WO2021182555A1 - Anticorps de détection de l'aldostérone et méthode de dosage immunologique de l'aldostérone - Google Patents

Anticorps de détection de l'aldostérone et méthode de dosage immunologique de l'aldostérone Download PDF

Info

Publication number
WO2021182555A1
WO2021182555A1 PCT/JP2021/009705 JP2021009705W WO2021182555A1 WO 2021182555 A1 WO2021182555 A1 WO 2021182555A1 JP 2021009705 W JP2021009705 W JP 2021009705W WO 2021182555 A1 WO2021182555 A1 WO 2021182555A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
aldosterone
complex
minutes
amino acid
Prior art date
Application number
PCT/JP2021/009705
Other languages
English (en)
Japanese (ja)
Inventor
一久 中島
小野　仁
Original Assignee
日立化成ダイアグノスティックス・システムズ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日立化成ダイアグノスティックス・システムズ株式会社 filed Critical 日立化成ダイアグノスティックス・システムズ株式会社
Priority to JP2022507271A priority Critical patent/JPWO2021182555A1/ja
Publication of WO2021182555A1 publication Critical patent/WO2021182555A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to an antibody for detecting aldosterone, an immunoassay method for aldosterone, and an immunoassay reagent for aldosterone.
  • Aldosterone is a mineral steroid hormone with a molecular weight of about 360 secreted from the outer layer of the adrenal cortex. Aldosterone secretion is predominantly dependent on the renin-angiotensin-aldosterone system. Aldosterone is a hormone that plays an important role in maintaining electrolyte homeostasis, circulating blood volume and blood pressure. Aldosterone has the function of increasing blood pressure by acting on the mineral corticoid receptors of the distal tubules of the kidney to promote the reabsorption of sodium and water.
  • Measurement of aldosterone concentration in biological samples is carried out for the differential diagnosis of primary aldosteronism, Bartter syndrome, Liddle's syndrome, hydroxylase deficiency, and selective hypoaldosteronism.
  • the renin activity Plasma Renin Activity: PRA
  • renin concentration Activity
  • PAC renin concentration
  • an LC-MS method As a method for measuring aldosterone, an LC-MS method, an immunoassay method, and the like are known.
  • the immunoassay method include a radioimmunoassay method (RIA method) and an enzyme-linked immunosorbent assay method (EIA method) (see Patent Document 1).
  • Patent Document 2 describes that an antibody that specifically binds to a complex of a steroid and an antibody is used as an anti-steroid antibody in a method for immunologically quantifying a steroid.
  • Patent Document 2 does not disclose a specific antibody that can be used as an antibody that specifically binds to the complex.
  • a main object of the present invention is to provide a technique for accurately detecting aldosterone in a sample.
  • the present invention provides the following [1]-[35].
  • CDR Complementarity determining regions
  • CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 16
  • CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 17,
  • poly represented by the amino acid sequence of SEQ ID NO: 18 containing CDR3 consisting of the peptide in the heavy chain.
  • the heavy chain contains a variable region consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 5
  • the light chain contains a variable region consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 6 [1] or [ 2] antibody.
  • Anti-aldosterone antibody that recognizes aldosterone A complex-recognizing antibody that specifically recognizes a complex of aldosterone and the anti-aldosterone antibody, To form a multiplex complex of aldosterone, the anti-aldosterone antibody, and the complex-recognizing antibody; and to measure the multiplex complex; Methods of immunoassaying aldosterone, including.
  • the complex recognition type antibody is represented by CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 10, CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 11, and the amino acid sequence of SEQ ID NO: 12.
  • CDR3 consisting of a polypeptide is contained in a heavy chain and is represented by CDR1 consisting of a polypeptide represented by the amino acid sequence of SEQ ID NO: 16, CDR2 consisting of a polypeptide represented by the amino acid sequence of SEQ ID NO: 17, and the amino acid sequence of SEQ ID NO: 18.
  • the complex recognition type antibody contains a variable region consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 5 in the heavy chain, and the variable region consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 6 in the light chain.
  • the method of any of [14]-[24] which is a sandwich ELISA (Enzyme-Linked ImmunoSorben Assay), a latex agglutination method, or an immunochromatography method.
  • An anti-aldosterone antibody that recognizes aldosterone A complex-recognizing antibody that specifically recognizes a complex of aldosterone and the anti-aldosterone antibody, Aldosterone immunoassay reagents, including.
  • the complex recognition type antibody is represented by CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 10, CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 11, and the amino acid sequence of SEQ ID NO: 12.
  • CDR3 consisting of a polypeptide is contained in a heavy chain and is represented by CDR1 consisting of a polypeptide represented by the amino acid sequence of SEQ ID NO: 16, CDR2 consisting of a polypeptide represented by the amino acid sequence of SEQ ID NO: 17, and the amino acid sequence of SEQ ID NO: 18.
  • the complex recognition type antibody contains a variable region consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 5 in the heavy chain, and the variable region consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 6 in the light chain.
  • the term "antibody” is used in the broadest sense and is a monoclonal antibody, polyclonal antibody, dimer, multimer, multispecific antibody (eg, double) as long as it exhibits the desired biological activity. It may be a specific antibody), an antibody fragment or an antibody modified product.
  • the antibody may be a mouse antibody, a rabbit antibody, a human antibody, a humanized antibody or a chimeric antibody, or may be an antibody derived from another species.
  • Antibodies can be in any class of immunoglobulin molecules (eg, IgG, IgE, IgM, IgD and IgA), in any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).
  • the terms "antibody” and "immunoglobulin” are used in a broad sense with compatibility.
  • an “antibody fragment” is a portion of an antibody that comprises a variable domain of an antibody, or at least an antigen-binding region.
  • Antibody fragments include, for example, Fab, Fab', F (ab') 2 , Fv fragment, linear antibody, single chain antibody (scFv), sc (Fv) 2 , Fab 3 , domain antibody (dAb), diabody. , Triabodies, tetrabodies, minibodies, and multispecific antibodies formed from antibody fragments thereof.
  • An “Fv fragment” is the smallest antibody fragment that contains a complete antigen recognition region and antigen binding region.
  • the "antibody modified product” is obtained by chemically modifying an antibody or antibody fragment, and examples thereof include antibodies to which various molecules such as polyethylene glycol (PEG) are bound.
  • PEG polyethylene glycol
  • the molecule that binds to the antibody is not limited.
  • Specifically recognizing a complex of aldosterone and an anti-aldosterone antibody means binding to a complex of aldosterone and an anti-aldosterone antibody with a higher affinity than for free aldosterone. Preferably, it means that it binds only to the complex of aldosterone and anti-aldosterone antibody, not to free aldosterone.
  • the affinity for free aldosterone, or the affinity for a complex of aldosterone and an anti-aldosterone antibody can be measured, for example, by an ELISA method or a method using the principle of surface plasmon resonance.
  • the affinity of the antibody that specifically recognizes the complex of aldosterone and the anti-aldosterone antibody according to the present invention for the complex of aldosterone and the anti-aldosterone antibody is 10 times, 20 times, or 30 times the affinity for free aldosterone. It is times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, preferably 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times. , 1000 times, and in some cases 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, 9000 times, 10000 times or more.
  • the present invention provides a technique for accurately detecting aldosterone in a sample.
  • KTM-611 antibody aldosterone-anti-aldosterone antibody complex
  • KTM-2012 antibody anti-aldosterone antibody
  • the horizontal axis shows the concentration of KTM-611 antibody or KTM-2012 antibody
  • the vertical axis shows absorption (main wavelength 450 nm, sub-wavelength 660 nm).
  • the squares show the results of the KTM-611 antibody
  • the circles show the results of the KTM-2012 antibody.
  • the antibody according to the present invention specifically binds to a complex of aldosterone and an anti-aldosterone antibody.
  • the structure of the antibody according to the present invention is not particularly limited as long as it is an antibody that specifically binds to a complex of aldosterone and an anti-aldosterone antibody, and is composed of, for example, two heavy chains and two light chains.
  • the antibody according to the present invention may be a monoclonal antibody, particularly a mouse monoclonal antibody.
  • examples of the monoclonal antibody include an antibody produced by a hybridoma, a recombinant antibody produced by a transformant transformed with a recombinant vector containing a DNA encoding a monoclonal antibody, and the like.
  • the antibody according to the present invention is prepared by fusing spleen B cells of an animal (for example, mouse) immunized with a complex of aldosterone and an anti-aldosterone antibody with myeloma cells (for example, P3-U1 cells) to prepare a hybridoma library. It can be obtained by selecting a hybridoma that produces the desired antibody from the library and purifying the antibody produced from the selected hybridoma. As the anti-aldosterone antibody, a commercially available antibody may be used.
  • aldosterone or a derivative thereof for example, keyhole limpet hemocyanin-binding aldosterone 3-carboxymethyloxime
  • myeloma cells for example, 240E cells.
  • the anti-aldosterone antibody produced may be used.
  • the antibody according to the present invention may be produced from the hybridoma KTM-611 internationally deposited on September 11, 2019, with the accession number NITE BP-03018.
  • Hybridoma KTM-611 is a depositary organization based on the provisions of Article 27-2 and 3 of the Patent Law Enforcement Regulations, and an international depositary authority based on the Budapest Treaty on International Approval of Deposits of Microorganisms. It has been internationally deposited at the Japan Biotechnology Center, Patented Microbial Deposit Center (NPMD) (Room 122, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture).
  • NPMD Patented Microbial Deposit Center
  • the antibody according to the present invention may be any of the following antibodies (1)-(6).
  • Contains antibodies (2)
  • Antibodies contained in (3) A heavy chain of CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 10, CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 11 and CDR3 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 12. Containing in, CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 16, CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 17 and CDR3 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 18 are light chains.
  • Amino acids contained in (4) One or several (2-20, preferably 2-10, more preferably 2-5, particularly preferably 2 or 3) amino acids are substituted in the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence.
  • One or several (2-20, preferably 2-10, more preferably 2-5, particularly preferably 2 or 3) amino acids are substituted in the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence.
  • An antibody in which the light chain contains a variable region consisting of the polypeptide represented by the deleted, inserted and / or added amino acid sequence.
  • One or several (2-20, preferably 2-10, more preferably 2-5, particularly preferably 2 or 3) amino acids are substituted in the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence.
  • the heavy chain contains a variable region consisting of the polypeptide represented by the deleted, inserted and / or added amino acid sequence, and one or several (2-20, preferably 2-20) in the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence.
  • the antibody according to the present invention is a recombinant antibody produced by a transformant transformed with a recombinant vector containing a DNA encoding any of the above antibodies (1) to (6). May be.
  • a DNA encoding the antibody according to the present invention is inserted into an expression vector capable of expressing the antibody according to the invention to prepare a recombinant vector, which is transformed with the recombinant vector. It can be produced by culturing the transformant.
  • Examples of the DNA encoding CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 10 include DNA of SEQ ID NO: 7, and DNA encoding CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 11.
  • Examples of the DNA include the DNA of SEQ ID NO: 8
  • examples of the DNA encoding CDR3 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 12 include the DNA of SEQ ID NO: 9.
  • Examples of the DNA encoding CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 16 include DNA of SEQ ID NO: 13, and DNA encoding CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 17.
  • Examples of the DNA include the DNA of SEQ ID NO: 14, and examples of the DNA encoding CDR3 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 18 include the DNA of SEQ ID NO: 15.
  • Examples of the DNA encoding the amino acid sequence of SEQ ID NO: 5 include the DNA of SEQ ID NO: 3, and examples of the DNA encoding the amino acid sequence of SEQ ID NO: 6 include the DNA of SEQ ID NO: 4.
  • the method for producing the antibody according to the present invention by the gene recombination method will be specifically described below. After obtaining the antibody produced by the hybridoma by the above-mentioned method, the following (I) to (V) are performed.
  • the cDNA encoding the VH and VL of the monoclonal antibody obtained by the above method can be obtained and the amino acid sequence can be analyzed as follows.
  • Total RNA is prepared from the hybridoma cells obtained by the above method, mRNA is extracted from the total RNA, and then cDNA is synthesized from the mRNA.
  • the DNA encoding VH or VL is amplified by PCR from the cDNA using a primer capable of specifically amplifying the DNA encoding VH or VL of the monoclonal antibody obtained by the above method.
  • the entire base sequence of VH or VL of the monoclonal antibody obtained by the above method is determined, and the entire amino acid sequence of VH or VL is estimated from the base sequence.
  • RNA ease kit for the preparation of total RNA from hybridoma cells, a kit of guanidine thiocyanate-cesium trifluoroacetate (Methos in Enzymol., 154, 3 (1987)) or RNA ease kit (manufactured by Qiagen) can be used.
  • the preparation of mRNA from total RNA can be performed by the Oligo (dT) immobilized cellulose column method (Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)), or Oligo-PuriT. It can be performed using a kit such as (manufactured by Takara Bio Inc.).
  • mRNA can also be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Global Life Science Technologies Japan). Synthesis of cDNA from mRNA can be performed using a kit such as PrimeScript (tm) II 1st strand cDNA Synthesis Kit (manufactured by Takara Bio Inc.). A method for specifically amplifying DNA encoding VH or VL of a monoclonal antibody can be carried out using a kit such as SMARTer RACE 5'/3'Kit (manufactured by Takara Bio Inc.).
  • the amplified DNA is inserted into a plasmid such as pBluescript SK (-) (manufactured by Agilent Technologies), and the base sequence of the DNA is determined by a commonly used base sequence analysis method or the like.
  • a base sequence analysis method for example, DNA processed by the dideoxy method (Proc. Natl. Acad. Sci. USA, 74, 5436 (1977)) or the like is used as an Applied Biosystems Sex Studio Genetic Analyzer (Thermo Fisher). ) And the like for analysis with an automatic base sequence analyzer.
  • the total amino acid sequences of VH and VL of the monoclonal antibody were estimated, respectively, and compared with the total amino acid sequences of VH and VL of the known antibody. And it can be confirmed whether it encodes the complete amino acid sequence of VL, respectively.
  • a database of the entire amino acid sequence for example, Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991), Immunology Today, vol. 18 (11), pp. 509 (1997) and the like can be mentioned.
  • the complete amino acid sequence of VH and VL of an antibody including the secretory signal sequence, the full amino acid sequence of VH and VL of a known antibody (Sequences of Proteins of Immunological Interest, US Dept.
  • the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and the subgroup to which they belong can be known.
  • the amino acid sequences of the VH and VL CDRs are also compared with the amino acid sequences of the VH and VL of known antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)). Can be done.
  • the BLAST method J. Mol. Biol., 215, 403 (1990) was performed on any database such as SWISS-PROT or PIR-Protein. ), BLASTP method (Nucleic Acids Res., Vol. 25 (17), pp. 3389-3402 (1997)), etc., and perform a homology search to determine whether the complete amino acid sequences of VH and VL are novel. You can check.
  • (III) Construction of Recombinant Antibody Expression Vector The amino acid sequences of CDR1, CDR2 and CDR3 of VH and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are used as the framework region of VH or VL of the antibody (hereinafter referred to as FR). Obtain the cDNA encoding the variable region transplanted into (notation).
  • the FR of the antibody VH or VL may be the same as or different from the animal species from which the VH and VL are derived.
  • a cDNA encoding a variable region in which the amino acid sequences of CDR1, CDR2 and CDR3 of VH and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are transplanted into FR of VH or VL of an antibody is inserted into a constant region-containing expression vector. Then, a recombinant antibody expression vector can be constructed.
