WO2019167128A1 - Sandwich immunoassay - Google Patents

Sandwich immunoassay Download PDF

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Publication number
WO2019167128A1
WO2019167128A1 PCT/JP2018/007295 JP2018007295W WO2019167128A1 WO 2019167128 A1 WO2019167128 A1 WO 2019167128A1 JP 2018007295 W JP2018007295 W JP 2018007295W WO 2019167128 A1 WO2019167128 A1 WO 2019167128A1
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Prior art keywords
antibody
target polypeptide
immunoassay
antigen
affinity substance
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PCT/JP2018/007295
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French (fr)
Japanese (ja)
Inventor
金子　直樹
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株式会社 島津製作所
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Application filed by 株式会社 島津製作所 filed Critical 株式会社 島津製作所
Priority to PCT/JP2018/007295 priority Critical patent/WO2019167128A1/en
Priority to US16/975,559 priority patent/US20210155680A1/en
Priority to CN201980015355.9A priority patent/CN111770935A/en
Priority to JP2020503468A priority patent/JP7434144B2/en
Priority to PCT/JP2019/006778 priority patent/WO2019167830A1/en
Priority to EP19760318.6A priority patent/EP3760640A4/en
Publication of WO2019167128A1 publication Critical patent/WO2019167128A1/en
Priority to JP2021190264A priority patent/JP2022031783A/en
Priority to JP2024023850A priority patent/JP2024056971A/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a sandwich type immunoassay method using a specific immune reaction, belonging to fields such as clinical diagnostics such as trace components and infectious microorganism antigens, biochemical research, and immunological research. More particularly, the present invention relates to a sandwich immunoassay useful for detecting and quantifying trace polypeptides in a sample.
  • An immunoassay is a method that uses a specific antigen-antibody reaction to measure the concentration of an analyte in a sample, and because it can measure trace components specifically and accurately, Widely used in chemical research.
  • the immunoassay includes enzyme immunoassay (EIA) using an enzyme as a labeling substance, radioimmunoassay (RIA) using a radioisotope, chemiluminescence immunoassay (CIA) using a chemiluminescent substance, Fluorescence immunoassay (FIA) using fluorescent materials, electrochemiluminescence immunoassay (ECLIA) using metal complexes, bioluminescence immunoassay (BLIA) using bioluminescent materials such as luciferase, antibody labeling PCR to amplify the detected nucleic acid by PCR, immunoturbidimetric method (TAI) to detect turbidity generated by immune complex formation, latex agglutination turbidimetric method to detect latex aggregated by immune complex formation (LA), an immunochromatography method for reacting on a cellulose membrane.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • CIA chemiluminescence immunoassay
  • sandwich ELISA Enzyme-Linked ImmunoSorbent I Assay
  • EIA Enzyme-Linked ImmunoSorbent I Assay
  • This is a method in which the detection antibody labeled with is reacted, and the concentration of the substance to be analyzed is determined using the color of the substrate of the labeled enzyme (for example, Patent Document 1: Japanese Patent Application Laid-Open No. 8-220098).
  • ECLIA also uses a sandwich method in which a biotinylated antibody and an antibody labeled with a ruthenium (Ru) complex that emits light by an electrochemical change are reacted with an analyte (for example, patent documents) 2: Japanese Translation of PCT International Publication No. 2014-509735). As described above, the sandwich method is widely used as an immunoassay.
  • ruthenium (Ru) complex that emits light by an electrochemical change
  • the sandwich method is also used as a method for specifically quantifying a specific peptide fragment, and the peptide fragment is specifically measured by sandwiching the peptide fragment to be analyzed between an N-terminal recognition antibody and a C-terminal recognition antibody.
  • a ⁇ produced by cleaving from amyloid precursor protein (Amyloid precursor protein; APP) with a protease
  • a ⁇ 1-42 is quantified by an immunoassay such as ELISA as a cerebrospinal fluid (CSF) biomarker of Alzheimer's disease (Non-patent Document 2).
  • Patent Document 3 International Publication WO2015 / 178398. These A ⁇ and A ⁇ -related peptides are quantified by performing mass spectrometry (MALDI-TOF-MASS) after immunoprecipitation (IP).
  • MALDI-TOF-MASS mass spectrometry
  • IP immunoprecipitation
  • Patent Document 4 International Publication WO2017 / 047529 describes three ratios for A ⁇ and A ⁇ related peptides (A ⁇ -like peptides), A ⁇ 1-39 / A ⁇ 1-42, A ⁇ 1-40 / A ⁇ 1-42, and APP669-711. It is disclosed that a numerical value obtained by combining two or more ratios selected from the group consisting of / A ⁇ 1-42 by a mathematical method is promising as a blood biomarker.
  • a ⁇ and A ⁇ -related peptides are quantified by performing mass spectrometry (MALDI-TOF-MASS) after immunoprecipitation (IP).
  • Patent Document 5 Japanese Patent Application Laid-Open No. 2004-23985 discloses a method for improving the measurement sensitivity of an immunoassay method by using a high concentration of ions before immunoassay for a protein that is difficult to extract from a sample. A method for extracting a sample with an ionic surfactant has been reported.
  • two antibodies that recognize each of two different sites (epitopes) of the polypeptide to be analyzed are analyzed simultaneously. After binding to the peptide, or after binding one of the antibodies, the other antibody is bound. That is, it is necessary that two antibodies simultaneously bind to two different sites (epitope) of the polypeptide to be analyzed, and the polypeptide to be analyzed is sandwiched between the two antibodies.
  • a sandwich immunoassay for measuring A ⁇ which is a peptide fragment
  • the peptide fragment is sandwiched between an N-recognizing antibody and a C-terminal recognizing antibody, but the primary antibody recognition sites (epitopes) are separated from each other.
  • the two epitopes are close in spatial distance (Non-patent Document 3). Therefore, it may cause a decrease in sensitivity of the sandwich immunoassay.
  • An object of the present invention is to provide a sandwich immunoassay that can detect and quantify a polypeptide to be analyzed with high sensitivity.
  • an object of the present invention is to provide a sandwich immunoassay that can detect and quantify A ⁇ and A ⁇ -related peptides to be analyzed with high sensitivity.
  • an object of the present invention is to provide a sandwich immunoassay kit for polypeptides.
  • the inventor performs a treatment to change the conformation of the polypeptide to be analyzed and to separate the two epitopes to be bound by the two antibodies used in the sandwich immunoassay from each other in terms of spatial distance.
  • the analysis target polypeptide can be made highly sensitive. It has been found that a sandwich immunoassay can be obtained that can be detected and quantified. As a result, the present invention has been completed.
  • the polypeptide includes a peptide and a protein.
  • the protein includes post-translationally modified proteins such as glycoproteins and phosphorylated proteins.
  • a ⁇ is used as an abbreviation for amyloid ⁇ peptide. That is, “A ⁇ ” includes A ⁇ 1-40 and A ⁇ 1-42.
  • a peptide other than A ⁇ produced by cleaving amyloid precursor protein (Amyloid precursor protein; APP) may be referred to as an A ⁇ -related peptide (or A ⁇ -like peptide).
  • a ⁇ and A ⁇ -related peptides (or A ⁇ -like peptides) generated by cleaving amyloid precursor protein (Amyloid precursor protein; APP) may be referred to as “APP-derived peptides”.
  • the present invention includes the following inventions.
  • the antigen affinity substance is selected from the group consisting of an antibody, a peptide, a low molecular compound, and a nucleic acid aptamer.
  • the sandwich immunoassay includes enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CIA), fluorescence immunoassay (FIA), and electrochemiluminescence immunoassay.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • CIA chemiluminescence immunoassay
  • FIA fluorescence immunoassay
  • electrochemiluminescence immunoassay ECLIA
  • bioluminescent immunoassay BIA
  • immuno-PCR immunoturbidimetry
  • TAI latex agglutination turbidimetry
  • LA latex agglutination turbidimetry
  • the antigen affinity substance acts on an intermediate site from the 4-residue site from the N-terminal to the 4-residue site from the C-terminal of the target polypeptide.
  • sample (9) The sample according to any one of (1) to (8) above, wherein the sample is a biological sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, and body secretions. Immunoassay.
  • a sandwich type immunoassay kit for polypeptides comprising:
  • an antigen affinity substance capable of binding to the target polypeptide is added to the sample. This alters the conformation of the target polypeptide and separates the two epitopes to which each of the two antibodies used in the sandwich immunoassay should bind in spatial distance. Therefore, the antigen-antibody reaction at two sites is successfully performed, and the target polypeptide can be detected and quantified with high sensitivity.
  • a ⁇ A ⁇ 1-40 and A ⁇ 1-42
  • a ⁇ -related peptides eg, A ⁇ 1-39, APP669-711
  • a sandwich immunoassay that can detect and quantify these A ⁇ and A ⁇ -related peptides, particularly A ⁇ -related peptides, with high sensitivity is desired.
  • a sandwich immunoassay that can detect and quantify these A ⁇ and A ⁇ -related peptides, particularly A ⁇ -related peptides, with high sensitivity is provided.
  • FIG. 1 is a graph showing the calibration curve of APP669-711 ELISA by adding anti-A ⁇ antibody 4G8 in Example 1.
  • the horizontal axis represents the concentration of APP669-711 (fmol / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • a calibration curve is shown for each concentration of anti-A ⁇ antibody 4G8.
  • FIG. 2 is a graph showing the reactivity of APP669-711 ELISA by adding anti-A ⁇ antibody 4G8 in Example 1.
  • the horizontal axis represents the concentration (ng / mL) of anti-A ⁇ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • the dots are connected by a line for each concentration of APP669-711.
  • FIG. 3 is a graph showing the reactivity of APP669-711 ELISA to A ⁇ 1-40 when anti-A ⁇ antibody 4G8 was added in Example 1.
  • the horizontal axis indicates the concentration of A ⁇ 1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm.
  • FIG. 4 is a graph showing the effect of addition of anti-A ⁇ antibody 4G8 in APP669-711 ELISA using 3 clones of N-terminal recognition antibody in Example 2.
  • the horizontal axis represents the concentration (ng / mL) of anti-A ⁇ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • the dots are connected by a line for each clone of the N-terminal recognition antibody.
  • FIG. 5 is a graph showing the reactivity of APP669-711 ELISA to A ⁇ 1-40 when anti-A ⁇ antibody 4G8 was added in Example 2. Three clones were used as N-terminal recognition antibodies.
  • FIG. 6 is a graph showing the effect of adding 4 clones of anti-A ⁇ antibody in APP669-711 ELISA in Example 3.
  • the horizontal axis represents the concentration of added anti-A ⁇ antibody (ng / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • the dots are connected by a line for each clone of the anti-A ⁇ antibody.
  • FIG. 7 is a graph showing the reactivity of APP669-711 ELISA to A ⁇ 1-40 when anti-A ⁇ antibody was added in Example 3.
  • Four clones were used as the anti-A ⁇ antibody.
  • the horizontal axis indicates the concentration of A ⁇ 1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm.
  • FIG. 8 is a graph showing the effect of addition of anti-A ⁇ antibodies 4G8 and 6E10 in A ⁇ 1-40 ELISA in Example 4.
  • the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm.
  • standard included in ELISA Kit and A ⁇ 1-40 of AnaSpec were used.
  • the immunoassay method of the present invention comprises a first antibody having an antigen-binding site capable of recognizing a target polypeptide in a sample, and an antigen-binding site capable of recognizing the target polypeptide, the first antibody having In a sandwich immunoassay method for a polypeptide using a second antibody having an antigen binding site different from the antigen binding site, an antigen affinity substance capable of binding to the target polypeptide is added to the sample.
  • the antigen-affinity substance is added to a sample containing the target polypeptide, and then the target polypeptide is reacted with the first antibody and the second antibody, or
  • the antigen affinity substance is added to the reaction system, or
  • the target polypeptide is reacted with one of the first antibody and the second antibody, and then the antigen affinity substance is added to a reaction system, and then the target polypeptide is converted into the first antibody. And can be reacted with either one of the second antibodies.
  • the conformation of the target polypeptide is changed, so that the two epitopes to which the two antibodies used in the sandwich immunoassay should each bind Spatial distances from each other. Therefore, the antigen-antibody reaction at two sites is successfully performed, and the target polypeptide can be detected and quantified with high sensitivity.
