WO2018030456A1

WO2018030456A1 - Reagent for measuring periostin included in sample, pretreatment agent for periostin measurement, method for measuring periostin, and method for improving sensitivity of periostin measurement

Info

Publication number: WO2018030456A1
Application number: PCT/JP2017/028895
Authority: WO
Inventors: 出原　賢治; 雅之 ▲高▼井; 純也小野
Original assignee: 株式会社シノテスト; 国立大学法人佐賀大学
Priority date: 2016-08-10
Filing date: 2017-08-09
Publication date: 2018-02-15
Also published as: JP2021073435A; JP7006599B2; JPWO2018030456A1; JP7066142B2

Abstract

[Problem] To provide a high-sensitivity periostin measurement reagent that has improved sensitivity, a pretreatment agent for periostin measurement, and a method for measuring periostin that are for measuring the periostin included in a sample using an antigen-antibody reaction or the like; and to provide a method for improving the sensitivity of measurement of the preiostin included in a sample. [Solution] The present invention is characterized in that, when the periostin included in a sample is measured using an antigen-antibody reaction between the periostin included in the sample and an antibody that binds specifically to periostin, the periostin is brought into contact with a surfactant or a reducing agent.

Description

Periostin measurement reagent contained in sample, pretreatment agent for periostin measurement, periostin measurement method, and method for improving sensitivity of periostin measurement

The present invention relates to a measurement reagent for periostin (also referred to as osteoblast-specific factor 2 or OSF2) that can be a marker for allergic diseases and other diseases, a pretreatment agent for measurement, a measurement method, and a method for improving measurement sensitivity. .
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).
And Idehara et al. Found that the accuracy of periostin measurement was improved by using an anti-periostin monoclonal antibody (see Patent Document 3), and Idehara et al. Detected a specific region of periostin. The examination method etc. of the pulmonary fibrosis or the interstitial pneumonia with which the precision was improved were found (refer patent document 4).

In addition, a polyclonal antibody, a monoclonal antibody against OSF2 (periostin), a diagnostic method using these antibodies, etc. are disclosed (see Patent Document 5), and a novel osteoblast-specific transcription factor named Osf2 / Cbfa1 An immunoassay using an anti-OSF2 (periostin) antibody is disclosed (see Patent Document 6), 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 7), and an antibody against periostin having anti-cell adhesion activity and a method for quantifying periostin using this antibody are disclosed (see Patent Document 8). .)

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

International Publication No. 02/052006 Pamphlet International Publication No. 09/148184 Pamphlet JP 2012-58048 A International Publication No. 13/035799 Pamphlet Japanese Patent Laid-Open No. 5-268882 Special Table 2002-502250 JP 2005-500059 Publication International Publication No. 07/077934 Pamphlet

In the above-described measurement using the antigen-antibody reaction of periostin contained in the sample, the sensitivity of the measurement was not sufficient.
For this reason, in the measurement using the antigen-antibody reaction of periostin contained in a sample, further improvement in sensitivity of measurement has been desired.
On the other hand, an object of the present invention is to provide a highly sensitive measurement reagent with improved sensitivity, a pretreatment agent for measurement, and a measurement method in measurement using an antigen-antibody reaction of periostin contained in a sample. And providing a method for improving the sensitivity of the measurement of periostin contained in a sample.
In the present invention, the sensitivity is the amount of change in the amount to be measured (ratio of the magnitude of the response to the change in the amount to be measured).

The present inventors have repeatedly investigated the measurement of periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample. As a result, the periostin and a surfactant or It has been found that the above problem can be solved by bringing a reducing agent into contact with the reducing agent, and the present invention has been completed.

That is, this invention consists of the following invention.
(1) A measurement reagent for periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and an antibody that specifically binds to periostin, including an antibody that specifically binds to periostin, A reagent for measuring periostin contained in a sample, comprising an activator or a reducing agent.
(2) A pretreatment agent for measuring periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample, which contains a surfactant or a reducing agent A pretreatment agent for measuring periostin,
(3) In a method for measuring periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample, the periostin is brought into contact with a surfactant or a reducing agent. A method for measuring periostin contained in a sample.
(4) In the measurement of periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample, the periostin is brought into contact with a surfactant or a reducing agent. And a method for improving the sensitivity of measurement of periostin contained in a sample.

The periostin measurement reagent, preiostin measurement pretreatment agent, periostin measurement method, and periostin measurement sensitivity improvement method of the present invention can improve the sensitivity of the measurement in measuring periostin contained in a sample. is there.
Thereby, the measurement reagent of periostin of the present invention, the pretreatment agent for periostin measurement, the periostin measurement method, and the method for improving the sensitivity of periostin measurement can obtain a large signal as a measurement value in the measurement.
Therefore, in the measurement reagent for periostin, the pretreatment agent for periostin measurement, the method for measuring periostin, and the method for improving the sensitivity of periostin measurement according to the present invention, it is possible to measure even very low concentrations of periostin.
In addition, in the measurement reagent for periostin of the present invention, the pretreatment agent for periostin measurement, the periostin measurement method, and the method for improving the sensitivity of periostin measurement, although the reagent blind test (reagent blank) is not changed, the sensitivity is improved. Accurate measurement of periostin can be performed.
Furthermore, in the measurement reagent for periostin, the pretreatment agent for measuring periostin, the method for measuring periostin, and the method for improving the sensitivity of periostin measurement, the signal obtained as a measurement value is large. It is possible to dilute, and as a result, it is possible to reduce the degree of interference with measurement due to matrix components of a sample such as serum.

It is the figure which showed the measurement result of periostin contained in the serum sample.

Hereinafter, the present invention will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone. The present invention can be implemented in various forms without departing from the gist thereof.
All publications cited herein, for example, prior art documents, publications, patent publications and other patent documents, are incorporated herein by reference.

[1] Anti-periostin antibody Antibody In the measurement reagent for periostin contained in the sample of the present invention, the pretreatment agent for measuring periostin, the method for measuring periostin contained in the sample, and the method for improving the sensitivity of the measurement of periostin contained in the sample, specifically periostin An antibody that binds (hereinafter sometimes referred to as “anti-periostin antibody”) is used to cause an antigen-antibody reaction with periostin contained in the sample (or used for the antigen-antibody reaction).

In the present invention, the anti-periostin antibody is not particularly limited as long as it is an antibody that can specifically bind to periostin.

Examples of the anti-periostin antibody include monoclonal antibodies, polyclonal antibodies, antisera, antibody fragments (such as Fab and F (ab ′) ₂ ) that can bind to periostin, single-chain antibodies (scFv), and the like. Can be mentioned.

This anti-periostin antibody is an antibody (such as a chimeric antibody, humanized antibody, or 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. Also good.

The anti-periostin antibody is preferably a monoclonal antibody.

In the present invention, two or more kinds of anti-periostin antibodies may be used.

2. Immunogen The immunogen for producing the anti-periostin antibody in the present invention will be described below.
As an immunogen for producing an anti-periostin antibody in the present invention, all or a part of periostin can be used.
That is, all or part of periostin such as periostin derived from humans or mammals such as cows, pigs, dogs, cats, mice or rats, or birds such as chickens, or periostin obtained by gene recombination can be used. .

By using all or part of the periostin as an immunogen, the anti-periostin antibody in the present invention can be obtained.
The immunogen for producing this anti-periostin antibody is 1 to several (usually 1 to 8, preferably 1 to 6, more preferably 1 to the whole or a 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 ˜4 (particularly preferably 1 to 2) amino acid residues.

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).
Therefore, the minimum unit of the amino acid sequence of the immunogen of the anti-periostin antibody in the present invention is 1 to several in the whole or part of the amino acid sequence of periostin, or in the whole or part of the amino acid sequence of these amino acid sequences. By deletion, substitution, insertion, addition or modification of 1 (usually 1 to 8, preferably 1 to 6, more preferably 1 to 4, particularly preferably 1 to 2) amino acid residues Since an amino acid sequence consisting of three consecutive amino acid residues can be considered in the obtained amino acid sequence, a tripeptide consisting of an amino acid sequence consisting of these three consecutive amino acid residues, or another amino acid or A peptide added, etc. can be considered as the minimum immunogen unit of the anti-periostin antibody in the present invention. .

Peptide or protein containing the whole or part of the amino acid sequence of periostin as the immunogen, or 1 to several (usually 1 to 8) in the whole or part of the amino acid sequence of periostin A peptide comprising an amino acid sequence obtained by performing deletion, substitution, insertion, addition or modification of amino acid residues (preferably 1 to 6, more preferably 1 to 4, particularly preferably 1 to 2) Alternatively, proteins and the like can be obtained by extracting, purifying, and the like from body fluids such as humans, cells, tissues, or organs by a known method.

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, as a method for obtaining human periostin, the following method (“G. Takayama et al., J. Allergy Clin. Immunol., 118, No. 1, pages 713 to 723, issued in 2006”) can be mentioned. it can.
(A) First, a recombinant periostin protein obtained by adding a V5 / His tag to periostin (base sequence of polynucleotide: Accession Number D13666 of nucleic acid database GenBank; amino acid sequence: Accession Number BAA02837 of nucleic acid database GenBank) in S2 cells which are insect cells Purify after expression.

(B) Specifically, 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), which is a plasmid expressing pMT / Bip / periostin-V5-HisA and a hygromycin resistance gene, is co-introduced into S2 cells by a known method and transformed.
A transformant is selected with hygromycin to obtain a stable transformant.
Then, in the transformant of S2 cell, periostin in which V5 epitope / His tag is bound is expressed at the carboxy terminus.

(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 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 human periostin protein.

Human 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 human periostin whose amount (concentration) has been clarified.

Furthermore, human periostin can also be obtained, for example, by the method described in “I. Takayama et al., J. Biochem., 146, 5, 713-723, 2009”.

The EMI region of human periostin is, for example, “I. Kii et al., J. Biol. Chem., 285, No. 3, pp. 2028-2039, 2010” or “T. Maruhashi et al., J. Biol. Biol. Chem., 285, No. 17, 13294-13303, published in 2010 ”and the like.

In addition, the R1 region, R2 region or R3 region of human periostin is obtained by the method described in “I. Takayama et al., J. Biochem, Vol. 146, No. 5, pages 713 to 723, issued in 2009”, etc. Can be acquired.

In addition, the amino acid sequences of the R4 region and C-terminal region of human periostin are described in “I. Takayama et al., J. Biochem, Vol. 146, No. 5, pages 713 to 723, published in 2009” and the like, respectively. It can be obtained by.

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.
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, 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 | etc.,.

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 the case of using a carrier when producing antibodies, 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.
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. Preparation method of anti-periostin antibody which is polyclonal antibody A polyclonal antibody which can specifically bind to periostin, that is, an anti-periostin antibody which is a polyclonal antibody can be prepared by the following operation.