  • the constant region-containing expression vector is an expression vector for animal cells in which DNA encoding CH and CL of an antibody is incorporated, and by inserting DNA encoding CH and CL of an antibody into the expression vector for animal cells, respectively. Can be built. In the present invention, CH and CL of any animal antibody can be used as CH and CL.
  • CH of the IgG1 subclass of the mouse antibody, CL of the ⁇ class, and the like can be used.
  • CDNA is used as the DNA encoding CH and CL of the antibody, but chromosomal DNA consisting of exons and introns can also be used.
  • the expression vector for animal cells any vector can be used as long as it can incorporate and express a gene encoding a constant region of an animal antibody from which CH and CL are derived.
  • pAGE107 (Cytotechnol., 3, 133 (1990), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27, 223 (1984)), pCI (GenBank Accession Number 19).
  • the promoters and enhancers include the initial promoter of SV40 (J. Biochem., 101, 1307 (1987)), Moloney mouse leukemia virus LTR (Biochem. Biophyss. Res. Commun.
  • the constant region of the antibody incorporated in 1987 may be a constant region of an antibody of the same species as the animal from which the heavy chain and the light chain are derived, or may be of a different animal species.
  • the constant region-containing expression vector has the points that the constant region-containing expression vector can be easily constructed, introduced into animal cells, and the expression levels of antibody heavy chain and light chain in animal cells are balanced. Therefore, a type (tandem type) constant region-containing expression vector (J. Immunol. Methods, 167, 271 (1994)) in which the DNA encoding the antibody heavy chain and the DNA encoding the antibody light chain are present on the same vector. ) Is used. It is also possible to use a type in which the DNA encoding the antibody heavy chain and the DNA encoding the antibody light chain are present on separate vectors. As a tandem type constant region-containing expression vector, pKANTEX93 (WO97 / 10354), pEE18 (Hybridoma, Vol.17, p.559 (1998)) and the like can be used.
  • the host cell transformed with the recombinant antibody expression vector is not particularly limited as long as it is a host cell capable of expressing the monoclonal antibody of the present invention.
  • COS-7 cell American Type Culture Collection (ATCC) number: CRL1651.
  • CHOK1 ATCC CCL-61
  • DUkXB11 ATCC CCL-9906
  • Pro-5 ATCC CCL-1781
  • CHO-S Life Technologies, Cat # 11619
  • mouse myeloma cell NSO mouse myeloma cell SP2 / 0-Ag14
  • mouse P3X63-Ag8.653 cell ATCC number: CRL1580
  • dihydrofolate reductase gene ATCC number: CRL1580
  • the DEAE-dextran method Methods in Nucleic Acids Res., CRC press (1991)
  • the electroporation method Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133
  • the lipofection method Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)
  • the amino acid sequence of FR of VH or VL includes, for example, the amino acid sequence of FR of an antibody registered in a database such as Protein Data Bank, or the common amino acid sequence of each subgroup of FR of an antibody (Sequences of Proteins of Immunological Interest). , US Dept. Health and Human Services (1991)) and the like.
  • an amino acid sequence of FR having as high a homology (at least 60% or more) as possible with the amino acid sequence of FR of VH or VL of the antibody from which CDR is derived is selected.
  • the amino acid sequences of CDR1 to 3 of VH and the amino acids of CDR1 to 3 of VL are transplanted to the amino acid sequence of FR of VH or VL of the selected antibody, respectively, and the amino acid sequence of VH or VL of the recombinant antibody is transplanted.
  • the designed amino acid sequence is converted into a DNA sequence in consideration of the frequency of use of codons found in the base sequence of the antibody gene (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)), and the gene is recombined. Design a DNA sequence encoding the VH or VL amino acid sequence of the antibody, respectively.
  • each heavy chain and light chain full-length synthetic DNA synthesized as one DNA based on the designed DNA sequence can also be used.
  • the amplified VH-encoding DNA and the VL-encoding DNA were inserted into separate plasmids such as pBluescript SK (-) (manufactured by Agilent Technologies), and the VH-encoding DNA was inserted.
  • plasmids such as pBluescript SK (-) (manufactured by Agilent Technologies)
  • the VH-encoding DNA was inserted.
  • the base sequence of the DNA is determined by a commonly used base sequence analysis method or the like. Examples of the base sequence analysis method include the above-mentioned methods.
  • the antigen-binding activity is lowered as compared with the antibody of the animal that is the basis of the CDR.
  • a humanized antibody has a lower antigen-binding activity than the original non-human antibody when only the non-human antibody VH and VL CDRs are transplanted into the human antibody VH and VL FR (BIO /). TECHNOLOGY, 9, 266 (1991)).
  • the amino acid residue directly involved in binding to the antigen the amino acid residue interacting with the amino acid residue of CDR, and the three-dimensional structure of the antibody.
  • the amino acid residues of FR of VH and VL of the antibody from which CDR is derived can be modified by carrying out the above-mentioned PCR reaction using synthetic DNA for modification.
  • the nucleotide sequence of the amplified product after the PCR reaction is determined in the same manner as described above, and it can be confirmed that the desired modification has been performed.
  • a cDNA encoding VH or VL of the recombinant antibody constructed above is inserted upstream of each gene encoding CH or CL of the antibody of the constant region-containing expression vector, and the recombinant antibody expression vector is inserted. Can be built.
  • the above constant region is contained. It can be inserted upstream of each gene encoding CH or CL inserted into the expression vector so that the recombinant antibody VH or VL is expressed in an appropriate form.
  • transformant examples include a transformant capable of transiently expressing a recombinant antibody, a transformant capable of stably expressing a recombinant antibody, and the like.
  • Method for producing a transformant that transiently expresses a recombinant antibody By introducing the recombinant antibody expression vector obtained in (III) above into an appropriate host cell, a transformant that transiently expresses the recombinant antibody can be obtained.
  • the host cell into which the recombinant antibody expression vector is introduced is not particularly limited as long as it is a host cell capable of expressing the recombinant antibody.
  • COS-7 cell American Type Culture Collection (ATCC) number: CRL1651)
  • CHOK1 ATCC CCL-61
  • DUkXB11 ATCC CCL-9906
  • Pro-5 ATCC CCL-1781
  • CHO-S Life Technologies, Cat # 11619
  • mouse myeloma cell NSO mouse myeloma cell SP2 / 0-Ag14
  • mouse P3X63-Ag8.653 cell ATCC number: CRL1580
  • dihydrofolate reductase gene ATCC number: CRL1580
  • the DEAE-dextran method Methods in Nucleic Acids Res., CRC press (1991)
  • the electroporation method Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133
  • the lipofection method Proc. Natl. Acad. Sci. USA, 84, 7413 (1987) or the like is used.
  • the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant were determined by the monoclonal antibody-Principles and practice, Third edition, Acadetic Press (1996), Antibi. It can be measured using A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987), and the like.
  • Method for producing a transformant that stably expresses a recombinant antibody By introducing the recombinant antibody expression vector obtained in (III) above into an appropriate host cell, a transformant that stably expresses the recombinant antibody can be obtained.
  • the recombinant antibody expression vector into the host cell for example, the above-mentioned method or the like can be used.
  • the host cell into which the recombinant antibody expression vector is introduced is not particularly limited as long as it is a host cell capable of stably expressing the recombinant antibody.
  • CHOK1 (ATCC CCL-61) and DUkXB11 (ATCC CCL) -9096), Pro-5 (ATCC CCL-781), CHO-S (Life Technologies, Cat # 11619), rat myeloma cells YB2 / 3HL. P2. G11.16Ag.
  • mouse myeloma cell NSO mouse myeloma cell SP2 / 0-Ag14
  • mouse P3X63-Ag8.653 cell ATCC number: CRL1580
  • dihydrofolate reductase gene ATCC number: CRL1580
  • the transformant that stably expresses the recombinant antibody is cultured in a medium for culturing animal cells containing a drug such as G418 sulfate (hereinafter referred to as G418). It can be selected (Japanese Patent Laid-Open No. 2-257891).
  • the medium for culturing animal cells includes RPMI1640 medium (manufactured by Thermo Fisher Scientific), GIT medium (manufactured by Kojin Bio), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), and Hybridoma.
  • RPMI1640 medium manufactured by Thermo Fisher Scientific
  • GIT medium manufactured by Kojin Bio
  • EX-CELL301 medium manufactured by JRH
  • IMDM medium manufactured by Invitrogen
  • Hybridoma Hybridoma.
  • -SFM medium manufactured by Thermo Fisher Scientific
  • a medium obtained by adding various additives such as FBS can be used.
  • the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by an ELISA method or the like.
  • the transformant can increase the expression level of the recombinant antibody by using a DHFR amplification system (Japanese Patent Laid-Open No. 2-257891) or the like.
  • the molecular weight of the whole heavy chain, light chain, or antibody molecule of the purified recombinant antibody can be determined by polyacrylamide gel electrophoresis (Nature, 227, 680 (1970)) or Western blotting (Monoclonal Antibodies-Principles and practice,). It can be measured using Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and the like.
  • the affinity of the purified antibody according to the present invention for the complex of aldosterone and anti-aldosterone antibody can be measured by using the method described above. It can also be measured by using the fluorescent antibody method (Cancer Immunol. Immunother., 36, 373 (1993)) or the like.
  • Hybridomas that produce antibodies examples include hybridomas KTM-611, which have been internationally deposited under accession number NITE BP-03018, and can be produced by the above-mentioned method.
  • DNA encoding the antibody examples include a DNA encoding any of the following antibodies (1) to (6).
  • Contains antibodies (2)
  • Antibodies contained in (3) A heavy chain of CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 10, CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 11 and CDR3 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 12. Containing in, CDR1 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 16, CDR2 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 17 and CDR3 consisting of the polypeptide represented by the amino acid sequence of SEQ ID NO: 18 are light chains.
  • Amino acids contained in (4) One or several (2-20, preferably 2-10, more preferably 2-5, particularly preferably 2 or 3) amino acids are substituted in the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence.
  • One or several (2-20, preferably 2-10, more preferably 2-5, particularly preferably 2 or 3) amino acids are substituted in the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence.
  • An antibody in which the light chain contains a variable region consisting of the polypeptide represented by the deleted, inserted and / or added amino acid sequence.
  • the heavy chain contains a variable region consisting of the polypeptide represented by the deleted, inserted and / or added amino acid sequence, and one or several (2-20, preferably 2-20) in the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence.
  • the DNA encoding the antibody according to the present invention is produced by a known DNA production method such as a method using a DNA synthesizer based on the base sequence determined by the method described in (I) or (II) above. be able to.
  • Recombinant vectors examples include the gene recombinant antibody expression vector in which a DNA encoding the antibody according to the present invention is introduced into an expression vector for animal cells.
  • the recombinant antibody expression vector can be produced by the method described in (III) above.
  • Transformant includes a transformant obtained by transforming a host cell by a known method using the recombinant vector containing the DNA encoding the antibody according to the present invention. be able to.
  • the transformant in the present invention can be produced by the method described in (IV) above.
  • Aldosterone Immunoassay Method An antibody that specifically recognizes a complex of aldosterone and an anti-aldosterone antibody according to the present invention can be used as an aldosterone immunoassay reagent and can be suitably used for an aldosterone immunoassay method.
  • the method for measuring aldosterone immunoassay is With aldosterone in the sample, Anti-aldosterone antibody that recognizes aldosterone, A complex-recognizing antibody that specifically recognizes a complex of aldosterone and the anti-aldosterone antibody, To form a multiplex complex of aldosterone, an anti-aldosterone antibody, and the complex recognition type antibody (hereinafter, referred to as "multiplex complex formation step”); and to measure the multiplex complex (hereinafter, hereinafter). , “Multiple complex measurement process”); including.
  • the concentration of aldosterone in the sample can be determined by including the following steps after the multiple complex measurement step. Using a known concentration of aldosterone as a sample, perform a multiple complex formation step and a multiple complex measurement step to create a calibration curve showing the relationship between the aldosterone concentration and the measured value (hereinafter referred to as "calibration curve preparation step"). ); And the concentration of aldosterone in the sample shall be determined from the calibration curve prepared in the calibration curve preparation step and the measured value obtained by the measurement in the multiple complex measurement step.
  • aldosterone in the sample in the present invention means the aldosterone present in the sample.
  • the aldosterone is not particularly limited as long as it can bind to an antibody, and may be in a free state by aldosterone alone, or may be in a complex form with another substance such as a protein.
  • the anti-aldosterone antibody in the present invention is not particularly limited as long as it is an antibody that specifically binds to aldosterone, and may be an antibody that binds to free aldosterone, an antibody that binds to a complex form of aldosterone, or the like. Further, as the anti-aldosterone antibody in the present invention, either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable. Further, in the present invention, not only a full-length antibody but also an antibody fragment can be used.
  • the antibody fragment examples include an antibody fragment obtained by removing an Fc moiety such as Fab obtained by treating an antibody with papain, F (ab') 2 obtained by pepsin treatment, and Fab' obtained by pepsin treatment-reduction treatment. Be done.
  • the "complex recognition type antibody” in the present invention is synonymous with a so-called anti-metatype antibody, and means an antibody that binds to a complex of an antigen and an antibody.
  • the complex recognition type antibody in this measurement method means an antibody that binds to a complex of aldosterone and an anti-aldosterone antibody, and may be an antibody that binds to any site of the complex of aldosterone and an anti-aldosterone antibody.
  • the "complex-recognizing antibody” in the present invention can bind to a plurality of types of complexes of aldosterone and an anti-aldosterone antibody.
  • the complex recognition type antibody in the present invention either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable. Further, in the present invention, not only a full-length antibody but also an antibody fragment can be used. Examples of the antibody fragment include the above-mentioned antibody fragment and the like.
  • the sample in the aldosterone immunoassay method of the present invention is not particularly limited as long as it is a sample that may contain aldosterone.
  • a sample that may contain aldosterone.
  • whole blood, plasma, serum, urine, spinal fluid, saliva, sheep water, urine, and sweat for example, whole blood, plasma, serum, urine, spinal fluid, saliva, sheep water, urine, and sweat.
  • Biological samples such as pancreatic fluid, and preferably whole blood, plasma, serum, and urine.
  • the method for measuring aldosterone immunoassay according to the present invention is generally not particularly limited as long as it is a measuring method using an immune reaction with an antibody. Examples include a latex agglutination method and an immunochromatography method.
  • the multiple complex formation step is a method of contacting an aldosterone, an anti-aldosterone antibody, and a complex recognition type antibody in a sample to form a multiple complex of aldosterone, an anti-aldosterone antibody, and a complex recognition type antibody. If so, there is no particular limitation.
  • the multiplex complex forming step may be carried out in an aqueous medium or by developing in an insoluble membrane (immonochromatography method), and is preferably carried out in an aqueous medium.
  • the reaction temperature of the reaction for forming the multiple complex is not particularly limited as long as it is a temperature that enables the immunoassay method for aldosterone of the present invention, and is usually 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C.
  • reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 seconds.
  • the concentration of the anti-aldosterone antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring the immunoassay of aldosterone of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0. 05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL, etc., 0.1
  • the range of ⁇ 20 ⁇ g / mL is preferable.
  • the concentration of the complex recognition type antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring aldosterone immunoassay of the present invention, and is 0.01, 0.02, 0.03, 0. 04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL, etc. , 0.1 to 20 ⁇ g / mL is preferred.