  • the antigen-affinity substance may be any substance that has an affinity and can bind to the target polypeptide, and examples thereof include antibodies, peptides, low-molecular compounds, and nucleic acid aptamers.
  • the bond here includes a bond by an intermolecular interaction such as electrostatic interaction, van der Waals force, hydrogen bond, hydrophobic interaction, dipole interaction, dispersion force and the like. Any one may be used as long as the conformation of the target polypeptide is changed and the two epitopes to which each of the two antibodies used in the sandwich immunoassay should bind are separated from each other by a spatial distance.
  • an antibody having an antigen binding site different from the antigen binding site of the first antibody and the second antibody may be used.
  • a peptide consisting of 2 to 12 amino acid residues may be used as the peptide as the antigen affinity substance.
  • iA ⁇ 5 5 amino acids
  • D3 (12 amino acids)
  • NH2- examples thereof include D-Trp-Aib-OH (2 amino acids).
  • the low molecular weight compound as the antigen affinity substance a low molecular weight compound capable of binding to a target polypeptide may be used, for example, a low molecular weight compound that binds to a protein as used in drug discovery. It is done.
  • Examples of the low molecular compound that binds to A ⁇ include Scyllo-inositol. Various inhibitors (for example, various types of Stemolecule TM low molecular weight compounds) are also included.
  • Examples of the nucleic acid aptamer as the antigen affinity substance include a DNA aptamer and an RNA aptamer.
  • the amount of the antigen affinity substance added is not particularly limited depending on the type and amount of the target polypeptide in the sample and the type of the antigen affinity substance. An amount that can change the conformation of the peptide may be used.
  • the amount of the antigen affinity substance added is about 0.1 ng / mL to about 100,000 ng / mL, preferably about 0.1 ng / mL to about 10,000 ng / mL, as the concentration in the sample containing the target polypeptide. Preferably, it is about 0.1 ng / mL to 3000 ng / mL.
  • a first antibody having an antigen-binding site capable of recognizing a target polypeptide, and an antigen-binding site capable of recognizing the target polypeptide, which is different from the antigen-binding site of the first antibody As the second antibody having a binding site, various antibodies used in sandwich immunoassays can be used.
  • an N-terminal recognition antibody of a target polypeptide can be used as the first antibody, and a C-terminal recognition antibody of the target polypeptide can be used as the second antibody.
  • the antigen affinity substance does not act near the N-terminus of the target polypeptide and does not act near the C-terminus of the target polypeptide. It is not preferable that competition or inhibition between the antigen affinity substance and the first antibody occurs with respect to the target polypeptide, and competition or inhibition between the antigen affinity substance and the second antibody may occur. It is not preferable.
  • the antigen-affinity substance preferably acts on an intermediate site from the 4-residue site from the N-terminal to the 4-residue site from the C-terminal of the target polypeptide. Furthermore, the antigen affinity substance preferably acts on an intermediate site from the 6-residue site from the N-terminal to the 6-residue site from the C-terminal of the target polypeptide.
  • the N-terminal recognition antibody of the target polypeptide as the first antibody is used as a solid-phased antibody (capture antibody).
  • the C-terminal recognition antibody of the target polypeptide as the antibody may be used as a detection antibody (labeled antibody).
  • the sandwich immunoassay of the present invention includes a radioisotope (RIA) using a radioisotope, a chemiluminescence immunoassay (CIA) using a chemiluminescent substance, in addition to a sandwich ELISA using an enzyme as a labeling substance.
  • RIA radioisotope
  • CIA chemiluminescence immunoassay
  • FIA Immunofluorescence assay
  • ELIA electrochemiluminescence immunoassay
  • BLIA bioluminescence immunoassay
  • TAI immunoturbidimetric method
  • LA turbidity method
  • the basic operation of the sandwich immunoassay method of the present invention can be performed according to a known operation except that an antigen affinity substance is added to a sample.
  • the present invention is particularly suitable when the target polypeptide is A ⁇ and an A ⁇ -related peptide.
  • a ⁇ (A ⁇ 1-40 and A ⁇ 1-42) and A ⁇ -related peptides (for example, A ⁇ 1-39, APP669-711) are present in vivo and are attracting attention as biomarkers of Alzheimer's disease.
  • a sandwich immunoassay that can detect and quantify these A ⁇ and A ⁇ -related peptides, particularly A ⁇ -related peptides, with high sensitivity has been desired.
  • the present invention provides a sandwich immunoassay that can detect and quantify these A ⁇ and A ⁇ -related peptides, particularly A ⁇ -related peptides, with high sensitivity. Examples of A ⁇ and A ⁇ -related peptides are shown below.
  • APP672-709 (A ⁇ 1-38) (SEQ ID NO: 1): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG APP674-711 (A ⁇ 3-40) (SEQ ID NO: 2): EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672-710 (A ⁇ 1-39) (SEQ ID NO: 3): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV APP672-711 (A ⁇ 1-40) (SEQ ID NO: 4): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV OxAPP672-711 (OxA ⁇ 1-40) (SEQ ID NO: 5): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL M VGGVV (Met 706 is oxidized) APP672-713 (A ⁇ 1-42) (SEQ ID NO: 6): DAEFRHDSGYEVH
  • Amyloid precursor protein is a single-membrane transmembrane protein consisting of 770 amino acids. Amyloid precursor protein (APP) is proteolyzed by ⁇ -secretase and ⁇ -secretase, and amyloid beta peptide (A ⁇ ) is produced by proteolysis. APP672-713 and A ⁇ 1-42 represent the same peptide (SEQ ID NO: 6). APP672-711 and A ⁇ 1-40 represent the same peptide (SEQ ID NO: 4).
  • the target polypeptide includes, without limitation, various A ⁇ -related peptides other than those exemplified above. The present invention is also applicable to various polypeptides other than A ⁇ and A ⁇ -related peptides.
  • the target polypeptide is contained in a biological sample.
  • Biological samples include body fluids such as blood, cerebrospinal fluid (CSF), urine, body secretions, saliva, and sputum; and feces.
  • Blood samples include whole blood, plasma and serum.
  • a blood sample can be prepared by appropriately treating whole blood collected from an individual. The treatment performed when preparing a blood sample from the collected whole blood is not particularly limited, and any clinically acceptable treatment may be performed. For example, centrifugation can be performed.
  • the blood sample may be stored at a low temperature such as freezing as appropriate in the middle of the preparation process or at a later stage of the preparation process. In the present invention, the biological sample is discarded without returning to the original individual.
  • Targeting a blood sample as a target sample is less invasive than when the sample is a solid or cerebrospinal fluid, and is also a target sample for screening for various diseases in general health checkups and medical checkups. This is also preferable.
  • the polypeptide sandwich sandwich immunoassay kit of the present invention is for performing the sandwich immunoassay described above, A first antibody having an antigen binding site capable of recognizing a target polypeptide; A second antibody having an antigen binding site capable of recognizing the target polypeptide and different from the antigen binding site of the first antibody; and an antigen affinity substance capable of binding to the target polypeptide including.
  • the kit can contain various components used for the operation of the sandwich immunoassay, for example, a diluent for preparing a sample solution, a washing solution, and the like.
  • Example 1 APP669-711 sandwich ELISA operation method
  • Experimental Example 1 a basic operation method of the sandwich ELISA of APP669-711 will be described below. Each operation in Examples 1 to 4 was performed based on this operation method.
  • N-terminal recognition antibody ITM was requested to produce an antibody that recognizes the N-terminus of APP669-711, and 3 clones (20-1A, 24-6G, 34-6E) were obtained.
  • N-terminal recognition antibody Solidification and blocking of N-terminal recognition antibody
  • the N-terminal recognition antibody is diluted with sodium carbonate buffer (pH 9.6) to a concentration of 20 ⁇ g / mL, and 50 ⁇ L of the resulting antibody diluted solution is added to each well of a 96-well plate and incubated at 4 ° C. for 2 hours.
  • the antibody dilution solution in the plate was removed, and 100 ⁇ L of 20% Blocking One (Nacalai Tesque) was added to each well, followed by incubation at 4 ° C. for 2 hours for blocking.
  • sample solution was prepared by setting the peptide to be measured APP669-711 to a predetermined concentration with 5% Blocking One in PBST.
  • an antibody used as an antigen affinity substance was adjusted to an arbitrary concentration with 5% Blocking One in PBST, and an equal amount was added to the sample solution.
  • HRP-labeled antibody that recognizes C-terminal
  • An HRP-labeled antibody (clone BA27) solution that recognizes the C-terminus contained in Human ⁇ Amyloid (1-40) ELISA Kit (Wako Pure Chemical Industries) was diluted 5-fold with 5% Blocking One in PBST.
  • the sample solution in the plate was removed and washed 3 times with 300 ⁇ L of PBST.
  • 50 ⁇ L each of HRP-labeled antibody (BA27) solution diluted 5-fold was added and incubated at 4 ° C. for 1 hour.
  • the solution in the plate was removed and washed 5 times with 300 ⁇ L PBST.
  • Amyloid ⁇ (A ⁇ ) 1-40 has a structure in which the C-terminal and N-terminal are close in spatial distance (Non-patent Document 3).
  • APP669-711 which is only 3 amino acids longer than the N-terminus of A ⁇ 1-40, is considered to be close to the C-terminus and N-terminus.
  • Anti-A ⁇ antibody 4G8 was used as an antibody (antigen affinity substance) that can bind to APP669-711.
  • the concentration of APP669-711 is 0 fmol / mL, 15.625 fmol / mL, 31.25 fmol / mL, 62.5 fmol / mL, 125 fmol / mL, 250 fmol / mL, 500 fmol / mL, 1000 fmol / mL, anti-A ⁇ antibody 4G8 concentration is 0, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 ng / mL It prepared so that it might become. These solutions were measured by APP669-711 sandwich ELISA.
  • the epitope of clone 4G8 is A ⁇ 18-22.
  • N-terminal recognition antibody clone 34-6E was used as the immobilized antibody.
  • the absorbance increased when anti-A ⁇ antibody 4G8 was added compared to the case where 4G8 was not added (0 ng / mL) (FIG. 1).
  • the concentration increased depending on the concentration up to an addition concentration of 100 ng / mL, and gradually decreased when the concentration was exceeded (FIG. 2).
  • the reason why the absorbance increased may be that anti-A ⁇ antibody 4G8 was immobilized on the plate, captured by APP669-711, and reacted with a C-terminal recognition HRP-labeled antibody.
  • anti-A ⁇ antibody 4G8 reacts, but N-terminal recognition antibody 34-6E does not react with a sample solution of A ⁇ 1-40 (0 to 1000 fmol / mL).
  • APP669-711 sandwich ELISA reactivity when / mL was added was evaluated. Since the epitope of anti-A ⁇ antibody 4G8 is A ⁇ 18-22, if anti-A ⁇ antibody 4G8 is immobilized, it exhibits reactivity. However, no response was shown in this measurement result (FIG. 3).
  • FIG. 1 is a graph showing a calibration curve of APP669-711 ELISA with anti-A ⁇ antibody 4G8 added.
  • the horizontal axis represents the concentration of APP669-711 (fmol / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • a calibration curve is shown for each concentration of anti-A ⁇ antibody 4G8.
  • FIG. 2 is a graph showing the reactivity of APP669-711 ELISA with the addition of anti-A ⁇ antibody 4G8.
  • the horizontal axis represents the concentration (ng / mL) of anti-A ⁇ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • the dots are connected by a line for each concentration of APP669-711.
  • FIG. 3 is a graph showing the reactivity of APP669-711 ELISA against A ⁇ 1-40 when anti-A ⁇ antibody 4G8 is added.
  • the horizontal axis indicates the concentration of A ⁇ 1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm.
  • the amount of anti-A ⁇ antibody 4G8 added was 10,000 ng / mL.
  • Example 2 Verification of effect of addition of anti-A ⁇ antibody 4G8 when N-terminal recognition antibody clone is changed
  • anti-A ⁇ antibody 4G8 as an antigen affinity substance to the sample solution of APP669-711
  • APP669-711 concentration is 500 fmol / mL
  • anti-A ⁇ antibody 4G8 concentration is 0, 10, 30, 100, 300 , 1000, 3000 ng / mL.
  • These solutions were measured by APP669-711 sandwich ELISA.