As the immunogen for producing this polyclonal antibody, anti-periostin antibody, the above-mentioned immunogen can be used.

The immunogen or the conjugate of the immunogen and a carrier (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).

In addition, as an immunized animal for immunizing the immunogen or the conjugate of the immunogen and the carrier, 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.

The reason for this is that periostin produced in the animal's body binds to anti-periostin antibody produced in the body by immunization with an immunogen such as periostin, thereby reducing the antibody activity of the anti-periostin antibody. This is because the possibility is low in the knockout animals.
In the knockout animal, since periostin is not produced in the body of the animal, it is easy to recognize the immunized periostin as a foreign substance, and thus the production of antibodies is increased.

Examples of animals 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 ").

By the way, 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. Preferably, 0.1 μg 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.
As the 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.

After the first immunization, 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.
Also in this case, the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
After the first immunization, 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.

By the above operation, a polyclonal antibody (anti-periostin antibody which is a polyclonal antibody) capable of binding to periostin can be obtained.

By the way, when an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier is present in the obtained polyclonal antibody. Is preferred.

As this removal treatment method, 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.

4). Preparation Method of Anti-Periostin Antibody that is Monoclonal Antibody A monoclonal antibody that can bind to periostin, that is, an anti-periostin antibody that is a monoclonal antibody can be prepared by the following procedure.

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.

Furthermore, it is also possible to prepare a monoclonal antibody from a cDNA library of antibody genes using the phage display method of McCaffety et al. (M. McCafferty et al., Nature, 348, 552-554, published in 1990). is there.

In addition, for example, preparation of a monoclonal antibody by a cell fusion method can be performed by the following operation.

(1) First, 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).

Examples of animals 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 ")).

By the way, 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. Preferably, 0.1 μg 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.
Known 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.

(2) After the first immunization, 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.
Also in this case, the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.

(3) After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA, etc. When the antibody titer reaches a plateau, 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.

(4) Three to five days after the final immunization, cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.

(5) Cell fusion of antibody-producing cells obtained from this immunized animal and myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). A cell line deficient in an enzyme such as hypoxanthine, guanine, phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferable. For example, P3-X63-Ag8, which is a HGPRT-deficient cell line derived from BALB / c mice. Strain (ATCC TIB9), P3-X63-Ag8-U1 strain (cancer research source bank [JCRB] 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8-653 strain ( JCRB 0028) or SP2 / O-Ag-14 stock (JCRB 002) ), Or the like can be used.

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 the myeloma cells are of HGPRT-deficient strain or TK-deficient strain, by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine, cells having antibody-producing ability and myeloma cells Only fused cells (hybridomas) can be selectively cultured and grown.

(6) The hybridoma culture supernatant thus obtained is used for humans or animals such as cows, pigs, dogs, cats, mice, rats or chickens (for example, humans when used for measurement of human periostin) Derived from bovine periostin, preferably bovine derived, and preferably used from dog periostin, preferably derived from dog periostin) Alternatively, a hybridoma that produces a “monoclonal antibody capable of binding to periostin (an anti-periostin antibody that is a monoclonal antibody)” is measured by an immunoassay such as an ELISA method or a Western blot method using a peptide or the like. Can be selected.

(7) 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 antibody that is a monoclonal antibody in the present invention can be isolated and obtained.

(8) The monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the antiperiostin antibody, which is a monoclonal antibody in the present invention, can be obtained from the culture supernatant. A serum medium or the like may be used, and 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, 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 antibodies can also be obtained.

(9) The thus obtained anti-periostin antibody, which is a monoclonal antibody, is obtained by methods such as salting out using ammonium sulfate, sodium sulfate, etc., ion exchange chromatography, gel filtration or affinity chromatography, or these methods. The anti-periostin antibody which is a purified monoclonal antibody can be obtained by combining, for example.

(10) As described in (6) above, the culture supernatant of the obtained hybridoma is subjected to ELISA or Western blotting using a protein or peptide comprising all or part of periostin of an animal such as human or dog. The hybridoma which produces the anti-periostin antibody which is a monoclonal antibody can be selected by measuring by immunological measuring methods, such as.

(11) From the hybridoma producing this “monoclonal antibody, anti-periostin antibody”, “monoclonal antibody, anti-periostin antibody” can be obtained as described in (7) to (9) above.

The obtained “anti-periostin antibody that is a monoclonal antibody” is an antibody that can specifically bind to periostin.

[2]. Surfactant General The measurement reagent for periostin contained in the sample of the present invention is a measurement reagent for periostin contained in a sample using an antigen-antibody reaction between an anti-periostin antibody and periostin contained in the sample, and is a surfactant or reducing agent It is characterized by containing.

Further, the pretreatment agent for measuring periostin according to the present invention is a pretreatment agent for measuring periostin contained in a sample using an antigen-antibody reaction between an anti-periostin antibody and periostin contained in the sample, and is a surfactant. Or it contains a reducing agent.

The method for measuring periostin contained in the sample of the present invention and the method for improving the sensitivity of the measurement of periostin contained in the sample are characterized by bringing the periostin into contact with a surfactant or a reducing agent.

The periostin measurement reagent and the pretreatment agent for periostin measurement contained in the sample of the present invention can improve the sensitivity of the measurement of periostin contained in the sample by containing a surfactant or a reducing agent. is there.

In the method for measuring periostin contained in the sample of the present invention and the method for improving the sensitivity of periostin measurement contained in the sample, the periostin is measured by bringing the periostin into contact with a surfactant or a reducing agent. The sensitivity can be improved.

Incidentally, in the method for measuring periostin contained in the sample of the present invention and the method for improving the sensitivity of the measurement of periostin contained in the sample, contact between the periostin and the surfactant, for example, an anti-periostin antibody and periostin contained in the sample The periostin is brought into contact with a surfactant before the antigen-antibody reaction or during the antigen-antibody reaction.

2. Surfactant in the Present Invention Examples of the surfactant in the present invention include amphoteric surfactants, nonionic surfactants, anionic surfactants, and cationic surfactants.

Examples of the surfactant include amphoteric surfactants such as carboxybetaine type amphoteric surfactants, glycine type amphoteric surfactants, amine oxide type amphoteric surfactants or 2-alkylimidazoline-derived amphoteric surfactants; Polyoxyethylene alkyl ether, polyoxypropylene alkyl ether, polyoxyethylene-polyoxypropylene-alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene-fatty acid ester, polyoxypropylene-fatty acid ester, polyoxyethylene alkylamine, Polyoxyethylene alkylamide, sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene Nonionic surfactant such as lysine fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene phytosterol, phytostanol, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; sulfate salt, sulfonate salt, fatty acid salt And anionic surfactants such as phosphate ester salts or amino acid type surfactants; or cationic surfactants such as amine salt type, trimethyl type, dialkyl type or benzyl type.

In addition, examples of the carboxybetaine-type amphoteric surfactant that is an amphoteric surfactant include alkyldimethylaminoacetic acid betaine and fatty acid amidopropyldimethylamino acid betaine.
Examples of this alkyldimethylaminoacetic acid betaine include lauryldimethylaminoacetic acid betaine.

Further, examples of the glycine-type amphoteric surfactant include alkyl diaminoethyl glycine and dialkyl diaminoethyl glycine.

Further, examples of the amine oxide type amphoteric surfactant include alkyl dimethyl amine oxide.

Examples of 2-alkylimidazoline-derived amphoteric surfactants include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine.

The amphoteric surfactant, lauryldimethylaminoacetic acid betaine, is a product name of Nissan Anon (registered trademark) BL or Nissan Anone (registered trademark) BL-SF, and palm oil dimethyl-aminoacetic acid betaine is Nissan Anone (registered trademark). ) Palm oil fatty acid-amidopropyldimethyl-aminoacetate betaine is a product name of Nissan Anon (registered trademark) BDF-R or Nissan Anone (registered trademark) BDF-SF; Dimethyl-aminoacetic acid betaine is the product name Nissan Anone (registered trademark) BDC-SF, Lauric acid-amidopropyldimethyl-aminoacetic acid betaine is the product name Nissan Anone (registered trademark) BDL-SF, and lauryl diaminoethyl-glycine. Sodium solution is Nissan NON (registered trademark) LG-R is a trade name of 2-alkyl-N-carboxymethyl-N-hydroxyethyl-imidazolinium betaine (imidazoline type). NISSAN ANON (registered trademark) GLM-R or NISSAN ANON (registered) (Trademark) GLM-R-LV (viscosity improving type), and laurylaminodiacetic acid-monosodium is Nissan Anon (registered trademark) LA or Nissan Anone (registered trademark) LA powder, respectively. It is commercially available from Co., Ltd. (Japan).

Examples of polyoxyethylene alkyl ethers that are nonionic surfactants include polyoxyethylene linear alkyl ethers (for example, polyoxyethylene octyl ether, polyoxyethylene nonyl ether, polyoxyethylene lauryl ether, polyoxyethylene lauryl ether, Oxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, or polyoxyethylene behenyl ether), or a branched alkyl ether of polyoxyethylene.

Examples of the polyoxyethylene alkylphenyl ether that is a nonionic surfactant include polyoxyethylene linear alkylphenyl ether (for example, polyoxyethylene octylphenyl ether [4- (1,1,3,3 -Tetramethylbutyl) phenyl-polyethylene glycol [trade name: Triton X-100], polyoxyethylene (40) isooctyl phenyl ether [trade name: Triton X-405], etc., polyoxyethylene nonylphenyl ether, polyoxy Ethylene lauryl phenyl ether or polyoxyethylene stearyl phenyl ether), or polyoxyalkylene branched alkylphenyl ether.

Examples of polyoxyethylene-fatty acid esters that are nonionic surfactants include polyoxyethylene linear fatty acid esters (for example, polyoxyethylene laurate, polyoxyethylene stearate, polyoxyethylene oleate, Or palm oil fatty acid polyoxyethylene), polyoxyethylene branched chain fatty acid ester, polyoxyethylene linear alkyl-substituted benzoic acid ester (for example, octyl benzoic acid polyoxyethylene, nonyl benzoic acid polyoxyethylene, lauryl benzoic acid poly Oxyethylene or polyoxyethylene stearyl benzoate), or branched alkyl-substituted benzoic acid esters of polyoxyethylene.

In addition, as a sulfate ester salt which is an anionic surfactant, an alkyl sulfate ester salt, a polyoxyethylene alkyl ether sulfate ester salt, etc. can be mentioned, for example.

Examples of the alkyl sulfate salt include sodium lauryl sulfate (sodium dodecyl sulfate), alkyl sulfate ester-triethanolamine salt, and 2-ethylhexyl sulfate sodium salt.