  • the anti-aldosterone antibody and the complex recognition type antibody may be brought into contact with aldosterone in the sample at the same time, but after the reaction between the anti-aldosterone antibody and aldosterone, the complex recognition type antibody is produced. It is more preferable to react. That is, the multiple complex forming step is a step of contacting aldosterone and an anti-aldosterone antibody in a sample to form a first complex (hereinafter referred to as "complex 1") of the anti-aldosterone antibody and aldosterone (hereinafter referred to as "complex 1").
  • complex 2 a second complex of aldosterone, an anti-aldosterone antibody and a complex recognition type antibody. It is preferable to include the step of forming (1-2).
  • “contacting the anti-aldosterone antibody and the complex recognition type antibody with the aldosterone in the sample at the same time” means that the aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition type antibody are reacted in one reaction. It includes a state in which the aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition type antibody can come into contact with each other by coexisting in the container. In this case, the anti-aldosterone antibody, the complex recognition type antibody, and the aldosterone may be added in any order and brought into contact with each other.
  • the step (1-1) is not particularly limited as long as it is a method in which the aldosterone in the sample and the anti-aldosterone antibody are brought into contact with each other to form a complex 1 of the aldosterone and the anti-aldosterone antibody.
  • the step (1-1) may be carried out in an aqueous medium or by developing in an insoluble membrane, and is preferably carried out in an aqueous medium.
  • step (1-1) the reaction temperature of the reaction in which the aldosterone in the sample is brought into contact with the anti-aldosterone antibody to form the complex 1 is a temperature that enables the method for measuring aldosterone immunoassay of the present invention.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 seconds.
  • the concentration of the anti-aldosterone antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring the immunoassay of aldosterone of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0. 05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL, etc., 0.1
  • the range of ⁇ 20 ⁇ g / mL is preferable.
  • step (1-2) the complex 1 of the aldosterone and the anti-aldosterone antibody is brought into contact with the complex recognition type antibody to form the complex 2 of the aldosterone, the anti-aldosterone antibody and the complex recognition type antibody.
  • the "complex 2" formed in the step (1-2) is the same as the "multiplex" formed in the multiple complex forming step.
  • the step (1-2) may be carried out in an aqueous medium or by developing in an insoluble membrane, and is preferably carried out in an aqueous medium.
  • the reaction temperature of the reaction in which the complex 1 is brought into contact with the complex recognition antibody to form the complex 2 enables the method for measuring aldosterone immunoassay of the present invention.
  • the temperature is not particularly limited, and is usually 1 ° C, 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 11 ° C, 12 ° C, 13 ° C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C , 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 31
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 seconds.
  • the concentration of the complex recognition type antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring aldosterone immunoassay of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0. 8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL, etc., 0
  • the range of 1 to 20 ⁇ g / mL is preferable.
  • the anti-aldosterone antibody or complex recognition antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized.
  • the insoluble carrier is not particularly limited as long as it is an insoluble carrier that immobilizes an anti-aldosterone antibody or a complex recognition type antibody and enables the immunoassay method for aldosterone of the present invention.
  • a slide glass or an ELISA plate microtiter plate).
  • Beads, latex particles, magnetic particles, filters, films, membranes and the like examples of the material of the insoluble carrier include glass, silicon, ceramic, cellulose, nitrocellulose, nylon, polycarbonate, polyethylene, polypurpyrene, polystyrene, polyethylene terephthalate, polyurethane and the like.
  • the method for immobilizing the anti-aldosterone antibody or the complex recognition type antibody on the insoluble carrier is not particularly limited as long as it is an immobilization method that enables the immunometric method for aldosterone of the present invention, and is, for example, a chemical binding method (covalent bond method).
  • a known method such as a method of immobilizing by bonding) or a method of physically adsorbing can be applied.
  • the biotinylated antibody in which biotin is bound to the antibody may be immobilized on a streptavidin plate coated with streptavidin.
  • the antibody may be immobilized on an insoluble carrier via a linker.
  • linker examples include molecules capable of covalently binding both a functional group on the surface of an insoluble carrier and a functional group possessed by an anti-aldosterone antibody or a complex recognition type antibody.
  • an anti-aldosterone antibody or a complex recognition type antibody can be used.
  • a molecule in which the second reaction active group is different from that of the second reaction active group is preferably used.
  • Examples of the functional group possessed by the functional group of the anti-aldosterone antibody or the complex recognition type antibody and the functional group retained on the surface of the insoluble carrier include a carboxyl group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group and an imino group. Examples thereof include an N-hydroxysuccinyl group and a maleimide group.
  • Examples of the reactive group in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide ester, pentafluorophenyl (PFP) ester, solarene, pyridyl disulfide, vinyl sulfone and the like. The basis of.
  • a washing step (B / F separation step) for washing the insoluble carrier after the reaction of the multiple complex forming step using a washing liquid.
  • the cleaning solution used in the cleaning step is not particularly limited as long as it is a cleaning solution that enables the method for measuring the immunity of aldosterone of the present invention, and examples thereof include a PBS solution and a PBS solution containing a surfactant.
  • the surfactant include nonionic surfactants such as Tween 20 and the like.
  • a washing step for washing the insoluble carrier after the reaction of the step (1-1) is added between the steps (1-1) and the step (1-2), if necessary. You may.
  • the washing step impurities and unreacted antibodies in the sample can be removed from the surface of the insoluble carrier, and only the complex 1 formed on the surface of the carrier can be separated.
  • the cleaning solution used in the cleaning step is not particularly limited as long as it is a cleaning solution that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned cleaning solution.
  • a method for separating the complex of aldosterone, the anti-aldosterone antibody and the complex recognition type antibody for example, a chromatography method, a high performance liquid chromatography method, an electrophoresis method, a capillary electrophoresis method , Capillary chip electrophoresis, for example, a method using an automatic immunoassay device such as LiBAsys (manufactured by Shimadzu Corporation) can be applied.
  • the multiple complex measuring step is not particularly limited as long as it is a method of measuring the multiple complex formed in the multiple complex forming step by using the following method.
  • the multiplex complex can be measured using a labeled third antibody in which a labeling substance is bound to an anti-aldosterone antibody or an antibody that binds to a complex recognition type antibody (hereinafter referred to as “third antibody”). That is, the labeled third antibody and the multiplex complex are brought into contact with each other to form a complex 3 of an anti-aldosterone antibody, an aldosterone, a complex recognition type antibody, and a labeled third antibody, and the complex is formed.
  • the third antibody include an anti-aldosterone antibody or an antibody that binds to the Fc region of a complex recognition type antibody.
  • the multiple complex measurement step is performed without performing the washing step after the multiple complex formation step.
  • the homogenius method include a latex agglutination method described later, the surface plasmon resonance, and the like.
  • the multiplex complex can be measured by measuring the agglutination generated by the immune reaction.
  • the method for measuring aggregation include a method for measuring absorbance, a method for measuring scattered light, and the like.
  • the reaction temperature of the reaction when the labeled third antibody is brought into contact with the multiplex complex to form the complex 3 is particularly limited as long as it is a temperature that enables the immunoassay method for aldosterone of the present invention.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 seconds.
  • the concentration of the labeled third antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring aldosterone immunoassay of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0. 8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL, etc., 0
  • the range of 1 to 20 ⁇ g / mL is preferable.
  • the multiplex complex measurement step can be performed by measuring the multiplex complex of aldosterone, an anti-aldosterone antibody, and a complex recognition type antibody using a labeling substance.
  • the labeling substance may be bound to either an anti-aldosterone antibody or a complex recognition type antibody.
  • Methods of binding a labeling substance to an anti-aldosterone antibody or complex recognition antibody are known in the art of immunoassay.
  • the labeling substance can be attached to the antibody via one or several amino acids, or via a linker with one or several amino acids.
  • various kits for binding a labeling substance to a protein are commercially available.
  • the labeling substance for example, a substance that specifically binds to enzymes, radioactive isotopes, fluorescent substances, luminescent substances, DNA, RNA, coenzymes or coenzymes used in ordinary immunoassay methods (biotin, Avidin), tags, substances that absorb in the ultraviolet to infrared regions, color-developing fine particles, fluorescent fine particles, metallic fine particles, magnetic substances, substances having properties as spin labeling agents, and the like.
  • the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.
  • Radioisotopes include, for example, 3 H, 14 C, 35 S, 32 P, 125 P and 131 I.
  • fluorescent substance examples include FITC (fluorescein isothiocyanate) and RITC (rhodamine B-isothiocyanate).
  • luminescent substance examples include aclidinium and its derivatives, ruthenium complex compounds, loffin and the like.
  • a multiplex complex of aldosterone, an anti-aldosterone antibody, and a complex-recognizing antibody can be detected.
  • the method for measuring the signal generated from the labeling substance may be appropriately selected depending on the labeling substance to be used.
  • the labeling substance is a coloring substance, that is, a substance that absorbs light of a certain wavelength
  • the labeling substance can be measured by measuring the absorbance using a spectrophotometer, a multi-well plate reader, or the like.
  • the labeling substance is a fluorescent substance
  • the labeling substance can be measured by measuring the fluorescence intensity using a fluorometer, a fluorescent multi-well plate reader, or the like.
  • the labeling substance When the labeling substance is a luminescent substance, the labeling substance can be measured by measuring the luminescence intensity using a luminescent photometer, a luminescent multi-well plate reader, or the like.
  • the labeling substance When the labeling substance is a radioisotope, the labeling substance can be measured by measuring the radioactivity with a scintillation counter, a ⁇ -well counter, or the like.
  • the labeling substance is an enzyme
  • the labeling substance can be measured by measuring the enzyme activity. For example, the labeling substance can be measured by reacting the substrate of the enzyme with the enzyme and measuring the produced substance.
  • the method for measuring aldosterone immunoassay according to the present invention is not limited to the method used, and can be applied to implementation by an automatic analyzer. There are no particular restrictions on the combination of reagents, etc. when measuring using the method or automatic analyzer, depending on the environment and model of the automatic analyzer to be applied, or taking other factors into consideration. A combination of appropriate reagents and the like may be selected and used. Furthermore, the method for measuring aldosterone immunoassay according to the present invention can also be applied to Micro-TAS (Micro-Total Analysis Systems: ⁇ -TAS, ⁇ comprehensive analysis system).
  • Micro-TAS Micro-Total Analysis Systems: ⁇ -TAS, ⁇ comprehensive analysis system
  • the aqueous medium in the present invention is not particularly limited as long as it is an aqueous medium that enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable.
  • the aqueous medium may contain salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride, lithium bromide, sodium bromide, potassium bromide, calcium bromide, magnesium bromide, ammonium bromide and the like. ..
  • Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like.
  • saccharides include mannitol and sorbitol.
  • Examples of preservatives include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), bioace, Proclin 300, Proxel GXL, and the like.
  • Examples of the protein include bovine serum albumin (hereinafter referred to as BSA) and the like.
  • Examples of the surfactant include nonionic surfactants and the like.
  • Examples of the protein stabilizer include a peroxidase stabilizing buffer (Peroxidase Stabilizing Buffer, manufactured by DakoCytomation) and the like.
  • the sandwich ELISA includes: [1] With aldosterone in the sample, Anti-aldosterone antibody that recognizes aldosterone, To form a complex of aldosterone and an anti-aldosterone antibody (corresponding to "step (1-1)"above); [2] With the complex A complex-recognizing antibody that specifically recognizes a complex of aldosterone and the anti-aldosterone antibody, To form a multiplex complex of aldosterone, an anti-aldosterone antibody, and a complex-recognizing antibody (corresponding to "step (1-2)"above); and [3] measure the multiplex complex. (Corresponding to the above-mentioned "multiple complex measurement step").
  • the concentration of aldosterone in the sample can be determined by including the following. [4] Using a known concentration of aldosterone as a sample, perform the steps [1]-steps [3] to prepare a calibration curve showing the relationship between the aldosterone concentration and the measured value of the multiplex complex; and [ 5] To determine the concentration of aldosterone in the sample from the calibration curve prepared in the calibration curve preparation step and the measured value obtained by the measurement in the step [3].
  • step [1] and [2] may be performed sequentially or simultaneously.
  • Performing step [1] and step [2] at the same time means that aldosterone, an anti-aldosterone antibody, and a complex recognition type antibody in a sample are allowed to coexist in one reaction vessel, and aldosterone and an anti-aldosterone antibody are allowed to coexist. It also includes making the complex-recognizing antibody in contact with each other.
  • the step [1] of the measurement method 1 is not particularly limited as long as it is a method in which the aldosterone in the sample and the anti-aldosterone antibody are brought into contact with each other to form a complex 1 of the aldosterone and the anti-aldosterone antibody.
  • the step [1] is preferably performed in an aqueous medium.
  • the reaction temperature of the reaction in which the aldosterone and the anti-aldosterone antibody are brought into contact with each other to form the complex 1 is a temperature that enables the aldosterone immunoassay method of the present invention. There is no particular limitation, and examples thereof include the above-mentioned temperature.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and the above-mentioned time can be mentioned.
  • the concentration of the anti-aldosterone antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • the complex 1 of aldosterone and the anti-aldosterone antibody and the complex recognition type antibody are brought into contact with each other to obtain the complex 2 of the aldosterone, the anti-aldosterone antibody and the complex recognition type antibody.
  • Step [2] is preferably carried out in an aqueous medium.
  • the reaction temperature of the reaction of bringing the complex 1 into contact with the complex-recognizing antibody to form the complex 2 enables the immunoassay method for aldosterone of the present invention.
  • the temperature is not particularly limited, and examples thereof include the above-mentioned temperature.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned time.
  • the concentration of the complex recognition antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • a cleaning step may be added between the steps [1] and [2]. Further, a cleaning step may be added between the steps [2] and the step [3]. Examples of the cleaning method and cleaning solution in the cleaning step include the above-mentioned method and cleaning solution.
  • the anti-aldosterone antibody may or may not be immobilized on the insoluble carrier, but it is preferably immobilized.
  • the method for immobilizing the anti-aldosterone antibody on the insoluble carrier include the above-mentioned methods.
  • the complex recognition type antibody may or may not be labeled with a labeling substance, but it is preferably labeled.
  • the labeling substance in the multiplex complex may be measured in step [3].
  • the method for binding the labeling substance to the complex recognition type antibody include the above-mentioned methods.
  • the labeling substance include the above-mentioned labeling substances and the like.
  • the step [3] of the measurement method 1 is not particularly limited as long as it is a step that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the method of the multiple complex measurement step described above.