  • As the solid-phase antibody three clones 20-1A, 34-6G and 34-6E of N-terminal recognition antibodies were used, respectively. As a result, an increase in absorbance was confirmed in all three clones, and the concentration of anti-A ⁇ antibody 4G8 that showed the highest absorbance in each clone was 100 ng / mL in common (FIG. 4). That is, the optimal anti-A ⁇ antibody 4G8 addition concentration was the same for all clones.
  • FIG. 4 is a graph showing the effect of addition of anti-A ⁇ antibody 4G8 in APP669-711 ELISA using 3 N-terminal recognition antibody clones.
  • the horizontal axis represents the concentration (ng / mL) of anti-A ⁇ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • the dots are connected by a line for each clone of the N-terminal recognition antibody.
  • FIG. 5 is a graph showing the reactivity of APP669-711 ELISA against A ⁇ 1-40 when anti-A ⁇ antibody 4G8 is added.
  • Three clones were used as N-terminal recognition antibodies.
  • the horizontal axis indicates the concentration of A ⁇ 1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm.
  • the amount of anti-A ⁇ antibody 4G8 added was 3000 ng / mL.
  • Example 3 Verification of APP669-711 sandwich ELISA reactivity improvement effect when the added anti-A ⁇ antibody is changed
  • APP669-711 By adding 4 clones of anti-A ⁇ antibody (4G8, 6E10, BAM90.1, or NAB228) as an antigen affinity substance to the sample solution of APP669-711, the concentration of APP669-711 is 500 fmol / mL, anti-A ⁇ Antibody 4G8 concentrations were adjusted to 0, 10, 30, 100, 300, 1000, and 3000 ng / mL. These solutions were measured by APP669-711 sandwich ELISA.
  • the epitope of clone 6E10 is A ⁇ 3-8
  • BAM90.1 is A ⁇ 20-23
  • NAB228 is a part of A ⁇ 1-11.
  • As the immobilized antibody N-terminal recognition antibody clone 34-6E was used.
  • FIG. 6 is a graph showing the effect of adding four clones of anti-A ⁇ antibody in APP669-711 ELISA.
  • the horizontal axis represents the concentration of added anti-A ⁇ antibody (ng / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm.
  • the dots are connected by a line for each clone of the anti-A ⁇ antibody.
  • FIG. 7 is a graph showing the reactivity of APP669-711 ELISA against A ⁇ 1-40 when anti-A ⁇ antibody is added.
  • Four clones were used as the anti-A ⁇ antibody.
  • the horizontal axis indicates the concentration of A ⁇ 1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm.
  • the amount of anti-A ⁇ antibody added was 3000 ng / mL.
  • Example 4 Verification of anti-A ⁇ antibody addition effect in A ⁇ 1-40 sandwich ELISA
  • Human ⁇ Amyloid (1-40) ELISA Kit Waako Pure Chemical Industries
  • the samples were standard contained in Human ⁇ Amyloid (1-40) ELISA Kit and A ⁇ 1-40 purchased from AnaSpec.
  • 4G8 concentration is 100 ng so that A ⁇ 1-40 concentration is 50 fmol / mL / mL or 6E10 concentration was adjusted to 300 ng / mL.
  • These solutions were operated according to the protocol in the instruction manual of Human ⁇ Amyloid (1-40) ELISA Kit, and the absorbance was measured. Further, as a reference, the operation was performed without adding 4G8 or 6E10, and the absorbance was measured (indicated as “non-spiked” in FIG.
  • an anti-A ⁇ antibody to be added as an antigen-affinity substance, which has an epitope that is located at a certain spatial distance from the epitope recognized by the antibody used in the sandwich ELISA.
  • FIG. 8 is a graph showing the effect of addition of anti-A ⁇ antibodies 4G8 and 6E10 in A ⁇ 1-40 ELISA.
  • the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm.
  • standard included in ELISA Kit and A ⁇ 1-40 of AnaSpec were used.
  • the analysis target polypeptides are APP669-711 and A ⁇ 1-40.
  • the present invention is also useful for sandwich ELISAs that analyze polypeptides and proteins other than these. Further, the present invention is not limited to the ELISA method, and can be similarly applied to a sandwich method using other labels.

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Abstract

Provided is a sandwich immunoassay whereby a polypeptide to be analyzed can be detected with high sensitivity and quantified. A sandwich immunoassay for a polypeptide, which uses a first antibody having an antigen binding site capable of recognizing a target polypeptide in a sample, and a second antibody having an antigen binding site which different from the antigen binding site of the first antibody and is capable of recognizing the target polypeptide, wherein the immunoassay includes adding an antigen affinity substance capable of binding to the target polypeptide to the sample. The target polypeptide is not limited, and is selected from the group consisting of Aβ and Aβ-related peptides, for example.

Description

サンドイッチ型免疫測定法Sandwich immunoassay
　本発明は、微量成分や感染微生物抗原等の臨床検査薬、生化学研究、及び免疫学研究等の分野に属し、特異的な免疫反応を利用したサンドイッチ型免疫測定法に関する。より詳しくは、本発明は、試料中の微量ポリペプチドを検出及び定量するのに有用なサンドイッチ型免疫測定法に関する。 The present invention relates to a sandwich type immunoassay method using a specific immune reaction, belonging to fields such as clinical diagnostics such as trace components and infectious microorganism antigens, biochemical research, and immunological research. More particularly, the present invention relates to a sandwich immunoassay useful for detecting and quantifying trace polypeptides in a sample.
　免疫測定法とは、特異的な抗原抗体反応を利用して試料中の分析対象物質の濃度を測定する方法であり、微量成分を特異的に、かつ、精度よく測定できることから、臨床検査や生化学研究などで広く利用されている。 An immunoassay is a method that uses a specific antigen-antibody reaction to measure the concentration of an analyte in a sample, and because it can measure trace components specifically and accurately, Widely used in chemical research.
　免疫測定法には、標識物質として酵素を利用する酵素免疫測定法（ＥＩＡ）、放射性同位元素を利用する放射免疫測定法（ＲＩＡ）、化学発光物質を利用する化学発光免疫測定法（ＣＩＡ）、蛍光発光物質を利用する蛍光免疫測定法（ＦＩＡ）、金属錯体を利用する電気化学発光免疫測定法（ＥＣＬＩＡ）、ルシフェラーゼなどの生物発光物質を利用する生物発光免疫測定法（ＢＬＩＡ）、抗体に標識された核酸をＰＣＲで増幅させて検出するイムノＰＣＲ、免疫複合体形成により発生する濁度を検出する免疫比濁法（ＴＡＩ）、免疫複合体形成により凝集するラテックスを検出するラテックス凝集比濁法（ＬＡ）、セルロース膜上で反応させるイムノクロマト法などがある。 The immunoassay includes enzyme immunoassay (EIA) using an enzyme as a labeling substance, radioimmunoassay (RIA) using a radioisotope, chemiluminescence immunoassay (CIA) using a chemiluminescent substance, Fluorescence immunoassay (FIA) using fluorescent materials, electrochemiluminescence immunoassay (ECLIA) using metal complexes, bioluminescence immunoassay (BLIA) using bioluminescent materials such as luciferase, antibody labeling PCR to amplify the detected nucleic acid by PCR, immunoturbidimetric method (TAI) to detect turbidity generated by immune complex formation, latex agglutination turbidimetric method to detect latex aggregated by immune complex formation (LA), an immunochromatography method for reacting on a cellulose membrane.
　それら免疫測定法の中で、分析対象物質の異なるエピトープを認識する２つの抗体を用いて分析対象物質を挟み込む方法をサンドイッチ法と呼ぶ。例えば、サンドイッチＥＬＩＳＡ（Enzyme-Linked ImmunoSorbent Assay）とはＥＩＡの一つで、９６ウェルプレート面に固相された捕捉抗体で分析対象物質を捕捉し、その分析対象物質にペルオキシダーゼやアルカリフォスファターゼなどの酵素で標識された検出抗体を反応させ、標識酵素の基質の発色を利用して分析対象物質の濃度を決定する方法である（例えば、特許文献１：特開平８－２２００９８号公報）。ＥＣＬＩＡにおいても、分析対象物質に対して、ビオチン化された抗体と、電気化学的変化で発光するルテニウム（Ｒｕ）錯体を標識した抗体とを反応させるサンドイッチ法を利用している（例えば、特許文献２：特表２０１４－５０９７３５号公報）。このように、免疫測定法ではサンドイッチ法が広く利用されている。 Among these immunoassays, a method of sandwiching an analysis target substance using two antibodies that recognize different epitopes of the analysis target substance is called a sandwich method. For example, sandwich ELISA (Enzyme-Linked ImmunoSorbent Ｉ Assay) is an EIA that captures an analyte with a capture antibody immobilized on the surface of a 96-well plate, and an enzyme such as peroxidase or alkaline phosphatase on the analyte. This is a method in which the detection antibody labeled with is reacted, and the concentration of the substance to be analyzed is determined using the color of the substrate of the labeled enzyme (for example, Patent Document 1: Japanese Patent Application Laid-Open No. 8-220098). ECLIA also uses a sandwich method in which a biotinylated antibody and an antibody labeled with a ruthenium (Ru) complex that emits light by an electrochemical change are reacted with an analyte (for example, patent documents) 2: Japanese Translation of PCT International Publication No. 2014-509735). As described above, the sandwich method is widely used as an immunoassay.
　特定のペプチド断片を特異的に定量する方法としてもサンドイッチ法は利用され、分析対象ペプチド断片をＮ末端認識抗体とＣ末端認識抗体で挟み込む方法でペプチド断片を特異的に測定している。例えば、アミロイド前駆タンパク質（Amyloid precursor protein； ＡＰＰ）からプロテアーゼによって切断されて生成されるＡβについては、異なる断片のペプチドが生体内に存在している。それらの中でＡβ１－４２は、アルツハイマー病の脳脊髄液（ＣＳＦ）バイオマーカーとして、ＥＬＩＳＡなどの免疫測定法で定量されている（非特許文献２）。 The sandwich method is also used as a method for specifically quantifying a specific peptide fragment, and the peptide fragment is specifically measured by sandwiching the peptide fragment to be analyzed between an N-terminal recognition antibody and a C-terminal recognition antibody. For example, regarding Aβ produced by cleaving from amyloid precursor protein (Amyloid precursor protein; APP) with a protease, peptides of different fragments exist in vivo. Among them, Aβ1-42 is quantified by an immunoassay such as ELISA as a cerebrospinal fluid (CSF) biomarker of Alzheimer's disease (Non-patent Document 2).
　Ａβについては、最近の研究で、Ａβ関連ペプチド（Ａβ様ペプチド）の一つであるＡＰＰ６６９－７１１とＡβ１－４２との比が血液バイオマーカーとして有望であることが報告されている（非特許文献１，特許文献３：国際公開ＷＯ２０１５／１７８３９８）。これらＡβ及びＡβ関連ペプチドは、免疫沈降法（ＩＰ）の後、質量分析（ＭＡＬＤＩ－ＴＯＦ－ＭＡＳＳ）を行うことにより定量されている。 Regarding Aβ, a recent study has reported that the ratio of APP669-711, which is one of Aβ-related peptides (Aβ-like peptides), and Aβ1-42 is promising as a blood biomarker (non-patent literature). 1, Patent Document 3: International Publication WO2015 / 178398). These Aβ and Aβ-related peptides are quantified by performing mass spectrometry (MALDI-TOF-MASS) after immunoprecipitation (IP).
　さらに、特許文献４：国際公開ＷＯ２０１７／０４７５２９には、Ａβ及びＡβ関連ペプチド（Ａβ様ペプチド）についての３つの比、Ａβ１－３９／Ａβ１－４２、Ａβ１－４０／Ａβ１－４２、及びＡＰＰ６６９－７１１／Ａβ１－４２からなる群より選ばれる２以上の比を数学的手法で組み合わせた数値が血液バイオマーカーとして有望であることが開示されている。これらＡβ及びＡβ関連ペプチドは、免疫沈降法（ＩＰ）の後、質量分析（ＭＡＬＤＩ－ＴＯＦ－ＭＡＳＳ）を行うことにより定量されている。 Further, Patent Document 4: International Publication WO2017 / 047529 describes three ratios for Aβ and Aβ related peptides (Aβ-like peptides), Aβ1-39 / Aβ1-42, Aβ1-40 / Aβ1-42, and APP669-711. It is disclosed that a numerical value obtained by combining two or more ratios selected from the group consisting of / Aβ1-42 by a mathematical method is promising as a blood biomarker. These Aβ and Aβ-related peptides are quantified by performing mass spectrometry (MALDI-TOF-MASS) after immunoprecipitation (IP).