Also, examples of the polyoxyethylene alkyl ether sulfate ester salt include sodium polyoxyethylene lauryl ether sulfate and polyoxyethylene alkyl ether sulfate triethanolamine.

Examples of sulfonates that are anionic surfactants include alkylbenzene sulfonates, α-sulfo fatty acid methyl ester salts, α-olefin sulfonates, dialkyl sulfosuccinates and other sulfonates. Can be mentioned.

Examples of the alkyl benzene sulfonate include linear alkyl benzene sulfonic acid, linear alkyl benzene sulfonic acid sodium, dodecyl benzene sulfonic acid, sodium dodecyl benzene sulfonate, and the like.

Examples of the dialkyl sulfosuccinate include sodium dialkyl sulfosuccinate and di-2-ethylhexyl-sodium sulfosuccinate.

Examples of other sulfonates include sodium alkylnaphthalene sulfonate, sodium alkyldiphenyl ether disulfonate, and sodium alkane sulfonate.

In addition, examples of fatty acid salts that are anionic surfactants include alkyl ether carboxylates.

Examples of amino acid type surfactants that are anionic surfactants include acyl-N-methyl taurine, fatty acid sodium methyl taurate, coconut oil fatty acid methyl tauric acid-sodium salt, and N-decanoyl-N-methyl. Tauric acid-sodium salt, N-lauroyl-N-methyl-β-alanine sodium salt, N-lauroyl-N-methyl-β-alanine triethanolamine salt, α-sulfo fatty acid methyl ester sodium salt, fatty acid amide ether sulfate -Ester sodium salt, N-oleyl-N-methylglycine, N-lauroyl-N-methylglycine-sodium salt and the like.

Each of the above surfactants is commercially available from NOF Corporation (Japan), Kao Corporation (Japan), Nikko Chemicals Corporation (Japan), Wako Pure Chemical Industries, Ltd. (Japan), etc. ing.

3. Concentration In the method for measuring periostin contained in the sample of the present invention and the method for improving the sensitivity of periostin measurement contained in the sample, the concentration of the surfactant when contacting the surfactant with periostin contained in the sample is: Although not particularly limited, it is preferably 0.01% (w / v) or more at the time of contact between the periostin and the surfactant.

In addition, the minimum of the preferable density | concentration of surfactant at the time of the contact with the said periostin and surfactant becomes like this. More preferably, it is 0.04% (w / v) or more, More preferably, it is 0.12% (w / v). ) Or more, particularly preferably 0.5% (w / v) or more.

Further, the concentration of the surfactant at the time of contact between the periostin and the surfactant is not particularly limited, but up to 20% (w / v) is sufficient in consideration of cost and the like.

In addition, the minimum of the preferable density | concentration of surfactant at the time of the contact of the said periostin and surfactant becomes like this. More preferably, it is 10% (w / v) or less, Most preferably, it is 5% (w / v) or less. .

In the case where the periostin contained in the sample is brought into contact with the surfactant, the measurement is performed by adding a surfactant to the periostin measurement reagent or the pretreatment agent for measuring periostin contained in the sample of the present invention. The periostin and the surfactant may be contacted by mixing the reagent or the pretreatment agent and the sample.
In this case, a surfactant is included in the measurement reagent or the pretreatment agent so that the concentration of the surfactant when the measurement reagent or the pretreatment agent and the sample are mixed is the above concentration. Is preferred.

For example, when the measurement reagent is composed of a first reagent and a second reagent, and the reagent component that causes the antigen-antibody reaction is contained in the second reagent, the periostin and the surfactant are added before the antigen-antibody reaction. When contacting, the surfactant may be contained in the first reagent. (The first reagent may be a pretreatment agent for measuring periostin.) When the periostin and the surfactant are brought into contact with each other during the antigen-antibody reaction, the surfactant is contained in the second reagent. Just do it.

In addition, for example, when the measurement reagent includes the first reagent, the second reagent, the third reagent, the fourth reagent, and the fifth reagent, and the reagent component that causes the antigen-antibody reaction is included in the fourth reagent, the antigen When the periostin and the surfactant are brought into contact before the antibody reaction, the surfactant may be contained in the first reagent, the second reagent, and / or the third reagent. (These first reagent, second reagent and / or third reagent may be a pretreatment agent for measuring periostin.) Also, when periostin and surfactant are brought into contact during the antigen-antibody reaction. The surfactant may be contained in the fourth reagent.

Further, for example, when the periostin and the surfactant are brought into contact with each other before the antigen-antibody reaction between the anti-periostin antibody and periostin contained in the sample, a pretreatment agent for measuring periostin (for example, a diluted solution of the sample) The periostin and the surfactant may be brought into contact with each other by mixing the pretreatment agent and the sample.
In this case, the surfactant is mixed so that the concentration of the surfactant when the periostin and the surfactant are brought into contact with each other before the antigen-antibody reaction is mixed with the pretreatment agent and the sample, etc. The pretreatment agent is preferably contained.

4). Use of Surfactant, etc. In the present invention, the surfactant may be measured using one type, or may be measured using a plurality of types.

In the present invention, for the purpose of improving the sensitivity of measurement of periostin contained in a sample, as the surfactant, an amphoteric surfactant, a nonionic surfactant or an anionic surfactant is preferable.

As the amphoteric surfactant, a betaine-type amphoteric surfactant is preferable, an alkyldimethylbetaine-type amphoteric surfactant is more preferable, and lauryldimethylaminoacetic acid betaine is particularly preferable.

Further, as the anionic surfactant, a sulfate ester salt is preferable, an alkyl sulfate ester salt is more preferable, and sodium lauryl sulfate is particularly preferable.

[3]. Reducing agent General The measurement reagent for periostin contained in the sample of the present invention is a measurement reagent for periostin contained in a sample using an antigen-antibody reaction between an anti-periostin antibody and periostin contained in the sample, and is a surfactant or reducing agent It is characterized by containing.

Incidentally, in the method for measuring periostin contained in the sample of the present invention and the method for improving the sensitivity of periostin measurement contained in the sample, it is contact between the periostin and the reducing agent. For example, an anti-periostin antibody and periostin contained in the sample Examples include contacting the periostin with a reducing agent before the antigen-antibody reaction or at the time of the antigen-antibody reaction.

2. Reducing agent in the present invention (1) General remarks The reducing agent in the present invention is not particularly limited as long as it has a reducing ability.

Examples of the reducing agent include thiol compounds, “oxo acids having sulfur as a central atom, or thioacids or salts thereof”.

(2) Thiol Compound In the present invention, examples of the thiol compound include dithiothreitol (DTT), N-acetyl-L-cysteine (NAC), thioglycerol, reduced glutathione, cysteine, dithioerythritol, bromide 2 -SH groups such as aminoethylisothiouronium, 2-thioglucose, thioglycolic acid, 2-mercaptoethanol, N-guanyl-L-cysteine, mercaptoacetic acid, mercaptosuccinic acid, 2-mercaptoethanesulfonic acid, or cysteamine The compound which has can be mentioned.

(3) Oxyacid having sulfur as central atom or thioacid or salt thereof In the present invention, for example, oxoacid having sulfur as central atom or thioacid or salt thereof can be exemplified by sulfoxylic acid [ H ₂ SO ₂ ], sulfurous acid [H ₂ SO ₃ ], thiosulfuric acid [H ₂ S ₂ O ₂ ], thiosulfuric acid [H ₂ S ₂ O ₃ ], dithionic acid [H ₂ S ₂ O ₄ ], disulfurous acid [H ₂ S ₂ O ₅ ], dithionic acid [H ₂ S ₂ O ₆ ], disulfuric acid [H ₂ S ₂ O ₇ ], or polythionic acid [H ₂ SxO ₆ (x = 3, 4...)], Etc. Can be mentioned.
The “sulfur-centered oxoacid or this thioacid or salt thereof” is dissociated from the “sulfur-centered oxoacid or this thioacid” as a proton. This is a compound in which part or all of hydrogen that can be substituted is replaced with an alkali metal, an alkaline earth metal, another metal, or a basic group.
For example, examples of the alkali metal include lithium, sodium, potassium, rubidium, and cesium, and examples of the alkaline earth metal include beryllium, magnesium, calcium, strontium, and barium. Examples of the group include an ammonium group.

3. Concentration In the method for measuring periostin contained in the sample of the present invention and the method for improving the sensitivity of the measurement of periostin contained in the sample, the concentration of the reducing agent when bringing the periostin contained in the sample into contact with the reducing agent is particularly limited. However, it is preferably 0.001 mM or more upon contact between the periostin and the reducing agent.

In addition, the minimum of the preferable density | concentration of the reducing agent at the time of the contact with the said periostin and a reducing agent becomes like this. More preferably, it is 0.01 mM or more, Especially preferably, it is 0.05 mM or more.

The concentration of the reducing agent at the time of contact between the periostin and the reducing agent is not particularly limited, but up to 50 mM is sufficient in consideration of cost and the like.

In addition, the minimum of the preferable density | concentration of the reducing agent at the time of the contact with the said periostin and a reducing agent becomes like this. More preferably, it is 20 mM or less, Most preferably, it is 10 mM or less.

Further, when the periostin contained in the sample is brought into contact with the reducing agent, the measuring reagent or the pretreatment agent for measuring periostin contained in the sample of the present invention is allowed to contain a reducing agent, and the measuring reagent or The periostin and the reducing agent may be brought into contact by mixing the pretreatment agent and the sample.
In this case, it is preferable to contain the reducing agent in the measurement reagent or the pretreatment agent so that the concentration of the reducing agent when the measurement reagent or the pretreatment agent and the sample are mixed is the above concentration. .

For example, when the measurement reagent includes the first reagent and the second reagent, and the reagent component that causes the antigen-antibody reaction is included in the second reagent, the periostin and the reducing agent are contacted before the antigen-antibody reaction. When it is used, the reducing agent may be contained in the first reagent. (Note that this first reagent may be a pretreatment agent for measuring periostin.) When the periostin and the reducing agent are brought into contact with each other during the antigen-antibody reaction, the reducing agent may be contained in the second reagent. Good.

In addition, for example, when the measurement reagent includes the first reagent, the second reagent, the third reagent, the fourth reagent, and the fifth reagent, and the reagent component that causes the antigen-antibody reaction is included in the fourth reagent, the antigen When the periostin and the reducing agent are brought into contact before the antibody reaction, the reducing agent may be contained in the first reagent, the second reagent, and / or the third reagent. (These first reagent, second reagent and / or third reagent may be a pretreatment agent for measuring periostin.) When contacting the periostin with a reducing agent during the antigen-antibody reaction The reducing agent may be contained in the fourth reagent.