  • the insoluble carrier on which the anti-aldosterone antibody is immobilized may be produced in the reaction solution of the antigen-antibody reaction.
  • the anti-aldosterone antibody to which one of the set of affinity substances (A) is bound and the insoluble carrier to which the other (a) of the set of affinity substances is bound are reacted in the reaction solution of the antigen-antibody reaction.
  • an insoluble carrier on which the anti-aldosterone antibody is immobilized can be produced in the reaction solution of the antigen-antibody reaction.
  • Examples of the combination of Aa include a combination of biotin-avidins (avidin, neutravidin, streptavidin, etc.), avidins (avidin, neutravidin, streptavidin, etc.)-biotin, and an anti-aldosterone antibody Fc. Region-An antibody combination that binds to the Fc region can be mentioned.
  • the sandwich ELISA according to this embodiment includes: [1] With aldosterone in the sample, Anti-aldosterone antibody that recognizes aldosterone, A complex-recognizing antibody that specifically recognizes a complex of aldosterone and the anti-aldosterone antibody, To form a multiplex complex of aldosterone, an anti-aldosterone antibody, and the complex recognition type antibody (corresponding to the "multiplex complex formation step”); and [2] measure the multiplex complex. (Corresponding to the above-mentioned "multiple complex measurement step").
  • the concentration of aldosterone in the sample can be determined by including the following. [3] Using a known concentration of aldosterone as a sample, perform steps [1] and [2] to prepare a calibration curve showing the relationship between the aldosterone concentration and the measured value of the multiplex complex; and [4]. ] To determine the concentration of aldosterone in the sample from the calibration curve created in the calibration curve preparation step and the measured value obtained by the measurement in step [2].
  • the aldosterone in the sample, the anti-aldosterone antibody that recognizes aldosterone, and the complex-recognizing antibody that specifically recognizes the complex of the anti-aldosterone antibody are brought into contact with each other.
  • "" Means that aldosterone, an anti-aldosterone antibody, and a complex-recognizing antibody that specifically recognizes a complex of the anti-aldosterone antibody are brought into contact with each other at the same time, or with aldosterone in a sample.
  • the anti-aldosterone antibody and the complex recognition type antibody are reacted and coexisted in one container so that the aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition type antibody can come into contact with each other.
  • Means to do. "The aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition antibody can coexist in one container so that the aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition antibody can come into contact with each other.
  • the aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition type antibody may be added in any order and brought into contact with each other.
  • the aldosterone, the anti-aldosterone antibody, and the complex recognition type antibody in the sample are brought into contact with each other, and a multiple complex of the aldosterone, the anti-aldosterone antibody, and the complex recognition type antibody is brought into contact with each other.
  • the step [1] is preferably performed in an aqueous medium.
  • the reaction temperature of the reaction in which the aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition type antibody are brought into contact with each other to form the multiple complex is the temperature of the reaction of the aldosterone of the present invention.
  • the temperature is not particularly limited as long as it enables the immunoassay, and examples thereof include the above-mentioned temperatures.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned time.
  • the concentration of the anti-aldosterone antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • the concentration of the complex recognition antibody in the reaction solution is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned concentrations.
  • a cleaning step may be added between the step [1] and the step [2] of the measurement method 2.
  • Examples of the cleaning method and cleaning solution in the cleaning step include the above-mentioned method and cleaning solution.
  • the complex recognition type antibody may or may not be immobilized on the insoluble carrier, but it is preferably immobilized.
  • the method for immobilizing the complex recognition type antibody on the insoluble carrier include the above-mentioned methods.
  • the anti-aldosterone antibody may or may not be labeled with a labeling substance, but it is preferably labeled.
  • the labeling substance in the multiplex complex may be measured in step [2].
  • the method for binding the labeling substance to the anti-aldosterone antibody include the above-mentioned methods.
  • the labeling substance include the above-mentioned labeling substances and the like.
  • the step [2] of the measurement method 2 is not particularly limited as long as it is a step that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the method of the multiple complex measurement step described above.
  • the insoluble carrier on which the complex recognition type antibody is immobilized may be produced in the reaction solution of the antigen-antibody reaction.
  • the complex recognition type antibody to which one of the set of affinity substances (B) is bound and the insoluble carrier to which the other (b) of the set of affinity substances is bound are contained in the reaction solution for the antigen-antibody reaction.
  • an insoluble carrier on which the complex recognition type antibody is immobilized can be produced in the reaction solution of the antigen-antibody reaction.
  • Examples of the combination of BB include the same combination as Aa described above.
  • the latex agglutination method according to this embodiment includes the following. [1] The aldosterone in the sample and the anti-aldosterone antibody to which the insoluble carrier particles are bound are brought into contact with each other in an aqueous medium to form a complex of the aldosterone and the anti-aldosterone antibody to which the insoluble carrier particles are bound (the above-mentioned "step”. (1-1) ”); [2] A complex recognition antibody to which insoluble carrier particles are bound is added to the solution after the step [1], and the complex is brought into contact with the complex recognition antibody to which the insoluble carrier particles are bound to cause aggregation. To generate (corresponding to the above-mentioned "step (1-2)”); and [3] to measure the agglutination (corresponding to the above-mentioned "multiple complex measurement step").
  • the sample can be diluted with the above-mentioned aqueous medium in advance and held, and then the step [1] can be performed.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • the temperature at which the sample is previously diluted with an aqueous medium and held is not particularly limited as long as it is a temperature that enables the measurement of aldosterone of the present invention, and is 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C.
  • the time for pre-diluting and holding the sample in an aqueous medium is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds. 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes , 18 minutes, 19 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes. Minutes, 170 minutes, 180 minutes, 190 minutes, 200 minutes, 210 minutes, 220 minutes, 230 minutes, 240 minutes and the like can be mentioned.
  • the concentration of aldosterone in the sample can be determined by including the following. [4] Using a known concentration of aldosterone as a sample, perform steps [1]-[3] to prepare a calibration curve showing the relationship between the aldosterone concentration and the measured value of the aggregation; and [5] step [5] Determine the concentration of aldosterone in the sample from the calibration curve prepared in 4] and the measured value of the aggregation obtained by the measurement in step [3].
  • the steps [1] and [2] of the measurement method 3 may be performed sequentially or simultaneously.
  • the aldosterone in the sample is brought into contact with the anti-aldosterone antibody to which the insoluble carrier particles are bound to form a complex of the aldosterone and the anti-aldosterone antibody to which the insoluble carrier particles are bound.
  • the step [1] is preferably performed in an aqueous medium.
  • the reaction temperature of the reaction in which the aldosterone in the sample and the anti-aldosterone antibody to which the insoluble carrier particles are bound are brought into contact with each other to form the complex is the immunoassay of the aldosterone of the present invention.
  • the temperature is not particularly limited as long as it enables the method, and examples thereof include the above-mentioned temperature.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and the above-mentioned time can be mentioned.
  • the concentration of the anti-aldosterone antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • the concentration of the insoluble carrier particles to which the anti-aldosterone antibody is bound in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and is 0.00005, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.
  • the step [2] of the measurement method 3 is a method of contacting a complex of an anti-aldosterone antibody to which aldosterone and insoluble carrier particles are bound and a complex recognition type antibody to which insoluble carrier particles are bound to form an aggregate. If so, there is no particular limitation. Step [2] is preferably carried out in an aqueous medium.
  • the reaction temperature of the reaction in which the complex is brought into contact with the complex-recognizing antibody to which the insoluble carrier particles are bound to form the aggregation is the immunoassay of aldosterone of the present invention. There is no particular limitation as long as the temperature enables the method, and examples thereof include the above-mentioned temperature.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned time.
  • the concentration of the complex recognition antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • the concentration of the insoluble carrier particles to which the complex recognition type antibody is bound in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and is 0.00005, 0.0001, 0.
  • the step [3] of the measurement method 3 is not particularly limited as long as it is a step that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the method of the multiple complex measurement step described above.
  • the latex agglutination method includes the following. [1] In an aqueous medium, the aldosterone in the sample, the complex recognition type antibody to which the insoluble carrier particles are bound, and the anti-aldosterone antibody to which the insoluble carrier particles are bound are brought into contact with each other to generate agglutination (the above-mentioned "multi”. Corresponds to "heavy complex formation step”); [2] Measuring the agglomeration (corresponding to the above-mentioned “multiple complex measurement step”).
  • the sample can be diluted with the above-mentioned aqueous medium in advance and held, and then the step [1] can be performed.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • the temperature at which the sample is previously diluted with an aqueous medium and held is not particularly limited as long as it enables the measurement of aldosterone of the present invention, and examples thereof include the above-mentioned temperature.
  • the time for diluting and holding the sample in an aqueous medium in advance is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned time.
  • the concentration of aldosterone in the sample can be determined by including the following. [3] Using a known concentration of aldosterone as a sample, perform the steps [1] and [2] to prepare a calibration curve showing the relationship between the aldosterone concentration and the measured value of the aggregation; and [4]. To determine the concentration of aldosterone in the sample from the calibration curve prepared in step [3] and the measured value of the agglomeration obtained by the measurement in step [2].
  • step [1] of the measuring method 4 "contacting the aldosterone in the sample, the complex recognition type antibody to which the insoluble carrier particles are bound, and the anti-aldosterone antibody to which the insoluble carrier particles are bound” is described in the sample. It means that the complex recognition type antibody to which the aldosterone is bound, the anti-aldosterone antibody to which the insoluble carrier particles are bound, and the anti-aldosterone antibody to which the insoluble carrier particles are bound are simultaneously contacted, or the aldosterone in the sample and the anti-insoluble carrier are brought into contact with each other at the same time.
  • Complex recognition type antibody to which particles are bound and anti-aldosterone antibody to which the insoluble carrier particles are bound are allowed to coexist in one reaction vessel, and aldosterone in a sample and the anti-aldosterone particles are bound to recognize the complex. It also means that the type antibody and the anti-aldosterone antibody to which the insoluble carrier particles are bound can be brought into contact with each other. "A aldosterone in a sample, a complex recognition type antibody to which the anti-insoluble carrier particles are bound, and an anti-aldosterone antibody to which the insoluble carrier particles are bound are allowed to coexist in one reaction vessel to obtain aldosterone in the sample.
  • the complex recognition type antibody to which the anti-insoluble carrier particles are bound and the anti-aldosterone antibody to which the insoluble carrier particles are bound can come into contact with each other, the aldosterone in the sample and the anti-aldosterone particles are present.
  • the bound complex recognition type antibody and the anti-aldosterone antibody to which the insoluble carrier particles are bound may be added in any order and brought into contact with each other.
  • step [1] of the measurement method 4 is a method in which the complex recognition type antibody to which the aldosterone, the insoluble carrier particles are bound, and the anti-aldosterone antibody to which the insoluble carrier particles are bound are brought into contact with each other to form an aggregate.
  • the step [1] is preferably carried out in an aqueous medium.
  • the reaction of the reaction in which the complex recognition type antibody to which the aldosterone, the insoluble carrier particles are bound, and the anti-aldosterone antibody to which the insoluble carrier particles are bound are brought into contact with each other to form the agglutination.
  • the temperature is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned temperatures.
  • the reaction time of the reaction is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned time.
  • the concentration of the complex recognition antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • the concentration of the anti-aldosterone antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • the concentration of the insoluble carrier particles to which the complex recognition antibody binds in the reaction solution is not particularly limited as long as it enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned concentrations.
  • the concentration of the insoluble carrier particles to which the anti-aldosterone antibody binds in the reaction solution is not particularly limited as long as it enables the method for measuring aldosterone immunoassay of the present invention, and examples thereof include the above-mentioned concentrations.
  • the total concentration of the insoluble carrier particles to which the complex recognition type antibody binds and the insoluble carrier particles to which the anti-aldosterone antibody binds in the reaction solution is particularly high as long as it is a concentration that enables the method for measuring aldosterone immunoassay of the present invention.
  • examples of the method of immobilizing the anti-aldosterone antibody or the complex recognition type antibody on the insoluble carrier include the above-mentioned methods.
  • the anti-aldosterone antibody to which the insoluble carrier particles are bound may be produced in the reaction solution of the antigen-antibody reaction, and in this case, one of the set of affinity substances (C).
  • the complex recognition type antibody to which the insoluble carrier particles are bound may be produced in the reaction solution of the antigen-antibody reaction, and in this case, the complex recognition to which one of the set of affinity substances (D) is bound.
  • a complex recognition type antibody to which the insoluble carrier particles are bound can be produced.
  • the anti-aldosterone antibody to which the insoluble carrier particles are bound and the complex recognition type antibody to which the insoluble carrier particles are bound are produced in the same reaction solution, a set of affinity substances consisting of C and c is produced. Is preferably different from the set of affinity substances consisting of D and d.
  • Examples of the set of affinity substances include a combination of avidins (avidin, streptavidin, neutravidin, etc.) and biotin, a combination of an Fc region of an antibody and an antibody that binds to the Fc region, and the like.
  • the combination of Cc and the combination of Dd include, for example, the same combination as Aa described above.
  • the step [2] of the measurement method 4 is not particularly limited as long as it is a step that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the method of the multiple complex measurement step described above.
  • the insoluble carrier particles of the measuring method 3 and the measuring method 4 are not particularly limited as long as they are insoluble carrier particles that enable the method for measuring the immunity of aldosterone of the present invention, and examples thereof include latex particles and magnetic particles. Particles are preferred. Examples of the material of the latex particles include polystyrene, polyvinyl chloride, polypropylene and the like.