　特許文献５：特開２００４－２３９８８５号公報には、免疫測定法の測定感度を改善する方法の一つとして、試料中から抽出しにくいタンパク質に対して、免疫測定法の前に高濃度のイオン性界面活性剤で試料を抽出する方法が報告されている。 Patent Document 5: Japanese Patent Application Laid-Open No. 2004-23985 discloses a method for improving the measurement sensitivity of an immunoassay method by using a high concentration of ions before immunoassay for a protein that is difficult to extract from a sample. A method for extracting a sample with an ionic surfactant has been reported.
特開平８－２２００９８号公報JP-A-8-220098 特表２０１４－５０９７３５号公報Special table 2014-509735 gazette 国際公開ＷＯ２０１５／１７８３９８International Publication WO2015 / 178398 国際公開ＷＯ２０１７／０４７５２９International Publication WO2017 / 047529 特開２００４－２３９８８５号公報JP 2004-239985 A
　試料中の分析対象ポリペプチドをサンドイッチ免疫測定法で特異的に定量するためには、分析対象ポリペプチドの２箇所の異なる部位（エピトープ）に対して、それぞれを認識する２つの抗体を同時に分析対象ペプチドへ結合させる、もしくは、どちらか一方の抗体を結合させた後に、もう一方の抗体を結合させる。つまり、分析対象ポリペプチドの２箇所の異なる部位（エピトープ）に２つの抗体が同時に結合し、分析対象ポリペプチドが２つの抗体によりサンドイッチされた状態とする必要がある。 In order to specifically quantify a polypeptide to be analyzed in a sample by sandwich immunoassay, two antibodies that recognize each of two different sites (epitopes) of the polypeptide to be analyzed are analyzed simultaneously. After binding to the peptide, or after binding one of the antibodies, the other antibody is bound. That is, it is necessary that two antibodies simultaneously bind to two different sites (epitope) of the polypeptide to be analyzed, and the polypeptide to be analyzed is sandwiched between the two antibodies.
　ポリペプチドの一次構造としては２つの抗体が各々結合すべき２つの異なる認識部位（エピトープ）が互いに離れていても、ポリペプチドのコンフォメーションにより空間距離的に前記２つのエピトープが近い場合がある。分析対象ポリペプチドの前記２つのエピトープが空間距離的に近い場合には、前記２つの抗体が同時に結合することが難しくなり、サンドイッチ免疫測定法の感度が低下する要因となる。例えば、ペプチド断片であるＡβを測定するサンドイッチ免疫測定法では、Ｎ認識抗体とＣ末認識抗体とでペプチド断片を挟み込むが、一次構造としては２つの抗体の認識部位（エピトープ）が互いに離れていても、空間距離的には２つのエピトープが近い（非特許文献３）。そのため、サンドイッチ免疫測定法の感度低下を引き起こす可能性がある。 As the primary structure of a polypeptide, there are cases where the two epitopes are close to each other in terms of spatial distance due to the conformation of the polypeptide even if two different recognition sites (epitopes) to which two antibodies are to be bound are separated from each other. When the two epitopes of the polypeptide to be analyzed are close in spatial distance, it becomes difficult for the two antibodies to bind at the same time, which causes a decrease in the sensitivity of the sandwich immunoassay. For example, in a sandwich immunoassay for measuring Aβ, which is a peptide fragment, the peptide fragment is sandwiched between an N-recognizing antibody and a C-terminal recognizing antibody, but the primary antibody recognition sites (epitopes) are separated from each other. However, the two epitopes are close in spatial distance (Non-patent Document 3). Therefore, it may cause a decrease in sensitivity of the sandwich immunoassay.
　本発明の目的は、分析対象ポリペプチドを高感度で検出及び定量できるサンドイッチ免疫測定法を提供することにある。とりわけ本発明の目的は、分析対象Ａβ及びＡβ関連ペプチドを高感度で検出及び定量できるサンドイッチ免疫測定法を提供することにある。さらに、本発明の目的は、ポリペプチドのサンドイッチ型免疫測定用キットを提供することにある。 An object of the present invention is to provide a sandwich immunoassay that can detect and quantify a polypeptide to be analyzed with high sensitivity. In particular, an object of the present invention is to provide a sandwich immunoassay that can detect and quantify Aβ and Aβ-related peptides to be analyzed with high sensitivity. Furthermore, an object of the present invention is to provide a sandwich immunoassay kit for polypeptides.
　本発明者は、鋭意検討の結果、分析対象ポリペプチドのコンフォメーションを変化させ、サンドイッチ免疫測定法で使用される２つの抗体が各々結合すべき２つのエピトープを空間距離的に互いに遠ざける処置を施すことにより、具体的には、分析対象ポリペプチドに親和性のある物質（抗原親和性物質）を該分析対象ポリペプチドに結合させてコンフォメーションを変化させることにより、分析対象ポリペプチドを高感度で検出及び定量できるサンドイッチ免疫測定法が得られることを見出した。それにより、本発明を完成するに至った。 As a result of intensive studies, the inventor performs a treatment to change the conformation of the polypeptide to be analyzed and to separate the two epitopes to be bound by the two antibodies used in the sandwich immunoassay from each other in terms of spatial distance. In particular, by binding a substance having affinity for the analysis target polypeptide (antigen affinity substance) to the analysis target polypeptide and changing the conformation, the analysis target polypeptide can be made highly sensitive. It has been found that a sandwich immunoassay can be obtained that can be detected and quantified. As a result, the present invention has been completed.
　本明細書において、ポリペプチドには、ペプチド、及びタンパク質が含まれる。前記タンパク質には、糖タンパク、リン酸化タンパク質などの翻訳後修飾されたタンパク質も含まれる。 In the present specification, the polypeptide includes a peptide and a protein. The protein includes post-translationally modified proteins such as glycoproteins and phosphorylated proteins.
　本明細書において、アミロイド・βペプチドの略称として「Ａβ」を用いる。すなわち、「Ａβ」とは、Ａβ１－４０、及びＡβ１－４２を含んでいる。アミロイド前駆タンパク質（Amyloid precursor protein；ＡＰＰ）が切断されることにより生じる前記Ａβ以外のペプチドをＡβ関連ペプチド（又は、Ａβ様ペプチド）と称することがある。アミロイド前駆タンパク質（Amyloid precursor protein；ＡＰＰ）が切断されることにより生じるＡβ及びＡβ関連ペプチド（又は、Ａβ様ペプチド）を、「ＡＰＰ派生型ペプチド」と称することがある。 In this specification, “Aβ” is used as an abbreviation for amyloid β peptide. That is, “Aβ” includes Aβ1-40 and Aβ1-42. A peptide other than Aβ produced by cleaving amyloid precursor protein (Amyloid precursor protein; APP) may be referred to as an Aβ-related peptide (or Aβ-like peptide). Aβ and Aβ-related peptides (or Aβ-like peptides) generated by cleaving amyloid precursor protein (Amyloid precursor protein; APP) may be referred to as “APP-derived peptides”.
　本発明は、以下の発明を含む。
（１）　試料中の標的ポリペプチドを認識可能な抗原結合部位を持つ第１の抗体、及び前記標的ポリペプチドを認識可能な抗原結合部位であって前記第１の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ第２の抗体を用いたポリペプチドのサンドイッチ型免疫測定法において、試料に標的ポリペプチドに結合可能な抗原親和性物質を添加することを含む、免疫測定法。 The present invention includes the following inventions.
(1) a first antibody having an antigen-binding site capable of recognizing a target polypeptide in a sample, and an antigen-binding site capable of recognizing the target polypeptide and having the first antibody A polypeptide sandwich immunoassay method using a second antibody having a different antigen binding site, comprising adding an antigen affinity substance capable of binding to a target polypeptide to a sample.
（２）　前記標的ポリペプチドを含む試料に前記抗原親和性物質を添加し、その後、前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体と反応させるか、又は、
　前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体と反応させている時に、反応系に前記抗原親和性物質を添加するか、又は、
　前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体のいずれか一方と反応させ、その後、反応系に前記抗原親和性物質を添加し、その後、前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体のいずれか他方と反応させる、上記（１）に記載の免疫測定法。 (2) adding the antigen-affinity substance to a sample containing the target polypeptide, and then reacting the target polypeptide with the first antibody and the second antibody, or
When the target polypeptide is reacted with the first antibody and the second antibody, the antigen affinity substance is added to the reaction system, or
The target polypeptide is reacted with one of the first antibody and the second antibody, and then the antigen affinity substance is added to a reaction system, and then the target polypeptide is converted into the first antibody. And the immunoassay method according to (1) above, which is reacted with either one of the second antibody.
（３）　前記抗原親和性物質が、抗体、ペプチド、低分子化合物、及び核酸アプタマーからなる群から選ばれる、上記（１）又は（２）に記載の免疫測定法。 (3) The immunoassay method according to (1) or (2) above, wherein the antigen affinity substance is selected from the group consisting of an antibody, a peptide, a low molecular compound, and a nucleic acid aptamer.
（４）　前記サンドイッチ型免疫測定法が、酵素免疫測定法（ＥＩＡ）、放射免疫測定法（ＲＩＡ）、化学発光免疫測定法（ＣＩＡ）、蛍光免疫測定法（ＦＩＡ）、電気化学発光免疫測定法（ＥＣＬＩＡ）、生物発光免疫測定法（ＢＬＩＡ）、イムノＰＣＲ、免疫比濁法（ＴＡＩ）、及びラテックス凝集比濁法（ＬＡ）からなる群から選ばれる、上記（１）～（３）のうちのいずれかに記載の免疫測定法。 (4) The sandwich immunoassay includes enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CIA), fluorescence immunoassay (FIA), and electrochemiluminescence immunoassay. (ECLIA), bioluminescent immunoassay (BLIA), immuno-PCR, immunoturbidimetry (TAI), and latex agglutination turbidimetry (LA), among the above (1) to (3) The immunoassay method according to any one of the above.
（５）　前記第１の抗体が、前記標的ポリペプチドのＮ末端認識抗体であり、前記第２の抗体が、前記標的ポリペプチドのＣ末端認識抗体である、上記（１）～（４）のうちのいずれかに記載の免疫測定法。 (5) The above (1) to (4), wherein the first antibody is an N-terminal recognition antibody of the target polypeptide, and the second antibody is a C-terminal recognition antibody of the target polypeptide. The immunoassay method according to any one of the above.
（６）　前記抗原親和性物質が、前記標的ポリペプチドのＮ末端付近には作用せず、且つ、前記標的ポリペプチドのＣ末端付近には作用しないものである、上記（５）に記載の免疫測定法。 (6) The immunity according to (5) above, wherein the antigen affinity substance does not act near the N-terminus of the target polypeptide and does not act near the C-terminus of the target polypeptide. Measurement method.
（７）　前記抗原親和性物質は、前記標的ポリペプチドのＮ末端から４残基部位からＣ末端から４残基部位までの中間部位に作用するものである、上記（５）又は（６）に記載の免疫測定法。 (7) In the above (5) or (6), the antigen affinity substance acts on an intermediate site from the 4-residue site from the N-terminal to the 4-residue site from the C-terminal of the target polypeptide. The immunoassay described.
（８）　前記標的ポリペプチドが、Ａβ及びＡβ関連ペプチドからなる群から選ばれる、上記（１）～（７）のうちのいずれかに記載の免疫測定法。 (8) The immunoassay method according to any one of (1) to (7) above, wherein the target polypeptide is selected from the group consisting of Aβ and Aβ-related peptides.
（９）　前記試料が、血液、脳脊髄液、尿、糞便、及び、体分泌液からなる群から選ばれる生体由来試料である、上記（１）～（８）のうちのいずれかに記載の免疫測定法。 (9) The sample according to any one of (1) to (8) above, wherein the sample is a biological sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, and body secretions. Immunoassay.