In addition, for example, when the periostin and the reducing agent are brought into contact before the antigen-antibody reaction between the anti-periostin antibody and periostin contained in the sample, a pretreatment agent for measuring periostin (for example, a diluted solution of the sample). The periostin and the reducing agent may be brought into contact with each other by mixing the pretreatment agent and the sample.
In this case, the pretreatment agent and the sample are mixed, etc., and the reducing agent is pretreated so that the concentration of the reducing agent at the time of contacting the periostin and the reducing agent before the antigen-antibody reaction is the above concentration. It is preferable to make it contain in an agent.

4). Use of Reducing Agent, etc. In the present invention, the reducing agent may be measured using one type or may be measured using a plurality of types.

In the present invention, the measurement may be performed using a combination of a reducing agent and a surfactant.

In the present invention, for the purpose of improving the sensitivity of measurement of periostin contained in a sample, the reducing agent is preferably a thiol compound, and dithiothreitol, N-acetyl-L-cysteine, thioglycerol, reduced glutathione, Cysteine or dithioerythritol is more preferable, dithiothreitol, N-acetyl-L-cysteine or thioglycerol is more preferable, and dithiothreitol is particularly preferable.

[4]. Samples Samples in the measurement reagent for periostin contained in the sample of the present invention, a pretreatment agent for periostin measurement, a method for measuring periostin contained in the sample, and a method for improving the sensitivity of periostin measurement contained in the sample include blood, serum Samples such as biological samples that may contain periostin, such as plasma, urine, semen, spinal fluid, saliva, ascites or amniotic fluid, or extracts of organs, tissues, or cells such as blood vessels or liver If there is any.

In addition, since it is preferable that the form of the sample used for the measurement is a liquid, if the sample is not a liquid, a process such as an extraction process or a solubilization process may be performed according to a known method to form a liquid sample.

In addition, the sample may be subjected to concentration treatment as necessary.

Further, the sample may be diluted by adding a diluent before the measurement.
For example, before the sample is brought into contact with and bound to the anti-periostin antibody, a dilution treatment may be performed by adding a diluent to the sample.
Various aqueous solvents can be used as the diluent.
For example, 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.
The pH of this buffer is preferably in the range of pH 5 to pH 10.

Further, when the sample is blood (whole blood), 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.

[5]. Measurement target substance Periostin measurement reagent contained in the sample of the present invention, pretreatment agent for periostin measurement, periostin measurement method contained in the sample, and periostin measurement sensitivity improvement method contained in the sample, the measurement target substance is periostin It is.

The periostin as a substance to be measured in the present invention is not particularly limited, and examples include periostin derived from humans or mammals such as cows, pigs, dogs, cats, mice or rats, or birds such as chickens. .

As the periostin as the measurement target substance, human periostin is particularly preferable.

[6]. Periostin measurement reagent General The measurement reagent for periostin contained in the sample of the present invention is a measurement reagent for periostin contained in a sample using an antigen-antibody reaction between an anti-periostin antibody and periostin contained in the sample, and is a surfactant or reducing agent It is characterized by containing.

The measurement reagent for periostin of the present invention can improve the sensitivity of the measurement in measuring periostin contained in a sample due to the above-described characteristics.

2. Periostin measurement reagent using antigen-antibody reaction Periostin measurement reagent contained in the sample of the present invention is a periostin measurement reagent contained in the sample using an antigen-antibody reaction between an anti-periostin antibody and periostin contained in the sample. It is characterized by containing a surfactant or a reducing agent, but if it is such, it is not particularly limited to its measurement principle, and it has the expected effect. is there.

As a measurement principle of a measurement reagent for measuring periostin contained in this sample using an antigen-antibody reaction with an anti-periostin antibody, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radiation Immunoassay (RIA), Luminescent immunoassay (LIA), Enzyme antibody method, Fluorescent antibody method, Immunochromatography method, Immunoturbidimetric method, Latex turbidimetric method, Latex agglutination method, Red blood cell agglutination method, Particle aggregation A carrier having a surface coated with a specific binding substance to a substance to be measured (test substance) described in a reaction method, JP-A-9-229936 and JP-A-10-132919, And a measurement method using particles in which a specific binding substance for a measurement target substance (test substance) is immobilized, or EL shown by Dahlback et al. A method (Enzyme-linked Ligandsorbent Assay) (Thromb.Haemost, 79, pp. 767-772 pp., 1998 issue;. WO 98/23963 pamphlet), and the like.

And any method, such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method), can be applied to the measurement using the measurement reagent of periostin of the present invention.
In addition, the measurement of the periostin measurement reagent of the present invention may be performed by a method, or may be performed using an apparatus such as an analyzer.

The measurement reagent for periostin of the present invention may comprise one measurement reagent.
In this case, the anti-periostin antibody is contained in the one measurement reagent.
In this case, a surfactant (or reducing agent) is also contained in the one measuring reagent.

The periostin measurement reagent of the present invention may be composed of two or more measurement reagents.
In this case, the anti-periostin antibody may be contained in one measurement reagent of two or more measurement reagents, or may be contained in two or more measurement reagents. .
In this case, the surfactant or reducing agent may also be contained in one of the two or more measurement reagents, or contained in two or more of the measurement reagents. It may be. At this time, the measurement reagent containing the surfactant or the reducing agent may be the pretreatment agent for measuring periostin of the present invention.

For example, when the measurement reagent for periostin of the present invention is composed of two measurement reagents, a first reagent and a second reagent, the anti-periostin antibody may be contained only in the first reagent, You may make it contain only in a 2nd reagent, Furthermore, you may make it contain in both a 1st reagent and a 2nd reagent.
For example, when the measurement reagent for periostin of the present invention is composed of two measurement reagents, the first reagent and the second reagent, the surfactant or the reducing agent is contained only in the first reagent. Alternatively, it may be contained only in the second reagent, or may be contained in both the first reagent and the second reagent.

In addition, various aqueous solvents can be used as the solvent for the measurement reagent for periostin of the present invention.

Examples of the aqueous solvent include water, physiological saline and the like, or tris (hydroxymethyl) aminomethane buffer [Tris buffer], phosphate buffer, or phosphate buffered saline. And various buffer solutions.

As for the pH of this buffer solution, 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.

In addition to the anti-periostin antibody and the like, the measurement reagent for periostin of the present invention includes proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or salts thereof; various metal ions such as calcium ions; Various salts such as calcium salts; Various sugars; Nonfat dry milk; Various animal sera such as normal rabbit serum; Various preservatives such as sodium azide or antibiotics; Activating substance; Reaction promoting substance; Sensitivity increasing substance such as polyethylene glycol; Or you may contain 1 type (s) or 2 or more types, such as a nonspecific reaction inhibitor, suitably.

The concentration of these in the periostin measurement reagent 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 preferred.

The periostin measuring reagent of the present invention can be sold alone or used for measuring periostin contained in a sample.
In addition, the measurement reagent for periostin of the present invention can be sold in combination with other reagents, or used for measurement of periostin 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.

By the way, the measurement reagent for periostin of the present invention may be a measurement reagent for periostin including a first reagent containing a surfactant or a reducing agent and an aqueous solvent and a second reagent containing an anti-periostin antibody. . In this case, the first reagent may be the pretreatment agent for measuring periostin of the present invention.

The periostin measurement reagent of the present invention may be a periostin measurement reagent including a first reagent containing an aqueous solvent and a second reagent containing an anti-periostin antibody and a surfactant or a reducing agent. .

The periostin measurement reagent of the present invention is preferably a measurement reagent kit.

3. Anti-periostin antibody In the reagent for measuring periostin of the present invention, the antibodies described in the above section “[1] Anti-periostin antibody” can be used. For example, the following antibodies can be used.
(I) an antibody capable of specifically binding to periostin (ii) a polyclonal antibody capable of specifically binding to periostin (iii) a monoclonal antibody capable of specifically binding to periostin

In the measurement reagent for periostin of the present invention, for example, when two molecules of antibody are reacted with one molecule of periostin by antigen-antibody reaction, both of these antibodies need to be anti-periostin antibodies.
For example, in the ELISA method sandwich reagent using 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 antibodies. It needs to be.

By the way, the anti-periostin antibody described above may contain not only one type but also a plurality of types.

The details of this anti-periostin antibody are as described in the above section “[1] Anti-periostin antibody”.

4). Measuring reagent based on immunological measuring method using labeled antibody Measuring principle of periostin of the present invention is a labeled antibody such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay or luminescence immunoassay. If the measurement principle is the immunological measurement method used, that is, the measurement method using an antigen-antibody reaction using a labeled antibody, it can be performed by the sandwich method or the competition method, etc. Both the immobilized antibody and the labeled antibody to be bound to periostin contained in the sample must be anti-periostin antibodies.

The periostin measurement reagent contained in the sample of the present invention contains a surfactant or a reducing agent.

In addition, as a solid phase carrier used in an immunoassay reagent using this labeled antibody, polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposome, gelatin, Use of a solid support in the form of microcapsules, beads, microplates (microtiter plates), test tubes, sticks or test pieces made of agarose, cellulose, sepharose, glass, metal, ceramics or magnetic materials. it can.
The antibody can be immobilized on the solid phase carrier by adsorbing and binding the anti-periostin antibody and the solid phase carrier by a known method such as physical adsorption, chemical binding, or a combination thereof.

In the case of physical adsorption, according to a known method, the anti-periostin antibody and the solid phase carrier are mixed and brought into contact with a solution such as a buffer solution, or the anti-periostin antibody dissolved in the buffer solution is brought into contact with the solid phase carrier. It can be performed by, for example.
In addition, in the case of chemical bonding, 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. “Shinsei Kagaku Kenkyu Ken 1 Protein IV”, published by Tokyo Kagaku Dojin, published in 1991, etc., the anti-periostin antibody and the solid phase carrier are divalent such as glutaraldehyde, carbodiimide, imide ester or maleimide. It can be carried out by mixing and bringing into contact with a cross-linking reagent and reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the anti-periostin antibody and the 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, bovine serum albumin (on the surface or inner wall surface of the solid phase carrier on which the anti-periostin antibody is immobilized, BSA), human serum albumin (HSA), casein, gelatin, protein such as ovalbumin or a salt thereof, a surfactant or non-fat dry milk, etc. are treated by a known method, etc. You may perform a blocking process (masking process).

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

The binding method of anti-periostin antibody and labeling substances such as enzymes is published in 1983 by the Japanese Society of Clinical Pathology, Special Issue on Clinical Pathology No. 53, Immunoassay for Clinical Examination -Technology and Applications, published in 1983. In accordance with known methods described in the “Studies on New Chemistry Experiment 1 Protein IV” edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1991, etc., and the anti-periostin antibody and the labeling substance are selected from two types such as glutaraldehyde, carbodiimide, imide ester or maleimide. The binding can be carried out by mixing and bringing into contact with a valent crosslinking reagent and reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the anti-periostin antibody and the labeling substance.