  • the particle size of the insoluble carrier particles of the measuring method 3 and the measuring method 4 is not particularly limited as long as it is a particle size that enables the method for measuring the immunity of aldosterone of the present invention, and the average particle size is 30 to 800 nm. The average particle size is preferably 100 to 500 nm, and more preferably 150 to 450 nm.
  • the insoluble carrier particles to which the anti-aldosterone antibody binds and the insoluble carrier particles to which the complex recognition type antibody binds may be the same or different, but the same is used. preferable. Further, in the measuring method 3 and the measuring method 4, the average particle size of the insoluble carrier particles to which the anti-aldosterone antibody binds and the average particle size of the insoluble carrier particles to which the complex recognition type antibody binds are the same but different. You may.
  • a step of mixing the sample with a sample diluent containing an aqueous medium may be included.
  • the aqueous medium include the above-mentioned aqueous medium and the like.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • the aqueous medium in the measuring methods 1 to 4 is not particularly limited as long as it is an aqueous medium that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned aqueous medium.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • the samples in the measuring methods 1 to 4 are not particularly limited as long as they are samples that may contain aldosterone. For example, whole blood, plasma, serum, urine, spinal fluid, saliva, sheep water, urine, sweat, pancreatic fluid, etc. Examples thereof include whole blood, plasma, serum and urine.
  • the immunochromatographic method includes the following. [1] Supplying a sample to the sample supply section of the test strip; [2] Contacting aldosterone in a sample with an anti-aldosterone antibody that is retained elution in the developing portion of the test strip to form a complex of aldosterone with the anti-aldosterone antibody; [3] In the detection unit, the complex is brought into contact with the complex-recognizing antibody to form a multiplex complex of aldosterone, an anti-aldosterone antibody, and a complex-recognizing antibody; and [4] the multiplex complex is formed. To measure.
  • the concentration of aldosterone in the sample can be determined by including the following. [5] Using a known concentration of aldosterone as a sample, perform the above steps [1]-[4] to prepare a calibration curve showing the relationship between the aldosterone concentration and the measured value of the multiplex complex; [6] The concentration of aldosterone in the sample is determined from the calibration curve prepared in the calibration curve preparation step and the measured value obtained by the measurement in the step [5].
  • the test strip used here has a membrane made of a porous body having a sample supply part, a development part and a detection part.
  • An anti-aldosterone antibody (referred to as a labeled anti-aldosterone antibody) to which a labeling substance is bound is held in a part of the developing portion so that it can be eluted. Further, it has a detection part in which a complex recognition type antibody is immobilized on the downstream side of the development part. Examples of the method for binding the labeling substance to the anti-aldosterone antibody include the above-mentioned methods.
  • the sample supply unit is a place where a liquid sample is supplied, and may be any substance and form that can absorb the sample and allow the liquid and aldosterone to pass through.
  • the unfolding section is a place where the sample containing aldosterone is unfolded by a capillary phenomenon, and is located downstream of the sample supply section.
  • the step [1] in the measurement method 5 is a step of supplying a sample to the sample supply unit.
  • the sample may be diluted with a sample diluent in advance and then supplied to the sample supply unit. Further, the diluted sample may be supplied to the sample supply unit after the sample is diluted with the sample diluent and held.
  • the diluent includes the above-mentioned aqueous medium and the like.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • the retention time of the diluted sample is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, Examples include 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes and the like.
  • the step [2] of the measurement method 5 is a step of contacting the aldosterone in the sample with the labeled anti-aldosterone antibody to form a complex of the aldosterone and the labeled anti-aldosterone antibody.
  • aldosterone and the anti-aldosterone antibody come into contact with each other by developing in a membrane containing an aqueous medium.
  • the step [3] of the measurement method 5 is a step of forming a multiple complex of a complex of aldosterone and a labeled anti-aldosterone antibody and a complex recognition type antibody immobilized on the detection section in the detection unit.
  • the multiplex complex is formed on a membrane.
  • the step [4] of the measurement method 5 is a step of measuring the multiplex complex in the detection unit.
  • the multiplex complex can be measured by measuring the labeling substance in the multiplex complex.
  • the time from the addition of the sample to the sample supply unit to the measurement of the multiple complex in the detection unit is particularly limited as long as the time enables the immunoassay method for aldosterone of the present invention.
  • the immunochromatographic method includes the following. [1] Supplying a sample to the sample supply section of the test strip; [2] In the detection unit, the aldosterone in the sample, the anti-aldosterone antibody, and the complex recognition type antibody are brought into contact with each other to form a multiple complex of aldosterone, the anti-aldosterone antibody, and the complex recognition type antibody; [3] To measure the multiplex complex.
  • the concentration of aldosterone in the sample can be determined by including the following. [4] Using a known concentration of aldosterone as a sample, perform the steps [1]-steps [3] to prepare a calibration curve showing the relationship between the aldosterone concentration and the measured value of the multiplex complex; and [ 5] To determine the concentration of aldosterone in the sample from the calibration curve prepared in the calibration curve preparation step and the measured value obtained by the measurement in the step [3].
  • the test strip used here has a membrane made of a porous body having a sample supply part, a development part and a detection part.
  • a complex-recognizing antibody (referred to as a labeled complex-recognizing antibody) to which a labeling substance is bound is held in a part of the developing portion so that it can be eluted. Further, it has a detection part in which an anti-aldosterone antibody is immobilized on the downstream side of the development part.
  • Examples of the method for binding the labeling substance to the complex recognition type antibody include the above-mentioned methods.
  • the sample supply unit is a place where a liquid sample is supplied, and may be any substance and form that can absorb the sample and allow the liquid and aldosterone to pass through.
  • the developing unit is a place where the sample containing aldosterone and the labeled complex recognition antibody are developed by the capillary phenomenon, and is located downstream of the sample supply unit.
  • the step [1] in the measurement method 6 is a step of supplying a sample to the sample supply unit.
  • the sample may be diluted with a sample diluent in advance and then supplied to the sample supply unit. Further, the diluted sample may be supplied to the sample supply unit after the sample is diluted with the sample diluent and held.
  • the diluent includes the above-mentioned aqueous medium and the like.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • the retention time of the diluted sample is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, Examples include 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes and the like.
  • the step [2] of the measurement method 6 is a step of forming a multiple complex of aldosterone, a labeled complex recognition type antibody, and an anti-aldosterone antibody immobilized on the detection unit in the detection unit.
  • step [2] first, a sample containing aldosterone and a labeled complex recognition antibody are mixed, and the sample containing aldosterone and the labeled complex recognition antibody are developed in the membrane. Then, in the detection unit, a multiplex complex of aldosterone, a labeled complex recognition type antibody, and an anti-aldosterone antibody immobilized on the detection unit is formed.
  • the step [3] of the measurement method 6 is a step of measuring the multiplex complex in the detection unit.
  • the multiplex complex can be measured by measuring the labeling substance in the multiplex complex.
  • the time from the addition of the sample to the sample supply unit to the measurement of the multiple complex in the detection unit is particularly limited as long as the time enables the immunoassay method for aldosterone of the present invention.
  • the membrane used in the measuring method 5 and the measuring method 6 is not particularly limited as long as it is a membrane that enables the immunoassay method for aldosterone of the present invention.
  • examples thereof include a membrane made of polystyrene, polyethylene terephthalate, polyurethane or the like.
  • examples of the method of binding the labeling substance to the complex recognition type antibody or the anti-aldosterone antibody include the above-mentioned methods.
  • the labeling substance used in the measuring method 5 and the measuring method 6 is not particularly limited as long as it is a labeling substance that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned labeling substances.
  • the aqueous medium is not particularly limited as long as it is an aqueous medium that enables the immunoassay method for aldosterone of the present invention, and examples thereof include the above-mentioned aqueous medium.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • the samples in the measuring method 5 and the measuring method 6 are not particularly limited as long as they are samples that may contain aldosterone. For example, whole blood, plasma, serum, urine, spinal fluid, saliva, sheep water, urine, sweat, etc. Examples thereof include biological samples such as pancreatic fluid, and preferably whole blood, plasma, serum and urine.
  • the aldosterone immunoassay reagent of the present invention is a reagent used in the aldosterone immunoassay method of the present invention, and comprises an anti-aldosterone antibody that recognizes aldosterone and a complex of aldosterone and the anti-aldosterone antibody. It is an aldosterone immunoassay reagent containing a complex recognition type antibody that specifically recognizes.
  • the anti-aldosterone antibody in the present invention is not particularly limited as long as it is an antibody that specifically binds to aldosterone, and examples thereof include the above-mentioned antibodies.
  • the anti-aldosterone antibody in the present invention either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable.
  • the present invention not only a full-length antibody but also an antibody fragment can be used. Examples of the antibody fragment include the above-mentioned antibody fragment and the like.
  • the complex recognition type antibody in the immunoassay reagent for aldosterone is 6. It is the same as the "complex recognition type antibody" in the aldosterone immunoassay method, and is not particularly limited as long as it is an antibody that specifically binds to the complex of aldosterone and an anti-aldosterone antibody. Examples thereof include an antibody that specifically recognizes a complex with an antibody.
  • the sample of the aldosterone immunoassay reagent is not particularly limited as long as it is a sample that may contain aldosterone, and examples thereof include the above-mentioned samples.
  • the concentration of the anti-aldosterone antibody in the aldosterone immunoassay reagent is not particularly limited as long as it is a concentration that enables the aldosterone immunoassay method of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0. 8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL, etc., 0
  • the range of 1 to 20 ⁇ g / mL is preferable.
  • the concentration of the complex recognition antibody in the aldosterone immunoassay reagent is not particularly limited as long as it enables the aldosterone immunoassay method of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0. 7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL, etc.
  • the range is preferably 0.1 to 20 ⁇ g / mL.
  • the anti-aldosterone antibody or complex recognition antibody may or may not be immobilized on an insoluble carrier, but is preferably immobilized.
  • the insoluble carrier include the above-mentioned insoluble carrier and the like.
  • the method for immobilizing the anti-aldosterone antibody or the complex recognition type antibody on the insoluble carrier include the above-mentioned methods. It is also possible to immobilize the antibody on an insoluble carrier using a very strong binding reaction such as the avidin-biotin reaction. Further, in the aldosterone immunoassay reagent, the anti-aldosterone antibody or the complex recognition type antibody on which the insoluble carrier is immobilized may be produced in the reaction solution of the above-mentioned multiple complex formation step.
  • the immunoassay reagent is an anti-aldosterone antibody or complex to which one (E) of a set of affinity substances is bound instead of the anti-aldosterone antibody or complex recognition type antibody on which an insoluble carrier is immobilized. It includes a recognition antibody and an insoluble carrier to which the other (e) of a set of affinity substances is bound.
  • the combination of one set of affinity substances that is, the combination of E and e include the same combination as Aa described above.
  • the aldosterone immunoassay reagent may contain a cleaning solution.
  • the cleaning liquid include the above-mentioned cleaning liquid and the like.
  • the aldosterone immunoassay reagent may contain a sample diluent for diluting the sample.
  • the sample diluent contains the above-mentioned aqueous medium.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, protein stabilizers and the like.
  • the anti-aldosterone antibody or the complex recognition type antibody may or may not be bound to the labeling substance, but it is preferable that the labeling substance is bound.
  • the labeling substance include the above-mentioned labeling substances and the like.
  • the aldosterone immunoassay reagent may be lyophilized or liquid. When a freeze-dried measurement reagent is used, it is dissolved in an aqueous medium to make it liquid and used for measurement before measurement. In the liquid measurement reagent, the anti-aldosterone antibody and the complex recognition type antibody are in a state of being dissolved in an aqueous medium.
  • the aqueous medium include the above-mentioned aqueous medium.
  • the aqueous medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, protein stabilizers and the like.
  • Measurement Reagent 1 Measurement Reagent 1 for Sandwich ELISA
  • Anti-aldosterone antibody immobilized on an insoluble carrier A complex-recognizing antibody that specifically recognizes a complex of aldosterone and anti-aldosterone, Aldosterone immunoassay reagents, including.
  • the measuring reagent 1 is a measuring reagent used in the above-mentioned measuring method 1.
  • Measurement Reagent 2 Measurement Reagent 2 for Sandwich ELISA
  • anti-aldosterone antibody A complex-recognizing antibody immobilized on an insoluble carrier that specifically recognizes a complex of aldosterone and anti-aldosterone, Aldosterone immunoassay reagents, including.
  • the measuring reagent 2 is a measuring reagent used in the above-mentioned measuring method 2.
  • Measurement reagent 3 Measuring reagent for latex agglutination method
  • Anti-aldosterone antibody to which insoluble carrier particles are bound and A complex-recognizing antibody that specifically recognizes a complex of aldosterone and anti-aldosterone to which insoluble carrier particles are bound, Aldosterone immunoassay reagents, including.
  • the measuring reagent 3 is a measuring reagent used in the above-mentioned measuring method 3 or measuring method 4.
  • Measurement Reagent 4 Measurement Reagent 1 for Lateral Flow Test
  • a test strip having a membrane made of a porous body with a sample supply part, a development part and a detection part, and Anti-aldosterone antibody retained in a part of the development area so that it can be eluted, A complex-recognizing antibody that specifically recognizes a complex of aldosterone and anti-aldosterone bound to the detection unit.
  • Aldosterone immunoassay reagents including.
  • the measuring reagent 4 is a measuring reagent used in the above-mentioned measuring method 5.
  • Measurement Reagent 5 Measuring Reagent 2 for Lateral Flow Test
  • a test strip having a membrane made of a porous body with a sample supply part, a development part and a detection part, and A complex-recognizing antibody that is retained in a part of the development area so that it can be eluted, With the anti-aldosterone antibody bound to the detection part, Aldosterone immunoassay reagents, including.
  • the measuring reagent 5 is a measuring reagent used in the above-mentioned measuring method 6.
  • the aldosterone immunoassay reagent of the present invention can also be in the form of a kit from the viewpoints of storage, transportation, distribution and the like.
  • the aldosterone immunoassay kit is used in the aldosterone immunoassay method of the present invention.
  • preferred embodiments of the aldosterone immunoassay kit according to the present invention will be illustrated.
  • examples of the anti-aldosterone antibody, the complex-recognizing antibody that specifically recognizes the complex of aldosterone and the anti-aldosterone antibody, the insoluble carrier, the insoluble carrier particles, and the labeling substance include the above-mentioned anti-aldosterone antibody.
  • Aldosterone antibody, a complex recognition type antibody that specifically recognizes a complex of aldosterone and the anti-aldosterone antibody, an insoluble carrier, an insoluble carrier particle, and a labeling substance are used.
  • Measurement kit 1 Measurement kit for sandwich ELISA 1