（１０）　標的ポリペプチドを認識可能な抗原結合部位を持つ第１の抗体、
　前記標的ポリペプチドを認識可能な抗原結合部位であって前記第１の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ第２の抗体、及び
　前記標的ポリペプチドに結合可能な抗原親和性物質
を含む、ポリペプチドのサンドイッチ型免疫測定用キット。 (10) a first antibody having an antigen-binding site capable of recognizing a target polypeptide,
A second antibody having an antigen binding site capable of recognizing the target polypeptide and different from the antigen binding site of the first antibody; and an antigen affinity substance capable of binding to the target polypeptide A sandwich type immunoassay kit for polypeptides, comprising:
　本発明のサンドイッチ型免疫測定法において、試料に標的ポリペプチドに結合可能な抗原親和性物質を添加する。このことにより、標的ポリペプチドのコンフォメーションが変化させられ、サンドイッチ免疫測定法で使用される２つの抗体が各々結合すべき２つのエピトープが空間距離的に互いに遠ざけられる。そのため、２つの部位での抗原抗体反応がうまく行われ、標的ポリペプチドを高感度で検出及び定量できる。 In the sandwich immunoassay method of the present invention, an antigen affinity substance capable of binding to the target polypeptide is added to the sample. This alters the conformation of the target polypeptide and separates the two epitopes to which each of the two antibodies used in the sandwich immunoassay should bind in spatial distance. Therefore, the antigen-antibody reaction at two sites is successfully performed, and the target polypeptide can be detected and quantified with high sensitivity.
　特定のポリペプチドＡβ（Ａβ１－４０、及びＡβ１－４２）や、Ａβ関連ペプチド（例えば、Ａβ１－３９、ＡＰＰ６６９－７１１）は、生体内に存在しており、アルツハイマー病のバイオマーカーとして注目されていた。これらＡβやＡβ関連ペプチド、特にＡβ関連ペプチドについて、高感度で検出及び定量できるサンドイッチ免疫測定法が望まれている。本発明によれば、これらＡβやＡβ関連ペプチド、特にＡβ関連ペプチドについて、高感度で検出及び定量できるサンドイッチ免疫測定法が提供される。 Specific polypeptides Aβ (Aβ1-40 and Aβ1-42) and Aβ-related peptides (eg, Aβ1-39, APP669-711) are present in vivo and are attracting attention as biomarkers of Alzheimer's disease. It was. A sandwich immunoassay that can detect and quantify these Aβ and Aβ-related peptides, particularly Aβ-related peptides, with high sensitivity is desired. According to the present invention, a sandwich immunoassay that can detect and quantify these Aβ and Aβ-related peptides, particularly Aβ-related peptides, with high sensitivity is provided.
図１は、実施例１において、抗Aβ抗体4G8添加によるAPP669-711 ELISAの検量線を示すグラフである。横軸はAPP669-711の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。抗Aβ抗体4G8の濃度毎に検量線が示されている。FIG. 1 is a graph showing the calibration curve of APP669-711 ELISA by adding anti-Aβ antibody 4G8 in Example 1. The horizontal axis represents the concentration of APP669-711 (fmol / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. A calibration curve is shown for each concentration of anti-Aβ antibody 4G8. 図２は、実施例１において、抗Aβ抗体4G8添加によるAPP669-711 ELISAの反応性を示すグラフである。横軸は抗Aβ抗体4G8の濃度(ng/mL)、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。APP669-711の濃度毎に点を線で繋いでいる。FIG. 2 is a graph showing the reactivity of APP669-711 ELISA by adding anti-Aβ antibody 4G8 in Example 1. The horizontal axis represents the concentration (ng / mL) of anti-Aβ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. The dots are connected by a line for each concentration of APP669-711. 図３は、実施例１において、抗Aβ抗体4G8添加時におけるAβ1-40に対するAPP669-711 ELISAの反応性を示すグラフである。横軸はAβ1-40の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。FIG. 3 is a graph showing the reactivity of APP669-711 ELISA to Aβ1-40 when anti-Aβ antibody 4G8 was added in Example 1. The horizontal axis indicates the concentration of Aβ1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. 図４は、実施例２において、Ｎ末端認識抗体３クローンを用いたAPP669-711 ELISAにおける抗Aβ抗体4G8添加効果を示すグラフである。横軸は抗Aβ抗体4G8の濃度(ng/mL)、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。Ｎ末端認識抗体のクローン毎に点を線で繋いでいる。FIG. 4 is a graph showing the effect of addition of anti-Aβ antibody 4G8 in APP669-711 ELISA using 3 clones of N-terminal recognition antibody in Example 2. The horizontal axis represents the concentration (ng / mL) of anti-Aβ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. The dots are connected by a line for each clone of the N-terminal recognition antibody. 図５は、実施例２において、抗Aβ抗体4G8添加時におけるAβ1-40に対するAPP669-711 ELISAの反応性を示すグラフである。Ｎ末端認識抗体として、３クローンを各々用いた。横軸はAβ1-40の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。FIG. 5 is a graph showing the reactivity of APP669-711 ELISA to Aβ1-40 when anti-Aβ antibody 4G8 was added in Example 2. Three clones were used as N-terminal recognition antibodies. The horizontal axis indicates the concentration of Aβ1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. 図６は、実施例３において、APP669-711 ELISAにおける抗Aβ抗体の４クローンの添加効果を示すグラフである。横軸は抗Aβ抗体の添加濃度(ng/mL)、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。抗Aβ抗体のクローン毎に点を線で繋いでいる。FIG. 6 is a graph showing the effect of adding 4 clones of anti-Aβ antibody in APP669-711 ELISA in Example 3. The horizontal axis represents the concentration of added anti-Aβ antibody (ng / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. The dots are connected by a line for each clone of the anti-Aβ antibody. 図７は、実施例３において、抗Aβ抗体添加時におけるAβ1-40に対するAPP669-711 ELISAの反応性を示すグラフである。抗Aβ抗体は４クローンを用いた。横軸はAβ1-40の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。FIG. 7 is a graph showing the reactivity of APP669-711 ELISA to Aβ1-40 when anti-Aβ antibody was added in Example 3. Four clones were used as the anti-Aβ antibody. The horizontal axis indicates the concentration of Aβ1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. 図８は、実施例４において、Aβ1-40 ELISAにおける抗Aβ抗体4G8及び6E10の添加効果を示すグラフである。縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。サンプルとして、ELISA Kit内に含まれているstandardと、AnaSpec社のAβ1-40を用いた。FIG. 8 is a graph showing the effect of addition of anti-Aβ antibodies 4G8 and 6E10 in Aβ1-40 ELISA in Example 4. The vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. As a sample, standard included in ELISA Kit and Aβ1-40 of AnaSpec were used.
　本発明の免疫測定法は、試料中の標的ポリペプチドを認識可能な抗原結合部位を持つ第１の抗体、及び前記標的ポリペプチドを認識可能な抗原結合部位であって前記第１の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ第２の抗体を用いたポリペプチドのサンドイッチ型免疫測定法において、試料に標的ポリペプチドに結合可能な抗原親和性物質を添加することを含む。 The immunoassay method of the present invention comprises a first antibody having an antigen-binding site capable of recognizing a target polypeptide in a sample, and an antigen-binding site capable of recognizing the target polypeptide, the first antibody having In a sandwich immunoassay method for a polypeptide using a second antibody having an antigen binding site different from the antigen binding site, an antigen affinity substance capable of binding to the target polypeptide is added to the sample.
　本発明の免疫測定法において、前記標的ポリペプチドを含む試料に前記抗原親和性物質を添加し、その後、前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体と反応させるか、又は、
　前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体と反応させている時に、反応系に前記抗原親和性物質を添加するか、又は、
　前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体のいずれか一方と反応させ、その後、反応系に前記抗原親和性物質を添加し、その後、前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体のいずれか他方と反応させることができる。 In the immunoassay method of the present invention, the antigen-affinity substance is added to a sample containing the target polypeptide, and then the target polypeptide is reacted with the first antibody and the second antibody, or
When the target polypeptide is reacted with the first antibody and the second antibody, the antigen affinity substance is added to the reaction system, or
The target polypeptide is reacted with one of the first antibody and the second antibody, and then the antigen affinity substance is added to a reaction system, and then the target polypeptide is converted into the first antibody. And can be reacted with either one of the second antibodies.
　試料に標的ポリペプチドに結合可能な抗原親和性物質を添加することにより、標的ポリペプチドのコンフォメーションが変化させられ、サンドイッチ免疫測定法で使用される２つの抗体が各々結合すべき２つのエピトープが空間距離的に互いに遠ざけられる。そのため、２つの部位での抗原抗体反応がうまく行われ、標的ポリペプチドを高感度で検出及び定量できる。 By adding an antigen affinity substance that can bind to the target polypeptide to the sample, the conformation of the target polypeptide is changed, so that the two epitopes to which the two antibodies used in the sandwich immunoassay should each bind Spatial distances from each other. Therefore, the antigen-antibody reaction at two sites is successfully performed, and the target polypeptide can be detected and quantified with high sensitivity.
　前記抗原親和性物質は、標的ポリペプチドに親和性を有し結合可能な物質であればよく、抗体、ペプチド、低分子化合物、及び核酸アプタマー等が挙げられる。ここでの結合とは、静電相互作用、ファンデルワールス力、水素結合、疎水性相互作用、双極子相互作用、分散力などの分子間相互作用による結合が含まれる。標的ポリペプチドのコンフォメーションが変化させられ、サンドイッチ免疫測定法で使用される２つの抗体が各々結合すべき２つのエピトープが空間距離的に互いに遠ざけられるものであればよい。 The antigen-affinity substance may be any substance that has an affinity and can bind to the target polypeptide, and examples thereof include antibodies, peptides, low-molecular compounds, and nucleic acid aptamers. The bond here includes a bond by an intermolecular interaction such as electrostatic interaction, van der Waals force, hydrogen bond, hydrophobic interaction, dipole interaction, dispersion force and the like. Any one may be used as long as the conformation of the target polypeptide is changed and the two epitopes to which each of the two antibodies used in the sandwich immunoassay should bind are separated from each other by a spatial distance.
　前記抗原親和性物質としての抗体としては、前記第１の抗体及び前記第２の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ抗体を用いるとよい。
前記抗原親和性物質としてのペプチドとしては、２～１２アミノ酸残基からなるペプチドを用いるとよく、例えば、Ａβに結合するペプチドであれば、iAβ5（５アミノ酸）、D3（１２アミノ酸）、NH2-D-Trp-Aib-OH（２アミノ酸）等が例示される。
前記抗原親和性物質としての低分子化合物としては、標的ポリペプチドに結合可能な低分子化合物を用いるとよく、例えば、創薬で使われているような、あるタンパク質に結合する低分子化合物が挙げられる。Ａβに結合する低分子化合物であれば、Scyllo-inositol等が例示される。各種の阻害剤（例えば、Stemolecule TM 低分子化合物の各種）も挙げられる。
前記抗原親和性物質としての核酸アプタマーとしては、DNAアプタマー、RNAアプタマー等が挙げられる。 As the antibody as the antigen affinity substance, an antibody having an antigen binding site different from the antigen binding site of the first antibody and the second antibody may be used.
A peptide consisting of 2 to 12 amino acid residues may be used as the peptide as the antigen affinity substance. For example, iAβ5 (5 amino acids), D3 (12 amino acids), NH2- Examples thereof include D-Trp-Aib-OH (2 amino acids).
As the low molecular weight compound as the antigen affinity substance, a low molecular weight compound capable of binding to a target polypeptide may be used, for example, a low molecular weight compound that binds to a protein as used in drug discovery. It is done. Examples of the low molecular compound that binds to Aβ include Scyllo-inositol. Various inhibitors (for example, various types of Stemolecule ™ low molecular weight compounds) are also included.
Examples of the nucleic acid aptamer as the antigen affinity substance include a DNA aptamer and an RNA aptamer.
　前記抗原親和性物質の添加量は、試料中の標的ポリペプチドの種類や量、及び抗原親和性物質の種類にもより、特に限定されることはなく、標的ポリペプチドに結合して該標的ポリペプチドのコンフォメーションを変化させられる量とすればよい。例えば、前記抗原親和性物質の添加量は、標的ポリペプチドを含んでいる試料中の濃度として、０．１ｎｇ／ｍＬ～１０００００ｎｇ／ｍＬ程度、好ましくは０．１ｎｇ／ｍＬ～１００００ｎｇ／ｍＬ程度、より好ましくは０．１ｎｇ／ｍＬ～３０００ｎｇ／ｍＬ程度とするとよい。 The amount of the antigen affinity substance added is not particularly limited depending on the type and amount of the target polypeptide in the sample and the type of the antigen affinity substance. An amount that can change the conformation of the peptide may be used. For example, the amount of the antigen affinity substance added is about 0.1 ng / mL to about 100,000 ng / mL, preferably about 0.1 ng / mL to about 10,000 ng / mL, as the concentration in the sample containing the target polypeptide. Preferably, it is about 0.1 ng / mL to 3000 ng / mL.