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; Kitagawa Tsuneki et al., Edited by "Protein Nucleic Acid Enzyme Volume No.31, Enzyme Immunoassay", Kyoritsu Shuppan, published in 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. Is formed by an antigen-antibody reaction. (For example, the periostin and the surfactant are brought into contact with each other before or during the antigen-antibody reaction to form a complex of “solid phase carrier = solid phase antibody = periostin = labeled antibody”.)

Then, the unbound labeled antibody is washed and separated, and the amount of labeled antibody bound to the solid phase carrier via “solid phase antibody = periostin” or the amount of unbound labeled antibody is included in the sample. Only the amount (concentration) of can be measured.

Specifically, in the case of a measurement reagent for enzyme immunoassay, for example, 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. .
In the case of a fluorescent immunoassay measurement reagent, the fluorescence intensity or the like by a fluorescent substance label is measured, and in the case of a radioimmunoassay measurement reagent, the radiation dose or the like by a radioactive substance label is measured.
In the case of a measurement reagent for luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.

The details of the measurement method using the antigen-antibody reaction using the labeled antibody with respect to the periostin measurement reagent of the present invention based on the measurement method using the antigen-antibody reaction using the labeled antibody are described in “[8 ]. "Measurement method of periostin" as described in "3. Immunological measurement method using labeled antibody".

5). Measuring reagent based on immunological measuring method by agglutination method The measuring reagent for periostin of the present invention is immunoturbidimetric method, latex turbidimetric method, latex agglutination method, erythrocyte agglutination method, particle agglutination method, etc. When the formation of immune complex aggregates is carried out by measuring the transmitted light or scattered light by an optical method or by a visual measurement method, that is, the complex aggregates by antigen-antibody reaction When the measurement method (aggregation reaction method) for measuring the production is used as the measurement principle, the antibody to be bound to periostin contained in the sample needs to be an anti-periostin antibody.

When the above-described aggregation reaction method is used as a measurement principle, the periostin measurement reagent of the present invention contains “anti-periostin antibody” or “solid phase carrier on which anti-periostin antibody is immobilized”.

In the case where the measurement reagent for periostin of the present invention is composed of two measurement reagents, the “anti-periostin antibody” or “solid phase carrier on which the anti-periostin antibody is immobilized” is preferably contained in the second reagent.
When the measurement reagent for periostin of the present invention is composed of two or more measurement reagents, a reagent other than the reagent containing “anti-periostin antibody” or “solid phase carrier on which anti-periostin antibody is immobilized” That is, the reagent containing neither the “anti-periostin antibody” nor the “solid phase carrier on which the anti-periostin antibody is immobilized” may be, for example, a reagent containing the aforementioned aqueous solvent.

In the measurement reagent by the agglutination reaction method, a phosphate buffer, glycine buffer, tris (hydroxymethyl) aminomethane buffer (Tris buffer), Good buffer, or the like can be used as a solvent. Furthermore, a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor may be included.

When the anti-periostin antibody is immobilized on a solid support, the solid support includes polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate. Copolymer, vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid polymer, acrylic acid Particles made of a material such as a polymer, latex, gelatin, liposome, microcapsule, erythrocyte, silica, alumina, carbon black, metal compound, metal, ceramic, or magnetic material can be used.

As a method for immobilizing the anti-periostin 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.

In the case of physical adsorption, according to a known method, the anti-periostin antibody and the solid phase carrier are mixed and brought into contact in a solution such as a buffer solution, or the anti-periostin antibody dissolved in the buffer solution is brought into contact with the solid phase carrier. It can be performed by, for example.

In addition, in the case of chemical binding method, the Japanese Society of Clinical Pathology “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination -Technology and Applications”, Clinical Pathology Publishing Society, published in 1983; In accordance with known methods described in ed. “Shinsei Kagaku Kenkyu Ken 1 Protein IV”, published by Tokyo Kagaku Dojin, published in 1991, etc., the anti-periostin antibody and the solid phase carrier are divalent such as glutaraldehyde, carbodiimide, imide ester or maleimide. It can be carried out by mixing and bringing into contact with a cross-linking reagent and reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the anti-periostin antibody and the 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, bovine serum albumin (on the surface or inner wall surface of the solid phase carrier on which the anti-periostin antibody is immobilized, BSA), human serum albumin (HSA), casein, gelatin, ovalbumin or a salt thereof, a surfactant, a non-fat dry milk, etc. You may perform a blocking process (masking process).

In the case of a measurement reagent based on the measurement principle of latex turbidimetry, the particle size of latex particles used as a solid phase carrier is not particularly limited, but the latex particles are bound via a measurement target substance (periostin), For reasons such as the degree of formation of aggregates and the ease of measurement of the generated aggregates, the average particle diameter of the latex particles is preferably 0.04 to 1 μm.

In addition, in the case of a measurement reagent based on the latex turbidimetric method, the concentration of the latex particles to which the anti-periostin antibody is immobilized includes the concentration of periostin in the sample and the anti-periostin antibody on the latex particle surface. Since the optimum concentration differs depending on various conditions such as the distribution density, the particle size of latex particles, and the mixing ratio of the sample and the measuring reagent, it cannot be generally stated.

However, in general, during the antigen-antibody reaction (during the measurement reaction) between the “anti-periostin antibody” mixed with the sample and the latex particles and the “periostin” contained in the sample, In general, the concentration of latex particles on which the “periostin antibody” is immobilized is 0.005 to 1% (w / v) in the reaction mixture. A concentration of “latex particles with an anti-periostin antibody immobilized” at such a concentration is included in the measurement reagent.

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. 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.

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, etc. 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, a sample and an anti-periostin antibody, or a sample and “an anti-periostin antibody immobilized on a solid phase carrier” Then, transmitted light and scattered light are measured by the endpoint method or the rate method.
In the case of visual measurement, the sample and the “anti-periostin antibody immobilized on a solid phase carrier” are reacted in the container such as a plate or microplate, and the state of aggregation is visually measured. .
In addition, you may measure using apparatuses, such as a microplate reader, instead of measuring visually.

Examples of measurement operation methods are given below.
For example, first, a measurement reagent containing “a solid phase carrier on which an anti-periostin antibody is immobilized” and “surfactant” or “reducing agent”, or a “solid phase carrier on which an anti-periostin antibody is immobilized” is measured. Prepare and prepare reagents and measurement reagents containing “surfactant” or “reducing agent”.

Next, for example, a sample is mixed with a measurement reagent containing a “solid phase carrier on which an anti-periostin antibody is immobilized” and a “surfactant” (or “reducing agent”), thereby producing an “anti-periostin antibody”. The solid phase carrier on which is immobilized and the sample are brought into contact. [At the same time, the periostin contained in the sample comes into contact with the surfactant (or reducing agent). ]
Alternatively, the measurement reagent containing “surfactant” (or “reducing agent”) and the sample are mixed to bring the periostin contained in the sample into contact with the surfactant (or reducing agent), and then The mixed solution is mixed with a reagent containing “a solid phase carrier on which an anti-periostin antibody is immobilized”, thereby bringing the “solid phase carrier on which an anti-periostin antibody is immobilized” into contact with the sample.

This causes an antigen-antibody reaction between the “anti-periostin antibody” of the “solid phase carrier on which the anti-periostin antibody is immobilized” and the “periostin” contained in the sample.

Then, a complex aggregate (… [periostin]-[antiperiostin] with “antiperiostin antibody immobilized” (antiperiostin antibody = solid phase carrier = antiperiostin antibody) and “periostin” produced from this is formed. Periostin antibody = solid phase carrier = anti-periostin antibody]-[periostin]-[anti-periostin antibody = solid phase carrier = anti-periostin antibody]-[periostin].

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.

Then, 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. The 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.

In addition, 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).

In addition, 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).

The measurement method for measuring the formation of complex aggregates by antigen-antibody reaction relates to the measurement reagent for periostin of the present invention based on the measurement principle, and details of the measurement method for measuring the formation of complex aggregates by the antigen-antibody reaction Is as described in “4. Immunological measurement method by agglutination” in “[8]. Periostin measurement method” below.

[7]. 1. Pretreatment agent for measuring periostin General The pretreatment agent for measuring periostin of the present invention is a pretreatment agent for measuring periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample. And a surfactant or a reducing agent.

The pretreatment agent for measuring periostin of the present invention can improve the sensitivity of the measurement in measuring periostin contained in a sample due to the above characteristics.

2. Pretreatment agent for measuring periostin The pretreatment agent for measuring periostin of the present invention is used for measuring periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample. It is a pretreatment agent and is characterized by containing a surfactant or a reducing agent. However, it is not particularly limited as long as it is such, and the desired effect is achieved. is there.

As a measurement principle of a measurement method for measuring periostin contained in this sample using an antigen-antibody reaction with an anti-periostin antibody, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radiation Immunoassay (RIA), Luminescent immunoassay (LIA), Enzyme antibody method, Fluorescent antibody method, Immunochromatography method, Immunoturbidimetric method, Latex turbidimetric method, Latex agglutination method, Red blood cell agglutination method, Particle aggregation A carrier having a surface coated with a specific binding substance to a substance to be measured (test substance) described in a reaction method, JP-A-9-229936 and JP-A-10-132919, And a measurement method using particles in which a specific binding substance for a measurement target substance (test substance) is immobilized, or EL shown by Dahlback et al. A method (Enzyme-linked Ligandsorbent Assay) (Thromb.Haemost, 79, pp. 767-772 pp., 1998 issue;. WO 98/23963 pamphlet), and the like.

The pretreatment agent for measuring periostin of the present invention can be applied to any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method).
In addition, the pretreatment agent for measuring periostin of the present invention may be applied to a method of use, or may be applied to measurement performed using an apparatus such as an analyzer.

The pretreatment agent for measuring periostin of the present invention may contain a surfactant or a reducing agent, or may be a surfactant or a reducing agent itself.

Further, the pretreatment agent for measuring periostin of the present invention may be a liquid or a solid.

In addition, when the pretreatment agent for measuring periostin of the present invention is a liquid, various aqueous solvents can be used as the solvent.

In addition to surfactants, pretreatment agents for measuring periostin of the present invention include proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or salts thereof, and various metals such as calcium ions. Ions; Various salts such as calcium salts; Various sugars; Nonfat dry milk; Various animal sera such as normal rabbit serum; Various preservatives such as sodium azide or antibiotics; Activating substances; Reaction promoting substances; Increased sensitivity of polyethylene glycol, etc. One or two or more substances such as substances; or nonspecific reaction suppressing substances may be appropriately contained.

The concentration of these in the pretreatment agent for measuring periostin 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 preferred.