  • Measurement kit 2 Measurement kit for sandwich ELISA 2
  • a first reagent containing a complex-recognizing antibody that specifically recognizes a complex of aldosterone and the anti-aldosterone antibody immobilized on an insoluble carrier and A second reagent containing an anti-aldosterone antibody, Aldosterone immunoassay kit, including.
  • the measurement kit 1 and the measurement kit 2 may contain a cleaning liquid.
  • the cleaning liquid include the above-mentioned cleaning liquid and the like.
  • a sample diluent for diluting the sample may be included.
  • Measurement kit 3 Measurement kit for latex agglutination method 1
  • the measurement kit 3 may contain a sample diluent for diluting the sample.
  • Measurement kit 4 Measurement kit for latex agglutination method 2
  • the first reagent containing an aqueous medium and A second reagent containing an anti-aldosterone antibody to which insoluble carrier particles are bound and a complex recognition type antibody to which insoluble carrier particles are bound, and Aldosterone immunoassay kit, including.
  • test strip having a membrane made of a porous body with a sample supply part, a development part and a detection part, and The anti-aldosterone antibody retained in the developing portion so as to be eluted, The complex recognition type antibody bound to the detection unit and Aldosterone immunoassay kit, including.
  • test strip having a membrane made of a porous body with a sample supply part, a development part and a detection part, and The complex-recognizing antibody retained in the developing portion so as to be eluted, With the anti-aldosterone antibody bound to the detection part, Aldosterone immunoassay kit, including.
  • the measurement kit 5 and the measurement kit 6 may include a sample diluent for diluting the sample.
  • the hybridoma cells prepared above were suspended in RPMI1640 medium (manufactured by Thermo Fisher Scientific) containing 10% FCS and HAT, and then seeded and cultured on a microwell plate. The culture supernatant of the grown hybridoma cells was obtained.
  • the absorbance of the reaction solution was measured at a main wavelength of 450 nm and a sub-wavelength of 660 nm, and a well having a high absorbance (a well in which a monoclonal antibody having a high binding ability exists) was selected. bottom.
  • An anti-aldosterone monoclonal antibody-producing hybridoma cell line (KTM-2012) was established by cloning the hybridoma cells in the selected wells by the limiting dilution method.
  • Example 1 Preparation of monoclonal antibody against aldosterone-anti-aldosterone antibody complex (1) Immunization against mice The KTM-2012 antibody obtained in Reference Example 1 (4) was pepsin (manufactured by Roche Diagnostics). After digestion with, an HPLC system (Hitachi) using a G3000SW column (manufactured by Tosoh Corporation; diameter: 21.5 mm; length: 60 cm) using 0.1 mol / L phosphate buffer (pH 7.4) as a mobile phase. F (ab') 2 was separated by (manufactured by Mfg. Co., Ltd.).
  • the F (ab') 2 and aldosterone (manufactured by Sigma-Aldrich) were mixed in a PBS solution so as to have a molar ratio of 1: 540, and left at 25 ° C. for 1 hour to prepare an antigen solution.
  • An emulsion in which an equal amount of the antigen solution and adjuvant were mixed was prepared, and the emulsion was subcutaneously injected into a Balb / c mouse (manufactured by Nippon SLC Co., Ltd.) three times in total.
  • Freund's Complete Adjuvant (manufactured by Sigma-Aldrich) was used as an adjuvant
  • Freund's Incomplete Adjuvant manufactured by Sigma-Aldrich
  • Blood was collected after immunization a total of 3 times at 2-week intervals, and the antibody titer in serum was evaluated by ELISA according to the procedure (2) described later. After confirming that the antibody titer in serum was elevated, a solution obtained by mixing equal amounts of PBS solution and the antigen solution was injected subcutaneously into the mouse, and the spleen was removed from the mouse 3 days later. ..
  • TMB-ONE manufactured by Kem-En-Tech Diagnostics
  • 50 ⁇ L was dispensed and reacted at 25 ° C. for 30 minutes.
  • the absorbance of the reaction solution was measured at a main wavelength of 450 nm and a sub-wavelength of 660 nm.
  • the spleen was excised from the mouse in which an increase in antibody titer was confirmed, and spleen cells were prepared according to a conventional method.
  • the prepared spleen cells were fused with mouse myeloma cells (P3-U1) by an electrofusion method using a super cell fusion device EGFG21 (manufactured by Neppagene) according to the protocol provided by the company to prepare a hybridoma.
  • the hybridoma cells prepared above are suspended in a GIT medium (manufactured by Kojin Bio) containing 10% BM Condimed H1 Hybridoma Cloning Supplement (10 ⁇ ) (manufactured by Roche Diagnostics) and HAT, and then placed on a microwell plate. It was sown and cultured. The culture supernatant of the grown hybridoma cells was obtained.
  • GIT medium manufactured by Kojin Bio
  • 10% BM Condimed H1 Hybridoma Cloning Supplement (10 ⁇ ) manufactured by Roche Diagnostics
  • TMB-ONE 50 ⁇ L was dispensed and reacted at 25 ° C. for 30 minutes. After adding 0.5 mol / L sulfuric acid to each well (50 ⁇ L) and stirring and mixing, the absorbance of the reaction solution was measured at a main wavelength of 450 nm and a sub-wavelength of 660 nm. Culture supernatants that did not react with the negative control and specifically reacted with the complex of KTM-2012 antibody and aldosterone were selected.
  • hybridoma cell line KTM-611 producing a monoclonal antibody against the aldosterone-anti-aldosterone antibody complex was established.
  • Example 2 Isolation and analysis of cDNA encoding the variable region of KTM-611 antibody (1) Preparation of mRNA from hybridoma cells producing a monoclonal antibody against the aldosterone-anti-aldosterone antibody complex Obtained hybridoma KTM- 611 2 ⁇ 10 6 to 2 ⁇ 10 Using RNAeasy Mini kit (manufactured by QIAGEN) from 7 cells, mRNA of mouse monoclonal antibody against aldosterone-anti-aldosterone antibody complex was prepared according to the attached instruction manual. bottom.
  • a PCR reaction was carried out using a universal primer mix and a mouse IgG ( ⁇ ) -specific primer (SEQ ID NO: 2) to amplify a cDNA fragment of the light chain variable region (VL region) of each antibody.
  • VL region the light chain variable region
  • the obtained PCR product was separated by agarose gel electrophoresis, and the VH gene fragment and the VL gene fragment were separated using the NucleoSpin Gel and PCR Clean-up Kit included in the kit, respectively. Extracted.
  • the VH gene or VL gene of each antibody was incorporated into the pRACE vector using the In-Fusion HD Cloning Kit included in the kit, and the obtained vector was used for E. coli.
  • coli DH5 ⁇ Compound Cells (manufactured by Takara Bio Inc.) was transformed.
  • a plasmid was extracted from the obtained transformant using QIAprep Spin Miniprepkit (manufactured by QIAGEN).
  • the nucleotide sequence of the cloned PCR product was subjected to a plasmid separation reagent kit (manufactured by KURABO), an automatic nucleic acid separator PI-50 (manufactured by KURABO), and a sequencer ABI PRISM3700 Genetic Analyzer (manufactured by Thermo Fisher Scientific). Analyzed.
  • the genetic sequence of the VH region is shown in SEQ ID NO: 3, and the genetic sequence of the VL region is shown in SEQ ID NO: 4.
  • SEQ ID NO: 3 The genetic sequence of the VH region is shown in SEQ ID NO: 4
  • SEQ ID NO: 4 The genetic sequence of the VL region is shown in SEQ ID NO: 4.
  • V region a region encoding a region (hereinafter referred to as V region).
  • the database of DNA Databank of JAPAN was obtained by the BLASTP method (Nucleic Acids Res., Vol. 25 (17), pp. 3389-3402 (1997)). searched. No exact matching amino acid sequence was found, confirming that the VH and VL of the KTM-611 antibody had a novel amino acid sequence.
  • the CDRs of the VH region and VL region of each monoclonal antibody were identified by comparing with the amino acid sequences of known antibodies.
  • amino acid sequences of CDR1, CDR2 and CDR3 in the VH region of the KTM-611 antibody are shown in SEQ ID NOs: 10, 11 and 12
  • amino acid sequences of CDR1, CDR2 and CDR3 in the VL region are shown in SEQ ID NOs: 16, 17 and 18, respectively.
  • Example 3 Reactivity test of monoclonal antibody against complex of aldosterone-anti-aldosterone antibody (1) Preparation of peroxidase-labeled antibody solution Peroxidase Labeling of KTM-611 antibody (200 ⁇ g) obtained in Example 1 (5). It was labeled with peroxidase using Kit-NH 2 (manufactured by Dojin Chemical Research Institute) according to the attached manual. The peroxidase-labeled KTM-611 antibody was suspended in the Storage Buffer attached to the kit, and the suspension was diluted with the sample diluent to prepare a solution at 20 ng / mL, which was used as a peroxidase-labeled antibody solution.
  • a well to which only the sample diluent was added was prepared, and a well in which the complex of the KTM-2012 antibody and aldosterone was not present (only the anti-aldosterone antibody was present) was also prepared.
  • the peroxidase-labeled antibody solution prepared in (1) was added to each well (50 ⁇ L) and reacted at 25 ° C. for 1 hour.
  • TMB-ONE 50 ⁇ L was dispensed and reacted at 25 ° C. for 30 minutes.
  • the absorbance of the reaction solution was measured at a main wavelength of 450 nm and a sub-wavelength of 660 nm.
  • a plate on which a goat anti-rabbit IgG polyclonal antibody (manufactured by Thermo Fisher Scientific Co., Ltd.) was immobilized was prepared in the same manner as described above, and a KTM-2012 antibody (anti-aldosterone antibody) solution was used instead of the KTM-611 antibody solution. The same measurement was performed using.
  • Example 4 Preparation of aldosterone measurement kit An aldosterone measurement kit containing the following magnetic particle suspension, a biotin-conjugated KTM-611 antibody solution, and an alkaline phosphatase-labeled KTM-2012 antibody solution was prepared. In the following Examples 4-7, reagents of the following manufacturers were used.
  • HEPES N- (2-Hydroxyethyl) -N'-(2-sulfoethyl) piperazin
  • Tween 20 manufactured by Kanto Chemical Co., Ltd.
  • BSA bovine serum albumin
  • BSA bovine serum albumin
  • Detaminer CL Aldosterone manufactured by Hitachi Chemical Diagnostics Systems Co., Ltd.
  • Detaminer Control CL for Aldosterone manufactured by Hitachi Chemical Diagnostics Systems Co., Ltd.
  • CL Analyzer Cleaning Solution manufactured by Hitachi Chemical Diagnostics Systems Co., Ltd.
  • Magnetic particle suspension As the magnetic particles, a commercially available magnetic particle [Dynabeads MyOne Streptavidin T1 (manufactured by Thermo Fisher Scientific Co., Ltd.)] to which streptavidin was bound was used to prepare a magnetic particle suspension having the following composition.
  • Biotin-bound KTM-611 antibody solution Using Biotin Labeling kit-NH2 (manufactured by Dojin Chemical Laboratory Co., Ltd.), a biotin-bound KTM-611 antibody in which biotin was bound to the KTM-611 antibody was prepared according to the instruction manual of the kit. Using the obtained biotin-bound KTM-611 antibody, a biotin-bound KTM-611 antibody solution having the following composition was prepared.
  • Alkaline phosphatase-labeled KTM-2012 antibody solution Alkaline phosphatase-labeled KTM-2012 antibody in which alkaline phosphatase was bound to KTM-2012 antibody was prepared using Alkaline Phosphatase Labeling kit-SH (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.) according to the instruction manual of the kit. Using the obtained alkaline phosphatase-labeled KTM-2012 antibody, an alkaline phosphatase-labeled KTM-2012 antibody solution having the following composition was prepared.
  • Alkaline Phosphatase Labeling kit-SH manufactured by Dojin Kagaku Kenkyusho Co., Ltd.
  • the minimum concentration (minimum detection sensitivity) As a method of defining the minimum concentration (minimum detection sensitivity) that can be measured as a measurement system, there is a method of statistically evaluating using an average value and a standard deviation (SD). Specifically, the average value when the sample prepared in (1) is quintuple-measured is -2 times the standard deviation (+ 2SD), which is the average value when the 0 pg / mL sample is quintuple-measured. If the deviation (-2SD) is high, the sample can be defined as a detectable concentration. The smaller the minimum detection sensitivity, the higher the measurement sensitivity. When a sample having an aldosterone concentration of 0 pg / mL was measured, the average amount of luminescence + 2SD was 1416 RLU.
  • the average -2SD of the luminescence amount when the sample having the aldosterone concentration of 0.25 pg / mL was measured was 1584 RLU, which was higher than the average of the luminescence amount + 2SD when the 0 pg / mL sample was measured. Therefore, it was confirmed that 0.25 pg / mL of aldosterone could be measured. Therefore, in this measurement method, the minimum detection sensitivity can be defined as 0.25 pg / mL. Further, as shown in FIG. 2, a concentration-dependent increase in the amount of luminescence was observed at a concentration of 0.25 pg / mL or more of aldosterone.
  • the minimum detection sensitivity in the competitive method is when the average luminescence amount + 2SD when the sample prepared in (1) is quintuple-measured is lower than the average luminescence amount-2SD when the 0 pg / mL sample is quintuple-measured.
  • the sample can be defined as a detectable concentration.
  • the average amount of luminescence -2SD was 48722 RLU.
  • the average luminescence amount + 2SD when the aldosterone concentration was measured in the 4.1 pg / mL sample was 49622, which was higher than the average luminescence amount -2SD when the 0 pg / mL sample was measured. It was confirmed that the aldosterone concentration of 1 pg / mL could not be measured.
  • the average + 2SD of the luminescence amount when the sample having the aldosterone concentration of 8.2 pg / mL was measured was 47846 RLU, which was lower than the average + 2SD of the luminescence amount when the 0 pg / mL sample was measured. Therefore, it was confirmed that 8.2 pg / mL of aldosterone could be measured. Therefore, the minimum detection sensitivity in competition can be defined as 8.2 pg / mL. From this result, it was found that the method for measuring aldosterone using the measurement kit of Example 4 has high measurement sensitivity.
  • the simultaneous reproducibility test is a method of determining the accuracy of the measurement method by continuously measuring the same sample a plurality of times and evaluating the variation in the measured values.
  • Control L of "Determiner Control CL for Aldosterone" as a measurement sample, 20 layers of the sample were measured using the measurement kit of Example 4 according to the method described in Example 5 (2).
  • a calibration curve was prepared using the standard aldosterone reagents A to D included in the "Detaminer CL aldosterone" kit, and the aldosterone concentration was determined from the obtained luminescence amount.
  • the same sample was measured 20 times using "Detaminer CL aldosterone" to determine the aldosterone concentration.
  • the CV of the measurement kit of Example 4 was 1.7% and “Detaminer CL”.
  • the CV of "aldosterone” was 3.5%.
  • Example 7 Cross-reactivity test Corticosterone (manufactured by Tokyo Chemical Industry Co., Ltd.), cortisol (manufactured by Tokyo Chemical Industry Co., Ltd.), cortisol (manufactured by Tokyo Chemical Industry Co., Ltd.) in the measurement of aldosterone using the kit of Example 4.
  • Corticosterone, cortisol, cortisone, progesterone, tetrahydrocorticosterone, dexamethasone, prednisolone, and spironolactone are all steroid hormones similar in structure to aldosterone.
  • each cross-reactive substance was diluted with degreased plasma [Human Plasma, Defibrinated, Delimited double charcoal stripped (manufactured by Golden West Biologicals)] and shown in Table 2. A substance solution was prepared.
  • Example 5 After measuring each cross-reactive substance solution according to the method described in Example 5 (2), the concentration of the cross-reactive substance in each solution was determined according to the method described in Example 6. The cross-reactivity rate for each cross-reactive substance was determined by the following formula. The results are shown in Table 2. In addition, as a comparative example, the same sample was measured using "Detaminer CL aldosterone", and the cross-reactivity rate for each cross-reactive substance was determined by the following formula. The results are shown in Table 2.
  • the cross-reactivity is an index showing the rate at which cross-reactive substances that should not be measured are measured
  • the lower the cross-reactivity the more specificly aldosterone can be detected, and the more accurate the measurement can be. It shows that it is.
  • Table 2 it was found that the measurement method using the kit of Example 4 has a low crossover rate as much as the measurement method using "Detaminer CL aldosterone" and can accurately measure aldosterone.
  • FIG. 3 shows a correlation equation between the measured value of aldosterone using the measurement kit of Example 4 (y-axis) and the measured value of aldosterone using "Detaminer CL aldosterone" (x-axis).
  • the slope of the correlation equation according to FIG. 3 was 0.91, which was close to 1.0, and the correlation coefficient was 0.998, which was close to 1.0. Therefore, an accurate method for measuring aldosterone. It turned out to be.
  • SEQ ID NO: 1 Nucleotide sequence of mouse IgG1-specific primer
  • Nucleotide sequence Sequence number 2 Nucleotide sequence of mouse IgG ( ⁇ ) -specific primer
  • Nucleotide sequence No. 3 KTM-611 VH region Gene sequence
  • SEQ ID NO: 4 KTM-611 VL region
  • Nucleotide sequence No. 5 KTM-611 VH region Amino acid sequence
  • SEQ ID NO: 6 KTM-611 VL region Amino acid sequence No. 7: KTM-611 VH region CDR1 Gene sequence No. 8: KTM-611 VH region CDR2 Gene sequence No.
  • KTM -611 VH region CDR3 gene sequence SEQ ID NO: 10: KTM-611 VH region CDR1 amino acid sequence SEQ ID NO: 11: KTM-611 VH region CDR2 amino acid sequence SEQ ID NO: 12: KTM-611 VH region CDR3 amino acid sequence SEQ ID NO: 13: KTM-611 VL region CDR1 gene sequence SEQ ID NO: 14: KTM-611 VL region CDR2 gene sequence SEQ ID NO: 15: KTM-611 VL region CDR3 gene sequence SEQ ID NO: 16: KTM-611 VL region CDR1 amino acid sequence SEQ ID NO: 17: KTM-611 VL region CDR2 amino acid sequence SEQ ID NO: 18: KTM-611 VL region CDR3 amino acid sequence