　本発明において、標的ポリペプチドを認識可能な抗原結合部位を持つ第１の抗体、及び前記標的ポリペプチドを認識可能な抗原結合部位であって前記第１の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ第２の抗体としては、サンドイッチ型免疫測定法で用いられている種々のものを用いることができる。 In the present invention, a first antibody having an antigen-binding site capable of recognizing a target polypeptide, and an antigen-binding site capable of recognizing the target polypeptide, which is different from the antigen-binding site of the first antibody As the second antibody having a binding site, various antibodies used in sandwich immunoassays can be used.
　例えば、前記第１の抗体として、標的ポリペプチドのＮ末端認識抗体を用いて、前記第２の抗体として、標的ポリペプチドのＣ末端認識抗体を用いることができる。 For example, an N-terminal recognition antibody of a target polypeptide can be used as the first antibody, and a C-terminal recognition antibody of the target polypeptide can be used as the second antibody.
　この場合には、前記抗原親和性物質が、標的ポリペプチドのＮ末端付近には作用せず、且つ、前記標的ポリペプチドのＣ末端付近には作用しないものが好ましい。標的ポリペプチドに対して、前記抗原親和性物質と前記第１の抗体との競合や阻害が起こることは好ましくないし、前記抗原親和性物質と前記第２の抗体との競合や阻害が起こることも好ましくない。 In this case, it is preferable that the antigen affinity substance does not act near the N-terminus of the target polypeptide and does not act near the C-terminus of the target polypeptide. It is not preferable that competition or inhibition between the antigen affinity substance and the first antibody occurs with respect to the target polypeptide, and competition or inhibition between the antigen affinity substance and the second antibody may occur. It is not preferable.
　より具体的には、前記抗原親和性物質は、前記標的ポリペプチドのＮ末端から４残基部位からＣ末端から４残基部位までの中間部位に作用するものであることが好ましい。さらに、前記抗原親和性物質は、前記標的ポリペプチドのＮ末端から６残基部位からＣ末端から６残基部位までの中間部位に作用するものであることが好ましい。 More specifically, the antigen-affinity substance preferably acts on an intermediate site from the 4-residue site from the N-terminal to the 4-residue site from the C-terminal of the target polypeptide. Furthermore, the antigen affinity substance preferably acts on an intermediate site from the 6-residue site from the N-terminal to the 6-residue site from the C-terminal of the target polypeptide.
　標識物質として酵素を利用するサンドイッチＥＬＩＳＡ（Enzyme-Linked ImmunoSorbent Assay）の場合、前記第１の抗体としての標的ポリペプチドのＮ末端認識抗体を固相化抗体（捕捉抗体）として用いて、前記第２の抗体としての標的ポリペプチドのＣ末端認識抗体を検出抗体（標識抗体）として用いるとよい。 In the case of sandwich ELISA (enzyme-linked immunosorbent assay) using an enzyme as a labeling substance, the N-terminal recognition antibody of the target polypeptide as the first antibody is used as a solid-phased antibody (capture antibody). The C-terminal recognition antibody of the target polypeptide as the antibody may be used as a detection antibody (labeled antibody).
　本発明のサンドイッチ型免疫測定法は、標識物質として酵素を利用するサンドイッチＥＬＩＳＡの他に、放射性同位元素を利用する放射免疫測定法（ＲＩＡ）、化学発光物質を利用する化学発光免疫測定法（ＣＩＡ）、蛍光発光物質を利用する蛍光免疫測定法（ＦＩＡ）、金属錯体を利用する電気化学発光免疫測定法（ＥＣＬＩＡ）、ルシフェラーゼなどの生物発光物質を利用する生物発光免疫測定法（ＢＬＩＡ）、抗体に標識された核酸をＰＣＲで増幅させて検出するイムノＰＣＲ、免疫複合体形成により発生する濁度を検出する免疫比濁法（ＴＡＩ）、免疫複合体形成により凝集するラテックスを検出するラテックス凝集比濁法（ＬＡ）、セルロース膜上で反応させるイムノクロマト法などにも適用できる。 The sandwich immunoassay of the present invention includes a radioisotope (RIA) using a radioisotope, a chemiluminescence immunoassay (CIA) using a chemiluminescent substance, in addition to a sandwich ELISA using an enzyme as a labeling substance. ), Immunofluorescence assay (FIA) using a fluorescent substance, electrochemiluminescence immunoassay (ECLIA) using a metal complex, bioluminescence immunoassay (BLIA) using a bioluminescent substance such as luciferase, antibody Immuno-PCR that detects and detects nucleic acid labeled by PCR, immunoturbidimetric method (TAI) that detects turbidity caused by immune complex formation, latex aggregation ratio that detects latex that aggregates due to immune complex formation The present invention can also be applied to a turbidity method (LA), an immunochromatography method for reacting on a cellulose membrane, and the like.
　本発明のサンドイッチ型免疫測定法の基本的操作は、試料に抗原親和性物質を添加すること以外は、公知の操作に従って行なうことができる。 The basic operation of the sandwich immunoassay method of the present invention can be performed according to a known operation except that an antigen affinity substance is added to a sample.
　本発明は、標的ポリペプチドがＡβ及びＡβ関連ペプチドである場合に特に好適である。Ａβ（Ａβ１－４０、及びＡβ１－４２）や、Ａβ関連ペプチド（例えば、Ａβ１－３９、ＡＰＰ６６９－７１１）は、生体内に存在しており、アルツハイマー病のバイオマーカーとして注目されている。これらＡβやＡβ関連ペプチド、特にＡβ関連ペプチドについて、高感度で検出及び定量できるサンドイッチ免疫測定法が望まれていた。本発明により、これらＡβやＡβ関連ペプチド、特にＡβ関連ペプチドについて、高感度で検出及び定量できるサンドイッチ免疫測定法が提供される。以下に、Ａβ及びＡβ関連ペプチドの例を示す。 The present invention is particularly suitable when the target polypeptide is Aβ and an Aβ-related peptide. Aβ (Aβ1-40 and Aβ1-42) and Aβ-related peptides (for example, Aβ1-39, APP669-711) are present in vivo and are attracting attention as biomarkers of Alzheimer's disease. A sandwich immunoassay that can detect and quantify these Aβ and Aβ-related peptides, particularly Aβ-related peptides, with high sensitivity has been desired. The present invention provides a sandwich immunoassay that can detect and quantify these Aβ and Aβ-related peptides, particularly Aβ-related peptides, with high sensitivity. Examples of Aβ and Aβ-related peptides are shown below.
ＡＰＰ６７２－７０９（Ａβ１－３８）（配列番号１）：
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG
ＡＰＰ６７４－７１１（Ａβ３－４０）（配列番号２）：
EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
ＡＰＰ６７２－７１０（Ａβ１－３９）（配列番号３）：
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV
ＡＰＰ６７２－７１１（Ａβ１－４０）（配列番号４）：
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
ＯｘＡＰＰ６７２－７１１（ＯｘＡβ１－４０）（配列番号５）：
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV （Met ７０６が酸化されている）
ＡＰＰ６７２－７１３（Ａβ１－４２）（配列番号６）：
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
ＡＰＰ６６９－７１１（配列番号７）：
VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672-709 (Aβ1-38) (SEQ ID NO: 1):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG
APP674-711 (Aβ3-40) (SEQ ID NO: 2):
EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
APP672-710 (Aβ1-39) (SEQ ID NO: 3):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV
APP672-711 (Aβ1-40) (SEQ ID NO: 4):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
OxAPP672-711 (OxAβ1-40) (SEQ ID NO: 5):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL M VGGVV (Met 706 is oxidized)
APP672-713 (Aβ1-42) (SEQ ID NO: 6):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
APP669-711 (SEQ ID NO: 7):
VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
　アミロイド前駆タンパク質（ＡＰＰ）は、膜一回貫通タンパク質で７７０残基のアミノ酸から成る。アミロイド前駆タンパク質（ＡＰＰ）は、βセクレターゼとγセクレターゼによってタンパク質分解を受け、タンパク質分解によってアミロイド・ベータペプチド（Ａβ）が産生される。ＡＰＰ６７２－７１３とＡβ１－４２とは同じペプチドを表す（配列番号６）。また、ＡＰＰ６７２－７１１とＡβ１－４０とは同じペプチドを表す（配列番号４）。本発明において、標的ポリペプチドには、上記例示のもの以外にも、種々のＡβ関連ペプチドが限定されることなく含まれる。また、本発明は、Ａβ及びＡβ関連ペプチド以外の各種ポリペプチドにも適用される。 Amyloid precursor protein (APP) is a single-membrane transmembrane protein consisting of 770 amino acids. Amyloid precursor protein (APP) is proteolyzed by β-secretase and γ-secretase, and amyloid beta peptide (Aβ) is produced by proteolysis. APP672-713 and Aβ1-42 represent the same peptide (SEQ ID NO: 6). APP672-711 and Aβ1-40 represent the same peptide (SEQ ID NO: 4). In the present invention, the target polypeptide includes, without limitation, various Aβ-related peptides other than those exemplified above. The present invention is also applicable to various polypeptides other than Aβ and Aβ-related peptides.
　本発明において、標的ポリペプチドは生体試料に含まれる。生体試料には、血液、脳脊髄液（ＣＳＦ）、尿、体分泌液、唾液、及び痰などの体液；　及び糞便が含まれる。血液試料には、全血、血漿及び血清などが含まれる。血液試料は、個体から採取された全血を、適宜処理することによって調製することができる。採取された全血から血液試料の調製を行う場合に行われる処理としては特に限定されず、臨床学的に許容されるいかなる処理が行われてよい。例えば遠心分離などが行われうる。また、血液試料は、その調製工程の中途段階又は調製工程の後段階において、適宜冷凍など低温下での保存が行われたものであってよい。なお、本発明において生体試料は、由来元の個体に戻すことなく破棄される。血液試料を対象試料とすることは、試料採取が固体や脳脊髄液である場合に対して低侵襲であること、また、一般の健康診断や人間ドック等における種々の疾患のスクリーニングのための対象試料であることからも好ましい。 In the present invention, the target polypeptide is contained in a biological sample. Biological samples include body fluids such as blood, cerebrospinal fluid (CSF), urine, body secretions, saliva, and sputum; and feces. Blood samples include whole blood, plasma and serum. A blood sample can be prepared by appropriately treating whole blood collected from an individual. The treatment performed when preparing a blood sample from the collected whole blood is not particularly limited, and any clinically acceptable treatment may be performed. For example, centrifugation can be performed. Further, the blood sample may be stored at a low temperature such as freezing as appropriate in the middle of the preparation process or at a later stage of the preparation process. In the present invention, the biological sample is discarded without returning to the original individual. Targeting a blood sample as a target sample is less invasive than when the sample is a solid or cerebrospinal fluid, and is also a target sample for screening for various diseases in general health checkups and medical checkups. This is also preferable.
　本発明のポリペプチドのサンドイッチ型免疫測定用キットは、上記のサンドイッチ型免疫測定を行うためのものであって、
　標的ポリペプチドを認識可能な抗原結合部位を持つ第１の抗体、
　前記標的ポリペプチドを認識可能な抗原結合部位であって前記第１の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ第２の抗体、及び
　前記標的ポリペプチドに結合可能な抗原親和性物質
を含む。
　キットには、サンドイッチ型免疫測定法の操作に用いる各種成分、例えば、試料溶液調製用の希釈液、洗浄溶液等を含ませることができる。 The polypeptide sandwich sandwich immunoassay kit of the present invention is for performing the sandwich immunoassay described above,
A first antibody having an antigen binding site capable of recognizing a target polypeptide;
A second antibody having an antigen binding site capable of recognizing the target polypeptide and different from the antigen binding site of the first antibody; and an antigen affinity substance capable of binding to the target polypeptide including.
The kit can contain various components used for the operation of the sandwich immunoassay, for example, a diluent for preparing a sample solution, a washing solution, and the like.