Moreover, the pretreatment agent for measuring periostin of the present invention can be sold alone.
In addition, the pretreatment agent for measuring periostin of the present invention can be sold in combination with other reagents, or used for measuring periostin contained in a sample.

The pretreatment agent for measuring periostin of the present invention can be brought into contact with the periostin and the surfactant or reducing agent contained in the sample by adding to the sample or mixing with the sample.

[8]. Method for measuring periostin 1. General The method for measuring periostin contained in the sample of the present invention is a method for measuring periostin contained in a sample using an antigen-antibody reaction between an anti-periostin antibody and periostin contained in the sample. It is characterized by contacting the agent.

The method for measuring periostin of the present invention can improve the sensitivity of the periostin in the measurement of periostin contained in the sample due to the above characteristics.

In the method for measuring periostin contained in the sample of the present invention, the measuring reagent described in the above section “[6]. Periostin measuring reagent” and the above-mentioned “[7] pretreatment for measuring periostin” are provided. The pretreatment agent described in the section of “Agent” can be used.

2. Method for measuring periostin using antigen-antibody reaction The method for measuring periostin contained in a sample of the present invention is a method for measuring periostin contained in a sample using an antigen-antibody reaction between an anti-periostin antibody and periostin contained in a sample. The periostin is brought into contact with a surfactant or a reducing agent. However, the anti-periostin antibody is not particularly limited to the measurement principle as long as the anti-periostin antibody is used. There is an effect.

Examples of measurement methods for measuring periostin contained in this sample using an antigen-antibody reaction with an anti-periostin antibody include, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), and radioimmunoassay. (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescent antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination method, erythrocyte agglutination method, particle agglutination method, A carrier having a surface coated with a specific binding substance to a measurement target substance (test substance) described in JP-A-9-229936 and JP-A-10-132919, and a measurement target A measurement method using particles in which a specific binding substance to a substance (test substance) is immobilized, or an ELSA method (E zyme-linked Ligandsorbent Assay) (Thromb.Haemost, 79, pp. 767-772 pp., 1998 issue;. mention may be made of WO 98/23963 pamphlet), and the like.

And 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.
In addition, 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.

3. Anti-periostin antibody In the method for measuring periostin of the present invention, the antibody described in the above section “[1] Anti-periostin antibody” can be used. For example, the following antibodies can be used.
(I) an antibody capable of specifically binding to periostin (ii) a polyclonal antibody capable of specifically binding to periostin (iii) a monoclonal antibody capable of specifically binding to periostin

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 antibodies.
For example, in 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 must be this anti-periostin antibody. There is.

By the way, the above-mentioned anti-periostin antibody is not limited to one type, and a plurality of types may be used simultaneously.

4). 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. When the measurement method, that is, the measurement method using an antigen-antibody reaction using a labeled antibody, is performed by 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 must be anti-periostin antibodies.

In the present invention, for example, the periostin and a surfactant (or a reducing agent) are brought into contact before or during the antigen-antibody reaction between the anti-periostin antibody and periostin contained in the sample.

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. Is formed by an antigen-antibody reaction. [For example, the periostin and a surfactant (or a reducing agent) are added before or during the antigen-antibody reaction to form a complex of “solid phase carrier = solid phase antibody = periostin = labeled antibody”. Make contact. ]

Specifically, in the case of enzyme immunoassay, for example, 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.
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 the luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.

5). 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. When measuring the transmitted light or scattered light by an optical method or by a measuring method that visually measures the product, that is, a measuring method that measures the formation of complex aggregates by an antigen-antibody reaction When carried out by (aggregation reaction method), the antibody to be bound to periostin contained in the sample must be an anti-periostin antibody.

In the present invention, for example, the periostin is brought into contact with a surfactant or a reducing agent before or during the antigen-antibody reaction between the anti-periostin antibody and periostin contained in the sample.

Examples of measurement operation methods are given below.
For example, first, a measurement reagent containing “solid phase carrier on which anti-periostin antibody is immobilized” and “surfactant” (or “reducing agent”) or “solid phase carrier on which anti-periostin antibody is immobilized” is contained. And a measurement reagent containing “surfactant” (or “reducing agent”) are prepared and prepared.

Next, for example, a sample is mixed with a measurement reagent containing a “solid phase carrier on which an anti-periostin antibody is immobilized” and a “surfactant” (or “reducing agent”), thereby producing an “anti-periostin antibody”. The solid phase carrier on which is immobilized and the sample are brought into contact. [At the same time, the periostin contained in the sample comes into contact with the surfactant (or reducing agent). ]

Alternatively, by mixing the measurement reagent containing “surfactant” (or “reducing agent”) with the sample, periostin contained in the sample and the surfactant (or reducing agent) are brought into contact, The mixed solution is mixed with a reagent containing “a solid phase carrier on which an anti-periostin antibody is immobilized”, thereby bringing the “solid phase carrier on which an anti-periostin antibody is immobilized” into contact with the sample.

In the following, a more specific description will be given, taking as an example the case of measuring periostin by a method based on the measurement principle of latex turbidimetry.

[A] When contacting before antigen-antibody reaction (1) First, the following is prepared and prepared as a reagent for measuring periostin.
First reagent:
Pretreatment agent for periostin measurement [buffer solution containing surfactant (or reducing agent) (aqueous solvent)]
Second reagent:
Buffer solution containing "latex particles immobilized with anti-periostin antibody"

(2) 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.
Thus, prior to the antigen-antibody reaction between the anti-periostin antibody immobilized on the latex particles and the periostin contained in the sample, the periostin contained in the sample and the surfactant (or reducing agent) are brought into contact with each other.
In addition, what is necessary is just to select the mixing ratio (quantity ratio) of a sample and a 1st reagent suitably.
In addition, 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.)

(3) After a certain time, a certain amount of the second reagent is added to and mixed with the mixed solution of the sample and the first reagent, and left as a reaction mixture at a constant temperature for a certain time.
Thereby, the “latex particles on which the anti-periostin antibody is immobilized” and the sample are brought into contact with each other.
In addition, what is necessary is just to select the addition amount of a 2nd reagent suitably.
In addition, 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 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.

Then, by this antigen-antibody reaction, "... [periostin]-[anti-periostin antibody = latex particle = anti-periostin antibody]-[periostin]-[anti-periostin antibody = latex particle = anti-periostin antibody]-[periostin] ..." etc. Are formed, and a composite aggregate of “latex particles with immobilized anti-periostin antibody” is generated via periostin.

(4) Then, in an analyzer or a spectrophotometer, 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.

(5) And “measured value obtained by measuring the sample [decrease in transmitted light intensity (increase in absorbance) or increase in scattered light intensity]” and “standard substance such as standard solution or standard serum [ Sample measured by comparing with the measured value [decrease in transmitted light intensity (increase in absorbance) or increase in scattered light intensity] obtained by measurement of a sample containing periostin of known concentration] The amount (concentration) of periostin contained in is calculated.

[B] When contacting at the time of antigen-antibody reaction (1) First, the following are prepared and prepared as a measurement reagent for periostin.
First reagent:
Buffer solution (aqueous solvent)
Second reagent:
Buffer solution containing “latex particles immobilized with anti-periostin antibody” and “surfactant” (or “reducing agent”)

(2) 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.
In addition, what is necessary is just to select the mixing ratio (quantity ratio) of a sample and a 1st reagent suitably.
In addition, 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.)

(3) After a certain time, a certain amount of the second reagent is added to and mixed with the mixed solution of the sample and the first reagent, and left as a reaction mixture at a constant temperature for a certain time.
Thereby, the “latex particles on which the anti-periostin antibody is immobilized” and the sample are brought into contact with each other. [Thus, in the antigen-antibody reaction between the anti-periostin antibody immobilized on the latex particles and the periostin contained in the sample, the periostin contained in the sample and the surfactant (or reducing agent) are brought into contact with each other. . ]
In addition, what is necessary is just to select the addition amount of a 2nd reagent suitably.
In addition, 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.

[9]. Method for improving sensitivity of periostin measurement General The method for improving the sensitivity of the measurement of periostin contained in a sample of the present invention is the measurement of periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample. It is characterized in that periostin is brought into contact with a surfactant or a reducing agent.

The method for improving the sensitivity of the measurement of periostin contained in the sample of the present invention can improve the sensitivity of the measurement in the measurement of periostin contained in the sample due to the above characteristics.

In the method for improving the sensitivity of the measurement of periostin contained in the sample of the present invention, the measurement reagent described in the above section “[6]. Periostin measurement reagent” and “[7]. The pretreatment agent described in the section “Pretreatment agent” can be used.

2. Measurement of periostin using antigen-antibody reaction The method for improving the sensitivity of periostin measurement contained in the sample of the present invention is based on the antigen-antibody reaction between the antibody that specifically binds to periostin and periostin contained in the sample. In the measurement of periostin contained, the periostin and a surfactant or a reducing agent are brought into contact with each other, and the measurement of periostin contained in this sample is particularly if an anti-periostin antibody is used. The measurement principle is not limited, and the desired effect is achieved.

In the present invention, examples of the measurement method for measuring periostin contained in this sample using an antigen-antibody reaction with an anti-periostin antibody include, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), Radioimmunoassay (RIA), Luminescent immunoassay (LIA), Enzyme antibody method, Fluorescent antibody method, Immunochromatography method, Immunoturbidimetric method, Latex turbidimetric method, Latex agglutination method, Red blood cell agglutination method, Particle A carrier having a surface coated with a specific binding substance fixed to a substance to be measured (test substance) described in the agglutination reaction method, JP-A-9-229936, JP-A-10-132919, etc. , And a measurement method using particles in which a specific binding substance to a measurement target substance (test substance) is immobilized, or Dahlbeack et al. ELSA Law (Enzyme-linked Ligandsorbent Assay) (Thromb.Haemost, 79, pp. 767-772 pp., 1998 issue;. WO 98/23963 pamphlet), and the like.

And any method, such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method), can be applied to the measurement of periostin in the present invention.
In addition, the measurement of periostin in the present invention may be performed by a method, or may be performed using an apparatus such as an analyzer.

3. Anti-periostin antibody In the measurement of periostin in the present invention, the antibody described in the above section “[1] Anti-periostin antibody” can be used. For example, the following antibodies can be used.
(I) an antibody capable of specifically binding to periostin (ii) a polyclonal antibody capable of specifically binding to periostin (iii) a monoclonal antibody capable of specifically binding to periostin

In the measurement of periostin in 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 antibodies.
For example, in 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 must be this anti-periostin antibody. There is.

4). Immunological measurement using labeled antibody The measurement of periostin in the present invention is an immunological measurement method using a labeled antibody such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay, or luminescent immunoassay, that is, When the measurement method using an antigen-antibody reaction using a labeled antibody is performed, it can be performed by the sandwich method or the competition method. However, when the sandwich method is used, the solid antibody to be bound to periostin contained in the sample is used. Both the phased antibody and the labeled antibody must be anti-periostin antibodies.