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Food Science & Technology (AREA)
  • Endocrinology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un anticorps qui reconnaît spécifiquement un complexe d'aldostérone et un anticorps anti-aldostérone, en tant que technique pour détecter avec précision l'aldostérone dans un échantillon. Cet anticorps comprend un anticorps qui comporte : une région déterminant la complémentarité (CDR) 1 comprenant un polypeptide représenté par la séquence d'acides aminés de SEQ ID No. : 10, une CDR 2 comprenant un polypeptide représenté par la séquence d'acides aminés de SEQ ID No. : 11, et une CDR 3 comprenant un polypeptide représenté par la séquence d'acides aminés de SEQ ID No. : 12 dans une chaîne lourde; et une CDR 1 comprenant un polypeptide représenté par la séquence d'acides aminés de SEQ ID No. : 16, une CDR 2 comprenant un polypeptide représenté par la séquence d'acides aminés de SEQ ID No. : 17 et une CDR 3 comprenant un polypeptide représenté par la séquence d'acides aminés de SEQ ID No. : 18 dans une chaîne légère.
PCT/JP2021/009705 2020-03-12 2021-03-11 Anticorps de détection de l'aldostérone et méthode de dosage immunologique de l'aldostérone WO2021182555A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022507271A JPWO2021182555A1 (fr) 2020-03-12 2021-03-11

