This measurement operation was performed in accordance with the procedure of (a) to (k) in 2 (2) of Example 3.

3. Measurement Result (1) Measurement Result of Periostin Concentration in Sample In each sample of patients with pulmonary fibrosis (total 37 cases) and healthy persons (total 66 cases) in 2 (1) and (2) above The measurement results of the periostin concentration are shown in FIG.

The measurement result in the measurement using the polyclonal antibody as the anti-periostin antibody in (2) above is shown on the left side of FIG.
Moreover, the measurement result in the measurement using a monoclonal antibody as the anti-periostin antibody of (2) above is shown on the right side of FIG.

  And in either of the left side and the right side of FIG. 3, the horizontal axis represents whether the sample is serum of a patient with pulmonary fibrosis or serum of a healthy person, and the vertical axis represents each measured value. It represents the concentration (ng / mL) of periostin contained in the sample.

(2) ROC curve based on measurement results Periostin concentration in each sample of affected individuals (37 cases in total) and healthy subjects (total of 66 cases) of pulmonary fibrosis in (1) and (2) above An ROC curve was created based on the measurement results.
This ROC curve is shown in FIG.
In FIG. 4, the horizontal axis represents “1-specificity” and the vertical axis represents “sensitivity”.

  In FIG. 4, the ROC curve of the measurement result in the measurement using the polyclonal antibody as the anti-periostin antibody of (2) is represented by a broken line, and the monoclonal antibody is used as the anti-periostin antibody of (2). The ROC curve of the measurement result in the measured measurement is represented by a solid line.

In addition, the area under the curve (AUC) of the ROC curve of the measurement result in the measurement using the polyclonal antibody was 0.776.
Moreover, the area under the curve (AUC) of the ROC curve of the measurement result in the measurement using the monoclonal antibody was 0.905.

4). Discussion (1) About the measurement result of the periostin concentration in the sample (a) From the measurement result in the case of using the polyclonal antibody on the left side of FIG. 3, not only in patients suffering from pulmonary fibrosis but also in healthy persons, It can be seen that the periostin concentration in the serum is widely distributed from a low value to a high value.

In the measurement result when using a polyclonal antibody on the left side of FIG. 3, the cut-off value for distinguishing between affected (positive) and unaffected (negative) has high specificity and higher sensitivity. The cut-off value was set so that the balance between the two would be improved, and the periostin concentration was set to 200 ng / mL.
At this time, the sensitivity of the measurement using this polyclonal antibody was 56.8%, and the specificity was 83.3%.

(B) From the measurement result when using a monoclonal antibody on the right side of FIG. 3, it can be seen that the distribution of periostin concentration in the serum of a healthy person is gathered in a lower value range.
In the measurement result when using a monoclonal antibody on the right side of FIG. 3, the cut-off value for distinguishing between affected (positive) and unaffected (negative) has high specificity and higher sensitivity. The cut-off value was set so as to improve the balance between the two so that it could be taken, and the periostin concentration was set to 82 ng / mL.
At this time, when this monoclonal antibody was used, the sensitivity of the measurement was 62.2%, and the specificity was 95.5%.

(C) In other words, as a result of this, when a monoclonal antibody is used as an anti-periostin antibody used for measurement of periostin, it is distinguished between affected (positive) and unaffected (negative) when using a monoclonal antibody. It can be seen that the cut-off value for reducing the periostin concentration from 59 ng / mL to 82 ng / mL could be reduced by 59%.

  It can be seen that the sensitivity of the measurement can be increased from 56.8% to 62.2% when the monoclonal antibody is used compared to the case where the polyclonal antibody is used as the anti-periostin antibody.

  Furthermore, similarly, it can be seen that the specificity of measurement could be increased from 83.3% to 95.5% by using a monoclonal antibody.

  From these, in the measurement using the antigen-antibody reaction of periostin contained in the sample, the sensitivity of measurement and the specificity of measurement can be improved by using a monoclonal antibody as an anti-periostin antibody, that is, It was confirmed that the accuracy of the measurement can be improved.

(2) About the ROC curve based on the measurement result From FIG. 4, the measurement result when using a monoclonal antibody is compared with the ROC curve (broken line) of the measurement result when using a polyclonal antibody as an anti-periostin antibody. It can be seen that the ROC curve (solid line) is improved in both sensitivity and specificity.

  Then, the area under the curve (AUC) increased from 0.776 to 0.905 when the monoclonal antibody was used (solid line) compared to when the polyclonal antibody was used as the anti-periostin antibody (dashed line). It can be seen that the accuracy of the measurement is improved.

  From the results of this ROC curve, it is possible to improve the measurement sensitivity and the measurement specificity by using a monoclonal antibody as the anti-periostin antibody in the measurement using the antigen-antibody reaction of periostin contained in the sample. It has been determined that what can be done, that is, the accuracy of the measurement can be improved.

Claims (1)

  An antiperiostin monoclonal antibody is used in measurement using an antigen-antibody reaction of periostin contained in a sample, and the measurement accuracy is improved.

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