　以下に実施例を示し、本発明を具体的に説明するが、本発明はこれら実施例に制限されるものではない。以下において％で示される物の量は、特に断りがない場合は、その物が固体である場合は重量基準、液体である場合は体積基準で示されている。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In the following, unless otherwise specified, the amount of a product indicated by% is indicated on a weight basis when the product is a solid, and on a volume basis when the product is a liquid.
［実験例１：APP669-711のサンドイッチELISAの操作方法］
　まず、実験例１では、APP669-711のサンドイッチELISAの基本操作方法について以下に示す。実施例１～４の各操作はこの操作方法に基づいて行った。 [Experimental example 1: APP669-711 sandwich ELISA operation method]
First, in Experimental Example 1, a basic operation method of the sandwich ELISA of APP669-711 will be described below. Each operation in Examples 1 to 4 was performed based on this operation method.
（Ｎ末端認識抗体）
　APP669-711のＮ末端を認識する抗体の作成をＩＴＭ社へ依頼し、３クローン（20-1A、24-6G、34-6E）を取得した。 (N-terminal recognition antibody)
ITM was requested to produce an antibody that recognizes the N-terminus of APP669-711, and 3 clones (20-1A, 24-6G, 34-6E) were obtained.
（Ｎ末端認識抗体の固相化とブロッキング）
　Ｎ末端認識抗体を炭酸ナトリウム緩衝液（pH9.6）で20 μg/mLの濃度に希釈し、得られた抗体希釈溶液を96 well plateの各wellに50 μLずつ加え、４℃、２時間インキュベーションすることでＮ末端認識抗体を固相化した。Plate中の抗体希釈溶液を除去し、20% Blocking One（ナカライテスク）を各wellに100 μLずつ入れて、４℃、２時間インキュベートしてブロッキングを行った。 (Solidification and blocking of N-terminal recognition antibody)
The N-terminal recognition antibody is diluted with sodium carbonate buffer (pH 9.6) to a concentration of 20 μg / mL, and 50 μL of the resulting antibody diluted solution is added to each well of a 96-well plate and incubated at 4 ° C. for 2 hours. As a result, the N-terminal recognition antibody was immobilized. The antibody dilution solution in the plate was removed, and 100 μL of 20% Blocking One (Nacalai Tesque) was added to each well, followed by incubation at 4 ° C. for 2 hours for blocking.
（サンプル溶液の調製）
　測定対象ペプチドAPP669-711を5% Blocking One in PBSTで所定の濃度にしてサンプル溶液を調製した。また、抗原親和性物質として用いる抗体を5% Blocking One in PBSTで任意の濃度に調製し、サンプル溶液に等量添加した。 (Preparation of sample solution)
A sample solution was prepared by setting the peptide to be measured APP669-711 to a predetermined concentration with 5% Blocking One in PBST. In addition, an antibody used as an antigen affinity substance was adjusted to an arbitrary concentration with 5% Blocking One in PBST, and an equal amount was added to the sample solution.
（サンプル溶液の添加）
　Plate中の20% Blocking Oneを除去し、PBST 300 μLで３回洗浄した。前記サンプル溶液をplateの各wellに50 μLずつ入れて、４℃、１時間インキュベートした。 (Addition of sample solution)
20% Blocking One in the plate was removed, and the plate was washed 3 times with 300 μL of PBST. 50 μL of the sample solution was placed in each well of the plate and incubated at 4 ° C. for 1 hour.
（Ｃ末端認識するHRP標識抗体の添加）
　Human βAmyloid(1-40)ELISA Kit（和光純薬）に含まれるＣ末端を認識するHRP標識抗体（クローンBA27）溶液を5% Blocking One in PBSTで５倍希釈した。Plate中のサンプル溶液を除去し、PBST 300 μLで３回洗浄した。５倍希釈したHRP標識抗体（BA27）溶液を50 μLずつ入れて、４℃、１時間でインキュベートした。Plate中の溶液を除去し、PBST 300 μLで５回洗浄した。 (Addition of HRP-labeled antibody that recognizes C-terminal)
An HRP-labeled antibody (clone BA27) solution that recognizes the C-terminus contained in Human βAmyloid (1-40) ELISA Kit (Wako Pure Chemical Industries) was diluted 5-fold with 5% Blocking One in PBST. The sample solution in the plate was removed and washed 3 times with 300 μL of PBST. 50 μL each of HRP-labeled antibody (BA27) solution diluted 5-fold was added and incubated at 4 ° C. for 1 hour. The solution in the plate was removed and washed 5 times with 300 μL PBST.
（酵素標識抗体添加と吸光度測定）
　ELISA POD基質TMBキット（ナカライ）を100 μL ずつ加えて、暗所で１５分インキュベーションして発色させた。２Ｎ硫酸を100 μL ずつ加えて発色反応を止めた。マイクロプレートリーダーで主波長４５０ｎｍ／副波長６５０ｎｍの吸光度（Absorbance）を測定した。 (Enzyme-labeled antibody addition and absorbance measurement)
100 μL of ELISA POD substrate TMB kit (Nacalai) was added, and color was developed by incubation for 15 minutes in the dark. The color reaction was stopped by adding 100 μL of 2N sulfuric acid. Absorbance at a main wavelength of 450 nm / subwavelength of 650 nm was measured with a microplate reader.
［実施例１］
［抗Aβ抗体4G8添加によるAPP669-711 サンドイッチELISA反応性評価］
　アミロイドβ（Aβ）1-40は空間距離的にＣ末端とＮ末端が近い構造を取っている（非特許文献３）。Aβ1-40のＮ末端から３アミノ酸長くなっただけのAPP669-711もＣ末端とＮ末端が近いと考えられる。そこで、APP669-711に結合可能な抗体をサンプルに添加することにより、コンフォメーションが変化し、サンドイッチELISAの反応性が向上するかどうかを検証した。APP669-711に結合可能な抗体（抗原親和性物質）として、抗Aβ抗体4G8を用いた。 [Example 1]
[APP669-711 sandwich ELISA reactivity evaluation by adding anti-Aβ antibody 4G8]
Amyloid β (Aβ) 1-40 has a structure in which the C-terminal and N-terminal are close in spatial distance (Non-patent Document 3). APP669-711, which is only 3 amino acids longer than the N-terminus of Aβ1-40, is considered to be close to the C-terminus and N-terminus. Thus, it was verified whether adding a antibody capable of binding to APP669-711 to the sample would change the conformation and improve the reactivity of the sandwich ELISA. Anti-Aβ antibody 4G8 was used as an antibody (antigen affinity substance) that can bind to APP669-711.
　APP669-711のサンプル溶液に対して、抗原親和性物質として抗Aβ抗体4G8を添加することで、APP669-711濃度が0 fmol/mL、15.625 fmol/mL、31.25 fmol/mL、62.5 fmol/mL、125 fmol/mL、250 fmol/mL、500 fmol/mL、1000 fmol/mL、抗Aβ抗体4G8濃度が0, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 ng/mLとなるように調製した。それらの溶液を、APP669-711 サンドイッチELISAで測定した。クローン4G8のエピトープはAβ18-22である。固相化抗体としてはＮ末端認識抗体クローン34-6Eを用いた。その結果、4G8添加なし（0 ng/mL）と比べて、抗Aβ抗体4G8を添加することにより吸光度が増加した（図１）。添加濃度100 ng/mLまでは濃度依存的に増加し、それを超えると徐々に低下した（図２）。 By adding anti-Aβ antibody 4G8 as an antigen affinity substance to the sample solution of APP669-711, the concentration of APP669-711 is 0 fmol / mL, 15.625 fmol / mL, 31.25 fmol / mL, 62.5 fmol / mL, 125 fmol / mL, 250 fmol / mL, 500 fmol / mL, 1000 fmol / mL, anti-Aβ antibody 4G8 concentration is 0, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 ng / mL It prepared so that it might become. These solutions were measured by APP669-711 sandwich ELISA. The epitope of clone 4G8 is Aβ18-22. N-terminal recognition antibody clone 34-6E was used as the immobilized antibody. As a result, the absorbance increased when anti-Aβ antibody 4G8 was added compared to the case where 4G8 was not added (0 ng / mL) (FIG. 1). The concentration increased depending on the concentration up to an addition concentration of 100 ng / mL, and gradually decreased when the concentration was exceeded (FIG. 2).
　吸光度が増加した理由として、抗Aβ抗体4G8がプレートに固相化され、それがAPP669-711を捕捉し、Ｃ末端認識HRP標識抗体とサンドイッチしたことで反応した可能性も考えられる。それを検証するため、抗Aβ抗体4G8は反応するが、Ｎ末端認識抗体34-6E では反応しないAβ1-40（0～1000 fmol/mL）のサンプル溶液を用いて、抗Aβ抗体4G8を10000 ng/mL添加したときのAPP669-711 サンドイッチELISA反応性を評価した。抗Aβ抗体4G8のエピトープはAβ18-22であるため、仮に抗Aβ抗体4G8が固相化されたら反応性を示す。しかし、本測定結果では反応を示していなかった（図３）。つまり、抗Aβ抗体4G8が固相化され、それがAPP669-711を捕捉したことで反応性が増したわけではないことが確認された。以上の結果から、抗Aβ抗体4G8をサンプル溶液に添加することで、APP669-711への特異性を保持したまま、サンドイッチELISAの反応性を向上させることが判明した。 The reason why the absorbance increased may be that anti-Aβ antibody 4G8 was immobilized on the plate, captured by APP669-711, and reacted with a C-terminal recognition HRP-labeled antibody. To verify this, anti-Aβ antibody 4G8 reacts, but N-terminal recognition antibody 34-6E does not react with a sample solution of Aβ1-40 (0 to 1000 fmol / mL). APP669-711 sandwich ELISA reactivity when / mL was added was evaluated. Since the epitope of anti-Aβ antibody 4G8 is Aβ18-22, if anti-Aβ antibody 4G8 is immobilized, it exhibits reactivity. However, no response was shown in this measurement result (FIG. 3). That is, it was confirmed that the reactivity was not increased by immobilizing the anti-Aβ antibody 4G8 and capturing APP669-711. From the above results, it was found that adding the anti-Aβ antibody 4G8 to the sample solution improves the reactivity of the sandwich ELISA while retaining the specificity to APP669-711.
　図１は、抗Aβ抗体4G8添加によるAPP669-711 ELISAの検量線を示すグラフである。横軸はAPP669-711の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。抗Aβ抗体4G8の濃度毎に検量線が示されている。 FIG. 1 is a graph showing a calibration curve of APP669-711 ELISA with anti-Aβ antibody 4G8 added. The horizontal axis represents the concentration of APP669-711 (fmol / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. A calibration curve is shown for each concentration of anti-Aβ antibody 4G8.
　図２は、抗Aβ抗体4G8添加によるAPP669-711 ELISAの反応性を示すグラフである。横軸は抗Aβ抗体4G8の濃度(ng/mL)、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。APP669-711の濃度毎に点を線で繋いでいる。 FIG. 2 is a graph showing the reactivity of APP669-711 ELISA with the addition of anti-Aβ antibody 4G8. The horizontal axis represents the concentration (ng / mL) of anti-Aβ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. The dots are connected by a line for each concentration of APP669-711.
　図３は、抗Aβ抗体4G8添加時におけるAβ1-40に対するAPP669-711 ELISAの反応性を示すグラフである。横軸はAβ1-40の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。抗Aβ抗体4G8の添加量は10000 ng/mLであった。 FIG. 3 is a graph showing the reactivity of APP669-711 ELISA against Aβ1-40 when anti-Aβ antibody 4G8 is added. The horizontal axis indicates the concentration of Aβ1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. The amount of anti-Aβ antibody 4G8 added was 10,000 ng / mL.
［実施例２：Ｎ末端認識抗体のクローンを変えたときの抗Aβ抗体4G8添加効果の検証］
　固相化抗体として使用するＮ末端認識抗体のクローンを変えたときに、抗Aβ抗体4G8添加の効果があるかどうかを検証するための実験を行った。 [Example 2: Verification of effect of addition of anti-Aβ antibody 4G8 when N-terminal recognition antibody clone is changed]
An experiment was conducted to verify whether the effect of adding anti-Aβ antibody 4G8 was effective when the clone of the N-terminal recognition antibody used as a solid-phased antibody was changed.