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020042510 2020-03-12
JP2020-042510 2020-03-12

Publications (1)

Publication Number Publication Date
WO2021182555A1 true WO2021182555A1 (fr) 2021-09-16

Family

ID=77671604

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/009705 WO2021182555A1 (fr) 2020-03-12 2021-03-11 Anticorps de détection de l'aldostérone et méthode de dosage immunologique de l'aldostérone

Country Status (2)

Country Link
JP (1) JPWO2021182555A1 (fr)
WO (1) WO2021182555A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63501096A (ja) * 1984-10-29 1988-04-21 エクス　インコ−ポレ−テツド 小型分子の免疫測定法
JPS63112599A (ja) * 1986-10-09 1988-05-17 ベーリングヴェルケ・アクチエンゲゼルシャフト 免疫複合体レセプター
WO2007055294A2 (fr) * 2005-11-14 2007-05-18 Bioarc Co., Ltd. Methode de determination simultanee de substances et support utilise dans ladite methode de determination
WO2019098314A1 (fr) * 2017-11-17 2019-05-23 富士レビオ株式会社 Liquide de traitement destiné à être utilisé dans la désorption d'un stéroïde inclus dans la cyclodextrine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63501096A (ja) * 1984-10-29 1988-04-21 エクス　インコ−ポレ−テツド 小型分子の免疫測定法
JPS63112599A (ja) * 1986-10-09 1988-05-17 ベーリングヴェルケ・アクチエンゲゼルシャフト 免疫複合体レセプター
WO2007055294A2 (fr) * 2005-11-14 2007-05-18 Bioarc Co., Ltd. Methode de determination simultanee de substances et support utilise dans ladite methode de determination
WO2019098314A1 (fr) * 2017-11-17 2019-05-23 富士レビオ株式会社 Liquide de traitement destiné à être utilisé dans la désorption d'un stéroïde inclus dans la cyclodextrine

Also Published As

Publication number Publication date
JPWO2021182555A1 (fr) 2021-09-16

Similar Documents

Publication Publication Date Title
US11372001B2 (en) Anti-human IgG4 monoclonal antibody and methods of making and using same
US20220268792A1 (en) Compositions and methods for the detection of host cell proteins
EP3572426A1 (fr) Anticorps anti-epha2 et détection immunologique d'epha2 faisant appel audit anticorps
JP7002190B2 (ja) 糖ペプチドと反応するモノクローナル抗体およびその用途
WO2021182555A1 (fr) Anticorps de détection de l'aldostérone et méthode de dosage immunologique de l'aldostérone
EP2541252A1 (fr) Procédé d'obtention d'un liant pour prépro-vasopressine ou fragments associés
CN113214393B (zh) Il-6抗体或其抗原结合片段及包含其的检测试剂盒
EP3252073A1 (fr) Anticorps monoclonal réagissant avec un glycopeptide et son utilisation
EP3239711A2 (fr) Procédé de mesure d'anticorps anti-médicament
US11327078B2 (en) Monoclonal antibody against APOA4, immunological measurement method, and kit for measurement
CN110579610A (zh) 用于检测t细胞活化的v域免疫抑制因子的试剂盒
CN116375870B (zh) 抗人cd40蛋白的人源化抗体、制备方法及其应用
WO2021182558A1 (fr) Procédé et kit pour immunoessai de substance cible dans un échantillon
US20220137071A1 (en) Method and kit for measuring app669-711
CN110161248B (zh) 检测抗Carp抗体的均相免疫检测试剂盒及其应用
JP2022122283A (ja) Ｄｕｐａｎ－２抗原の測定方法、測定試薬及び測定キット
EP2495564B1 (fr) PROCÉDÉ D'IMMUNOESSAI DE PEPTIDE DE 5,9 kDa
JP2011016773A (ja) 抗ポリヒスチジンタグ抗体及びそれを用いた非特異反応の回避方法
JP2022122282A (ja) Ｄｕｐａｎ－２抗原の測定方法、測定試薬及び測定キット
CA3105726A1 (fr) Anticorps anti-bcl-2 et dosage immunologique pour le diagnostic du cancer
JP2024119083A (ja) Ｈｍｇｂ１の免疫学的測定時にｈｍｇｂ２の測りこみを抑制する方法及びｈｍｇｂ１を特異的に測定する免疫学的測定方法及びｈｍｇｂ１を特異的に測定する免疫学的測定試薬
EP3540437A1 (fr) Composition d'anticorps anti-équol et utilisation correspondante
JP4803943B2 (ja) 肝細胞増殖因子活性化因子阻害因子−１に対する抗体とその用途
CN116535504A (zh) 人NT-proBNP特异性单克隆抗体制备方法和应用
JP2005314397A (ja) 抗コンドロモジュリン−１特異的抗体及びその利用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21767690

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022507271

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21767690

Country of ref document: EP

Kind code of ref document: A1