In the present invention, the periostin is brought into contact with a surfactant or a reducing agent before the antigen-antibody reaction between the anti-periostin antibody and periostin contained in the sample or during the antigen-antibody reaction.

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. Is formed by an antigen-antibody reaction. [For example, the periostin and a surfactant (or a reducing agent) are contacted before or during the antigen-antibody reaction to form a complex of “solid phase carrier = solid phase antibody = periostin = labeled antibody”. . ]

5). Immunological measurement by the agglutination method The measurement of periostin in the present invention is carried out by producing immune complex aggregates such as immunoturbidimetry, latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination. In the case of measuring the transmitted light or scattered light by an optical method or by a measuring method that visually measures, that is, a measuring method (aggregation reaction method) that measures the formation of complex aggregates by an antigen-antibody reaction. ), The antibody to be bound to periostin contained in the sample needs to be an anti-periostin antibody.

Examples of measurement operation methods are given below.
For example, first, it contains a “solid phase carrier on which an anti-periostin antibody is immobilized” and a measurement reagent containing “surfactant” (or “reducing agent”) or “a solid phase carrier on which an anti-periostin antibody is immobilized”. And a measurement reagent containing “surfactant” (or “reducing agent”) are prepared and prepared.

Next, for example, a measurement reagent containing “an anti-periostin antibody immobilized solid phase carrier” and “surfactant” (or “reducing agent”) is mixed with a sample, whereby “anti-periostin antibody” The solid phase carrier on which is immobilized and the sample are brought into contact. [At the same time, the periostin contained in the sample comes into contact with the surfactant (or “reducing agent”). ]

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

[Example 1] (Confirmation of effect of the present invention-1) [Surfactant]
Periostin in human serum was measured to confirm the effects of the periostin measurement reagent, the pretreatment agent for periostin measurement, the periostin measurement method, and the periostin measurement sensitivity improvement method contained in the sample of the present invention.

1. Preparation of Periostin S2 cells which are recombinant periostin proteins obtained by adding V5 / His tag to periostin (nucleotide sequence: nucleotide sequence of Accession Number D13666 of Nucleic Acid Database GenBank, amino acid sequence: amino acid sequence of Accession Number BAA02837 of Nucleic Acid Database GenBank) And then 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 / periostin-V5-HisA.

Next, 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.

Next, a transformant was selected with hygromycin to obtain a stable transformant.
And in the transformant of S2 cell, periostin which the V5 / His tag couple | bonded was expressed at the carboxy terminus.

And purification of S2 recombinant periostin protein which 6 histidine couple | bonded with this C terminal was performed 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 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. Was mixed with nickel resin (Ni-NTA Agarose, Qiagen, Hilden, Germany) to bind the S2 recombinant periostin protein to the resin.
Next, the resin was washed to remove contaminants, and 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.

2. Anti-periostin monoclonal antibody [1] Preparation of anti-periostin monoclonal antibody-1st time An anti-periostin monoclonal antibody was prepared according to the following procedure. (First time)

(1) To 1 volume of the periostin solution prepared in 1 above, Titer Max Gold (Funakoshi Co., Tokyo, Japan) as a chemical synthesis adjuvant was mixed at a ratio of 1 volume.

(2) Next, 10-50 μg of a mixture of the above-mentioned periostin solution and Titer Max Gold is injected subcutaneously as an immunogen into the footpads of female rats, and 10 days to 2 weeks later, the rat footpads are immunized again. As a raw material, a mixture of 10-50 μg of the above-described periostin solution and Titer Max Gold was injected subcutaneously.
Here, Wistar rats [female, 6-8 weeks old] (Charles River Japan, Yokohama, Kanagawa, Japan) were used as rats.

(3) 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) are mixed in a ratio of 1: 1 to 10: 1. Then, polyethylene glycol (PEG 1500, Roche, Switzerland) was added by a general method to perform 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 (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.

Specifically, this ELISA method was performed as follows.
1 μg / 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.

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.

One clone was established from the grown fused cell lines and was named SS18A strain.

(4) 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).

1 volume of 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.

That is, a rat anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS18A)”) was obtained from the SS18A strain monoclonal antibody-producing cell line.

[2] Preparation of anti-periostin monoclonal antibody-second time At a different time from the above-mentioned [1], the procedure is repeated as described in (1) to (4) of [1]. Prepared. (Second time)

As a result, one clone was established from the grown fused cell lines and was designated as SS17B strain.

A rat anti-periostin monoclonal antibody (hereinafter referred to as “anti-periostin monoclonal antibody (SS17B)”) was obtained from the SS17B monoclonal antibody-producing cell line.

3. Sample Amphoteric surfactant lauryldimethylaminoacetate betaine [trade name: Nissan Anon (registered trademark) BL] was 0.5% (w / v), 1% (w / v), or 2% (w / v), respectively. 50 mM tris (hydroxymethyl) aminomethane containing at a concentration of v) and containing 0.5% (w / v) casein, 100 mM sodium chloride and 0.1% (w / v) sodium azide A buffer [Tris buffer] (pH 8.0) was prepared and used as a pretreatment agent for periostin measurement.

, 5.0 μL of each of 42 human serum samples was added to and mixed with 6.0 mL of each of these three types of periostin measurement pretreatment agents.

In addition, when the pretreatment agent for measuring periostin and a human serum sample are added and mixed, that is, when the periostin and the surfactant are brought into contact with each other before the antigen-antibody reaction, each of the surfactants (lauryldimethylamino) The concentration of betaine acetate) is 0.5% (w / v) when the concentration of the surfactant (lauryldimethylaminoacetic acid betaine) in the pretreatment agent for periostin measurement is 0.5% (w / v). Yes, when the concentration of the surfactant in the pretreatment agent for measuring periostin is 1% (w / v), it is 1% (w / v), and the concentration of the surfactant in the pretreatment agent for measuring periostin is When the concentration is 2% (w / v), it is 2% (w / v).

In addition, a 50 mM tris (hydroxymethyl) aminomethane buffer [Tris buffer containing 0.5% (w / v) casein, 100 mM sodium chloride and 0.1% (w / v) sodium azide. (PH 8.0) was prepared, and this was used as a pretreatment agent for measuring periostin having a lauryldimethylaminoacetic acid betaine concentration of 0% (w / v).

To 1.0 mL of this pretreatment agent for periostin measurement having a lauryldimethylaminoacetic acid betaine concentration of 0% (w / v), 5.0 μL of each of the 42 human serum samples was added and mixed.
Through the above operation, a total of 168 types of serum samples were prepared.

4). Measurement The periostin contained in each of the three serum samples was measured by the enzyme immunoassay (ELISA method) using an anti-periostin monoclonal antibody as follows.

(1) “Anti-periostin monoclonal antibody (SS18A)” in [2] above is added to phosphate buffered saline (PBS) [137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM diphosphate. Aqueous solution containing potassium hydrogen and 8.04 mM disodium hydrogen phosphate (pH 7.4)] and diluted to 2 μg / mL, and this was diluted with a 96-well microtiter plate (Thermo Fisher Scientific Inc., Illinois) , USA), and 100 μL was injected into each well and allowed to stand at 25 ° C. for 18 to 24 hours to immobilize “anti-periostin monoclonal antibody (SS18A)” in each well of the microtiter plate.

(2) Next, after removing the liquid in each well of the microtiter plate of (1) above, the blocking solution [0.5% casein, 100 mM sodium chloride, and 0.1% sodium azide was added. 200 μL of 50 mM Tris (hydroxymethyl) aminomethane buffer [Tris buffer] (pH 8.0)] contained therein was injected into each well and allowed to stand at 4 ° C. for 18 to 24 hours.

(3) Next, each well of the microtiter plate of (2) was washed three times with a washing solution [phosphate buffered saline (PBS) containing 0.05% Tween 20].

(4) Next, 100 μL of each of the three serum samples was injected into the wells of the microtiter plate of (3) and allowed to stand at 25 ° C. for 18 to 24 hours for reaction.
This allowed periostin contained in the sample to bind to the “anti-periostin monoclonal antibody (SS18A)” immobilized on the well.

(5) Next, each well of the microtiter plate of (4) was washed 5 times with the washing solution.

(6) POD-labeled anti-periostin monoclonal antibody obtained by binding peroxidase (POD) to the “anti-periostin monoclonal antibody (SS17B)” in [2] above, labeled with 0.5% casein and 100 mM sodium chloride. Dilute to 50 ng / mL with 50 mM Tris (hydroxymethyl) aminomethane buffer [Tris buffer] (pH 8.0) and add 100 μL to each well of the microtiter plate of (5). And left to stand at 25 ° C. for 90 minutes to carry out the reaction.

Thus, “anti-periostin monoclonal antibody (SS17B)” labeled with POD was bound to periostin bound to the above-mentioned solid-phased “anti-periostin monoclonal antibody (SS18A)”.

(7) Next, each well of the microtiter plate of (6) was washed 5 times with the washing solution.

(8) Next, 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 μL of 20 mM citrate buffer (pH 3.9)] was injected into each well of the microtiter plate of (7), and allowed to stand at 25 ° C. for 10 minutes to cause the reaction to develop color.

(9) Thereafter, 0.7N sulfuric acid was injected into each well of the microtiter plate of (8) to stop this reaction.

(10) Next, using a spectrophotometer, the absorbance at 450 nm (primary wavelength) and 550 nm (subwavelength) was measured for each well of the microtiter plate of (9).
Then, an absorbance difference value obtained by subtracting the absorbance at 550 nm (sub wavelength) from the measured absorbance at 450 nm (major wavelength) was obtained.

Note that the value of the absorbance difference is based on the concentration of periostin contained in the serum sample.

5). Measurement Result The measurement result of periostin contained in the serum sample in the above 4 is shown in FIG.

In this figure, the horizontal axis represents the concentration (unit:% (w / v)) of the surfactant (lauryldimethylaminoacetic acid betaine) when periostin and the surfactant are contacted, and the vertical axis represents the serum sample. Measurement result of periostin contained [absorbance difference value obtained by subtracting absorbance at 550 nm (sub wavelength) from absorbance at 450 nm (major wavelength) [unit: ΔAbs. ]] Of the difference in absorbance when the concentration of the surfactant is 0% (w / v) [unit: ΔAbs. ] [Relative sensitivity [unit:%]].

From this figure, compared to the case where the periostin contained in the serum sample is not brought into contact with the surfactant [that is, the concentration of the surfactant is 0% (w / v)], the periostin contained in the serum sample and the interface It can be seen that when the activator is brought into contact, the absorbance obtained by the measurement increases, that is, the sensitivity of the measurement increases.

Therefore, it was confirmed from the measurement results in this example that the sensitivity of measurement of periostin contained in the sample can be improved in the present invention.

[Example 2] (Confirmation of effect of the present invention-2) [Surfactant]
Periostin in human serum was measured to confirm the effects of the periostin measurement reagent, the pretreatment agent for periostin measurement, the periostin measurement method, and the periostin measurement sensitivity improvement method contained in the sample of the present invention.

1. Sample Amphoteric surfactant lauryldimethylaminoacetic acid betaine [trade name: Nissan Anon (registered trademark) BL] was 0.5% (w / v), 1% (w / v), 2% (w / v), respectively. ) At a concentration of 3% (w / v), 4% (w / v), or 5% (w / v) and 0.5% (w / v) casein, 100 mM sodium chloride and 0 A 50 mM tris (hydroxymethyl) aminomethane buffer [Tris buffer] (pH 8.0) containing 1% (w / v) sodium azide was prepared and used as a pretreatment agent for periostin measurement.

To each 6.0 mL of the six types of periostin measurement pretreatment agents, 5.0 μL of one human serum sample was added and mixed.

In addition, when the pretreatment agent for measuring periostin and a human serum sample are added and mixed, that is, when the periostin and the surfactant are brought into contact with each other before the antigen-antibody reaction, each of the surfactants (lauryldimethylamino) The concentration of betaine acetate) is 0.5% (w / v) when the concentration of the surfactant (lauryldimethylaminoacetic acid betaine) in the pretreatment agent for periostin measurement is 0.5% (w / v). Yes, when the concentration of the surfactant in the pretreatment agent for measuring periostin is 1% (w / v), it is 1% (w / v), and the concentration of the surfactant in the pretreatment agent for measuring periostin is When the concentration is 2% (w / v), it is 2% (w / v), and when the concentration of the surfactant in the pretreatment agent for periostin measurement is 3% (w / v), 3% ( w / v) When the concentration of the surfactant in the pretreatment agent for measuring periostin is 4% (w / v), the concentration of the surfactant in the pretreatment agent for measuring periostin is 4% (w / v). Is 5% (w / v) when 5% (w / v).

To 1.0 mL of the pretreatment agent for measuring periostin having a lauryldimethylaminoacetic acid betaine concentration of 0% (w / v), 5.0 μL of one human serum sample was added and mixed.
A total of 7 types of serum samples were prepared by the above operation.

2. Measurement For periostin contained in each of the serum samples of 1 above, the enzyme immunoassay method (ELISA method) using an anti-periostin monoclonal antibody was used, as described in Measurement 4 (1)-(10) of Example 1 above. Measurements were made.

3. Measurement Results Table 1 shows the measurement results of periostin contained in the serum sample in 1 above.

In this table, periostin contained in the serum sample at each concentration (unit:% (w / v)) of the surfactant (lauryldimethylaminoacetic acid betaine) when periostin and the surfactant are brought into contact with each other Measurement result [absorbance difference value obtained by subtracting absorbance at 550 nm (sub-wavelength) from absorbance at 450 nm (major wavelength) [unit: ΔAbs. ], And the absorbance difference value when the surfactant concentration is 0% (w / v) [unit: ΔAbs. ] [Relative sensitivity [unit:%]] respectively.

From this table, compared to the case where the periostin contained in the serum sample is not brought into contact with the surfactant [that is, the concentration of the surfactant is 0% (w / v)], the periostin contained in the serum sample and the interface It can be seen that when the activator is brought into contact, the absorbance obtained by the measurement increases, that is, the sensitivity of the measurement increases.

For example, when the surfactant concentration when periostin is brought into contact with the surfactant is 0.5% (w / v), the relative sensitivity is 171%, and the surfactant concentration is 3% (w / v). The relative sensitivity at v) is 399%, increasing.

Therefore, it was confirmed again from the measurement results in this example that the sensitivity of measurement of periostin contained in the sample can be improved in the present invention.

[Example 3] (Confirmation of effect of the present invention-3) [Surfactant]
The effect of measuring the periostin in human serum and measuring the periostin contained in the sample of the present invention, the pretreatment agent for measuring periostin, the method for measuring periostin contained in the sample, and the method for improving the sensitivity of the periostin measurement I confirmed.

1. Sample containing sodium lauryl sulfate, an anionic surfactant, at a concentration of 0.04% (w / v), 0.08% (w / v), or 0.12% (w / v), respectively 50 mM Tris (hydroxymethyl) aminomethane buffer [Tris buffer] containing 0.5% (w / v) casein, 100 mM sodium chloride and 0.1% (w / v) sodium azide ( pH 8.0) was prepared and used as a pretreatment agent for measuring periostin.

, 5.0 μL of one human serum sample was added to and mixed with 1.0 mL of each of these three types of preiostin measurement pretreatment agents.

Each of the surfactants (sodium lauryl sulfate) when periostin measurement pretreatment agent and human serum sample were added and mixed, that is, when periostin and surfactant were brought into contact before the antigen-antibody reaction. ) Is 0.04% (w / v) when the concentration of the surfactant (sodium lauryl sulfate) in the pretreatment agent for periostin measurement is 0.04% (w / v), and the periostin When the concentration of the surfactant in the pretreatment agent for measurement is 0.08% (w / v), it is 0.08% (w / v), and the concentration of the surfactant in the pretreatment agent for periostin measurement is When the concentration is 0.12% (w / v), it is 0.12% (w / v).

In addition, a 50 mM tris (hydroxymethyl) aminomethane buffer [Tris buffer containing 0.5% (w / v) casein, 100 mM sodium chloride and 0.1% (w / v) sodium azide. (PH 8.0) was prepared and used as a pretreatment agent for measuring periostin having a sodium lauryl sulfate concentration of 0% (w / v).

To 1.0 mL of the pretreatment agent for measuring periostin having a sodium lauryl sulfate concentration of 0% (w / v), 5.0 μL of one human serum sample was added and mixed.
By the above operation, a total of 4 types of serum samples were prepared.

3. Measurement Results Table 2 shows the measurement results of periostin contained in the serum sample in 1 above.

In this table, measurement of periostin contained in a serum sample at each concentration (unit:% (w / v)) of the surfactant (sodium lauryl sulfate) when periostin and the surfactant are brought into contact with each other Results [Value of absorbance difference obtained by subtracting absorbance at 550 nm (sub wavelength) from absorbance at 450 nm (major wavelength) [unit: ΔAbs. ], And the absorbance difference value when the surfactant concentration is 0% (w / v) [unit: ΔAbs. ] [Relative sensitivity [unit:%]] respectively.

For example, when the surfactant concentration when periostin is brought into contact with the surfactant is 0.04% (w / v), the relative sensitivity is 151%, and the surfactant concentration is 0.08% ( The relative sensitivity at w / v) is 377%, increasing.

[Example 4] (Confirmation of effect of the present invention-4) [Reducing agent]
Periostin in human serum was measured, and the effects of the periostin measurement reagent, the pretreatment agent for periostin measurement, the periostin measurement method, and the periostin measurement sensitivity improvement method were confirmed.

1. Sample A dithiothreitol as a reducing agent is contained at a concentration of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, or 1.0 mM, respectively, and 0.5% (w / v) casein, 100 mM Preparation of 50 mM Tris (hydroxymethyl) aminomethane buffer [Tris buffer] (pH 8.0) containing sodium chloride and 0.1% (w / v) sodium azide It was.

To each 6.0 mL of the five types of periostin measurement pretreatment agents, 5.0 μL of one human serum sample was added and mixed.

In addition, when the pretreatment agent for measuring periostin and the human serum sample are added and mixed, that is, when the periostin and the reducing agent are contacted before the antigen-antibody reaction, each of the reducing agents (dithiothreitol) The concentration is 0.01 mM when the concentration of the reducing agent (dithiothreitol) in the pretreatment agent for periostin measurement is 0.01 mM, and the concentration of the reducing agent in the pretreatment agent for periostin measurement is 0.05 mM. Is 0.05 mM, and when the concentration of the reducing agent in the pretreatment agent for periostin measurement is 0.1 mM, it is 0.1 mM, and the concentration of the reducing agent in the pretreatment agent for periostin measurement is 0. 0.5 mM when the concentration is 5 mM, and 1.0 mM when the concentration of the reducing agent in the pretreatment agent for periostin measurement is 1.0 mM. A.

In addition, a 50 mM tris (hydroxymethyl) aminomethane buffer [Tris buffer containing 0.5% (w / v) casein, 100 mM sodium chloride and 0.1% (w / v) sodium azide. (PH 8.0) was prepared and used as a pretreatment agent for measuring periostin with a dithiothreitol concentration of 0 mM.

To 1.0 mL of the pretreatment agent for measuring periostin with a dithiothreitol concentration of 0 mM, 5.0 μL of one sample of human serum was added and mixed.
A total of 6 types of serum samples were prepared by the above operation.

3. Measurement Results Table 3 shows the measurement results of periostin contained in the serum samples in 2 above.

In this table, the measurement result of periostin contained in a serum sample at each concentration (unit: mM) of the reducing agent (dithiothreitol) when periostin and the reducing agent are brought into contact [450 nm (main wavelength) Value of difference in absorbance obtained by subtracting absorbance at 550 nm (subwavelength) from absorbance at pH [unit: ΔAbs. ], And the absorbance difference value when the concentration of the reducing agent is 0 mM [unit: ΔAbs. ] [Relative sensitivity [unit:%]] respectively.

From this table, when the periostin contained in the serum sample is not brought into contact with the reducing agent [ie, the concentration of the reducing agent is 0 mM], when the periostin contained in the serum sample is brought into contact with the reducing agent, It can be seen that the absorbance obtained by the measurement increases, that is, the sensitivity of the measurement increases.

For example, the relative sensitivity when the concentration of the reducing agent when contacting the periostin and the reducing agent is 0.01 mM is 131%, and the relative sensitivity when the concentration of the reducing agent is 0.05 mM is 827%, The relative sensitivity when the concentration of the reducing agent is 0.5 mM is 1064%, which is increased.

Claims

A reagent for measuring periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and an antibody that specifically binds to periostin, including an antibody that specifically binds to periostin, comprising a surfactant or A reagent for measuring periostin contained in a sample, comprising a reducing agent.
A pretreatment agent for measuring periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample, which contains a surfactant or a reducing agent A pretreatment agent for measuring periostin.
In a method for measuring periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample, the periostin is brought into contact with a surfactant or a reducing agent. A method for measuring periostin contained in a sample.
In the measurement of periostin contained in a sample using an antigen-antibody reaction between an antibody that specifically binds to periostin and periostin contained in the sample, the periostin is brought into contact with a surfactant or a reducing agent, A method for improving the sensitivity of measuring periostin contained in a sample.