　APP669-711のサンプル溶液に対して、抗原親和性物質として抗Aβ抗体4G8を添加することで、APP669-711濃度が500 fmol/mL、抗Aβ抗体4G8濃度が0, 10, 30, 100, 300, 1000, 3000 ng/mLとなるように調製した。それらの溶液を、APP669-711 サンドイッチELISAで測定した。固相化抗体はＮ末端認識抗体の３クローン20-1A、34-6G及び34-6Eをそれぞれ用いた。その結果、３クローン全てにおいて吸光度の増加が確認され、各クローンにおいて最も高い吸光度を示した抗Aβ抗体4G8添加濃度は共通して100 ng/mLだった（図４）。つまり、どのクローンでも最適な抗Aβ抗体4G8添加濃度は同じであった。 By adding anti-Aβ antibody 4G8 as an antigen affinity substance to the sample solution of APP669-711, APP669-711 concentration is 500 fmol / mL, anti-Aβ antibody 4G8 concentration is 0, 10, 30, 100, 300 , 1000, 3000 ng / mL. These solutions were measured by APP669-711 sandwich ELISA. As the solid-phase antibody, three clones 20-1A, 34-6G and 34-6E of N-terminal recognition antibodies were used, respectively. As a result, an increase in absorbance was confirmed in all three clones, and the concentration of anti-Aβ antibody 4G8 that showed the highest absorbance in each clone was 100 ng / mL in common (FIG. 4). That is, the optimal anti-Aβ antibody 4G8 addition concentration was the same for all clones.
　なお、各クローンを固相化したAPP669-711 サンドイッチELISAにおいて、抗Aβ抗体4G8を3000 ng/mL添加したときのAβ1-40（0～1000 fmol/mL）のサンプル溶液への反応性を評価したが、反応は認められなかった（図５）。つまり、抗Aβ抗体4G8が固相化され、それがAPP669-711を捕捉することで反応性が増したわけではないことが確認された。 In addition, in the APP669-711 sandwich ELISA in which each clone was immobilized, the reactivity of Aβ1-40 (0 to 1000 fmol / mL) to the sample solution when anti-Aβ antibody 4G8 was added at 3000 μng / mL was evaluated. However, no reaction was observed (FIG. 5). That is, it was confirmed that the anti-Aβ antibody 4G8 was immobilized and the reactivity was not increased by capturing APP669-711.
　図４は、Ｎ末端認識抗体３クローンを用いたAPP669-711 ELISAにおける抗Aβ抗体4G8添加効果を示すグラフである。横軸は抗Aβ抗体4G8の濃度(ng/mL)、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。Ｎ末端認識抗体のクローン毎に点を線で繋いでいる。 FIG. 4 is a graph showing the effect of addition of anti-Aβ antibody 4G8 in APP669-711 ELISA using 3 N-terminal recognition antibody clones. The horizontal axis represents the concentration (ng / mL) of anti-Aβ antibody 4G8, and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. The dots are connected by a line for each clone of the N-terminal recognition antibody.
　図５は、抗Aβ抗体4G8添加時におけるAβ1-40に対するAPP669-711 ELISAの反応性を示すグラフである。Ｎ末端認識抗体として、３クローンを各々用いた。横軸はAβ1-40の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。抗Aβ抗体4G8の添加量は3000 ng/mLであった。 FIG. 5 is a graph showing the reactivity of APP669-711 ELISA against Aβ1-40 when anti-Aβ antibody 4G8 is added. Three clones were used as N-terminal recognition antibodies. The horizontal axis indicates the concentration of Aβ1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. The amount of anti-Aβ antibody 4G8 added was 3000 ng / mL.
［実施例３：添加する抗Aβ抗体を変えたときのAPP669-711 サンドイッチELISA反応性向上効果の検証］
　サンプルに添加する抗Aβ抗体のクローンを変えたときに、APP669-711 サンドイッチELISAの反応性が向上する効果があるかどうかを検証するための実験を行った。 [Example 3: Verification of APP669-711 sandwich ELISA reactivity improvement effect when the added anti-Aβ antibody is changed]
An experiment was conducted to verify whether the anti-Aβ antibody clone added to the sample had an effect of improving the reactivity of the APP669-711 sandwich ELISA.
　APP669-711のサンプル溶液に対して、抗原親和性物質として抗Aβ抗体４クローン（4G8、6E10、BAM90.1、又はNAB228）を添加することで、APP669-711濃度が500 fmol/mL、抗Aβ抗体4G8濃度が0, 10, 30, 100, 300, 1000, 3000 ng/mLとなるように調製した。それらの溶液を、APP669-711 サンドイッチELISAで測定した。クローン6E10のエピトープはAβ3-8、BAM90.1はAβ20-23であり、NAB228はAβ1-11の中の一部がエピトープとなる。固相化抗体はＮ末端認識抗体クローン34-6Eを用いた。測定の結果、添加した抗Aβ抗体４クローン全てにおいて吸光度の増加が認められた（図６）。各クローンで最も高い吸光度を示した濃度は、4G8では100 ng/mL、6E10では300 ng/mL、BAM90.1では1000 ng/mL、NAB228では3000 ng/mLであった。つまり、クローンによって最適な濃度は異なっていた。 By adding 4 clones of anti-Aβ antibody (4G8, 6E10, BAM90.1, or NAB228) as an antigen affinity substance to the sample solution of APP669-711, the concentration of APP669-711 is 500 fmol / mL, anti-Aβ Antibody 4G8 concentrations were adjusted to 0, 10, 30, 100, 300, 1000, and 3000 ng / mL. These solutions were measured by APP669-711 sandwich ELISA. The epitope of clone 6E10 is Aβ3-8, BAM90.1 is Aβ20-23, and NAB228 is a part of Aβ1-11. As the immobilized antibody, N-terminal recognition antibody clone 34-6E was used. As a result of the measurement, an increase in absorbance was observed in all 4 clones of the added anti-Aβ antibody (FIG. 6). The concentrations that showed the highest absorbance in each clone were 100 ng / mL for 4G8, 300 ng / mL for 6E10, 1000 ng / mL for BAM90.1, and 3000 ng / mL for NAB228. In other words, the optimum concentration was different depending on the clone.
　なお、APP669-711 サンドイッチELISAにおいて、各クローンの抗Aβ抗体を3000 ng/mL添加したときのAβ1-40（0～1000 fmol/mL、具体的には、0 fmol/mL、500 fmol/mL、1000 fmol/mL）のサンプル溶液への反応性を評価したが、反応は認められなかった（図７）。つまり、どのクローンを添加した場合においても、固相化され、APP669-711を捕捉することで反応性が増したわけではないことが確認された。 In APP669-711 sandwich ELISA, Aβ1-40 (0 to 1000 fmol / mL, specifically 0 fmol / mL, 500 fmol / mL, when 3000 ng / mL of anti-Aβ antibody of each clone was added, The reactivity to the sample solution (1000 fmol / mL) was evaluated, but no reaction was observed (FIG. 7). That is, it was confirmed that in any case of adding any clone, the reactivity was not increased by capturing the APP669-711 by immobilizing it.
　図６は、APP669-711 ELISAにおける抗Aβ抗体の４クローンの添加効果を示すグラフである。横軸は抗Aβ抗体の添加濃度(ng/mL)、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。抗Aβ抗体のクローン毎に点を線で繋いでいる。 FIG. 6 is a graph showing the effect of adding four clones of anti-Aβ antibody in APP669-711 ELISA. The horizontal axis represents the concentration of added anti-Aβ antibody (ng / mL), and the vertical axis represents the absorbance (Absorbance) at 450 nm / 650 nm. The dots are connected by a line for each clone of the anti-Aβ antibody.
　図７は、抗Aβ抗体添加時におけるAβ1-40に対するAPP669-711 ELISAの反応性を示すグラフである。抗Aβ抗体は４クローンを用いた。横軸はAβ1-40の濃度（fmol/mL）、縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。抗Aβ抗体の添加量は3000 ng/mLであった。 FIG. 7 is a graph showing the reactivity of APP669-711 ELISA against Aβ1-40 when anti-Aβ antibody is added. Four clones were used as the anti-Aβ antibody. The horizontal axis indicates the concentration of Aβ1-40 (fmol / mL), and the vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. The amount of anti-Aβ antibody added was 3000 ng / mL.
［実施例４：Aβ1-40 サンドイッチELISAにおける抗Aβ抗体添加効果の検証］
　Human βAmyloid(1-40)ELISA Kit（和光純薬）を用いて、Aβ1-40 サンドイッチELISA についても、抗Aβ抗体を添加して反応性が向上するかどうかを検証するための実験を行った。 [Example 4: Verification of anti-Aβ antibody addition effect in Aβ1-40 sandwich ELISA]
Using the Human βAmyloid (1-40) ELISA Kit (Wako Pure Chemical Industries), an experiment was conducted to verify whether anti-Aβ antibody was added to improve the reactivity of the Aβ1-40 sandwich ELISA.
　サンプルはHuman βAmyloid(1-40)ELISA Kit内に含まれているstandardと、AnaSpec社から購入したAβ1-40とした。それぞれのサンプルのAβ1-40 (Standard in kit、又はAnaSpec product)に、抗Aβ抗体クローン4G8又は6E10を添加することで、Aβ1-40濃度が50 fmol/mLとなるように、4G8濃度が100 ng/mL又は6E10濃度が300 ng/mLとなるように調製した。それらの溶液をHuman βAmyloid(1-40)ELISA Kitの取り扱い説明書のプロトコールに従って操作を行い、吸光度を測定した。また、基準として、4G8も6E10も添加しないで操作を行い、吸光度を測定した(図８において、"non-spiked"と表記されている)。その結果、クローン4G8添加では基準に対して吸光度の増加が認められたが、クローン6E10添加では基準に対して吸光度に変化を与えなかった。6E10はエピトープがAβ3-8であり、Aβ1-40のＮ末端に近いため、ELISAの固相化抗体として使用しているＮ末端認識抗体が立体構造的に6E10の結合を妨害していると考えられる。一方、クローン4G8はエピトープがAβ18-22であり、Ｎ末端認識抗体とは空間距離的に離れているため、Aβ1-40 サンドイッチELISAの反応性向上に寄与したと考えられる。 The samples were standard contained in Human βAmyloid (1-40) ELISA Kit and Aβ1-40 purchased from AnaSpec. By adding anti-Aβ antibody clone 4G8 or 6E10 to Aβ1-40 (Standard inkit or AnaSpec product) of each sample, 4G8 concentration is 100 ng so that Aβ1-40 concentration is 50 fmol / mL / mL or 6E10 concentration was adjusted to 300 ng / mL. These solutions were operated according to the protocol in the instruction manual of Human βAmyloid (1-40) ELISA Kit, and the absorbance was measured. Further, as a reference, the operation was performed without adding 4G8 or 6E10, and the absorbance was measured (indicated as “non-spiked” in FIG. 8). As a result, when clone 4G8 was added, an increase in absorbance relative to the reference was observed, but when clone 6E10 was added, there was no change in absorbance relative to the reference. Since 6E10 has an epitope of Aβ3-8 and is close to the N-terminus of Aβ1-40, it is considered that the N-terminal recognition antibody used as an immobilized antibody in ELISA is structurally hindering the binding of 6E10. It is done. On the other hand, since clone 4G8 has an epitope of Aβ18-22 and is spaced apart from the N-terminal recognition antibody by spatial distance, it is considered that it contributed to the improvement of the reactivity of Aβ1-40 sandwich ELISA.
　このため、抗原親和性物質として添加する抗Aβ抗体については、サンドイッチELISAで用いる抗体が認識するエピトープからある程度空間距離的に離れている部位をエピトープとする抗体を選択する必要があると考えられる。 For this reason, it is considered necessary to select an anti-Aβ antibody to be added as an antigen-affinity substance, which has an epitope that is located at a certain spatial distance from the epitope recognized by the antibody used in the sandwich ELISA.
　図８は、Aβ1-40 ELISAにおける抗Aβ抗体4G8及び6E10の添加効果を示すグラフである。縦軸は４５０ｎｍ／６５０ｎｍの吸光度（Absorbance）を示す。サンプルとして、ELISA Kit内に含まれているstandardと、AnaSpec社のAβ1-40を用いた。 FIG. 8 is a graph showing the effect of addition of anti-Aβ antibodies 4G8 and 6E10 in Aβ1-40 ELISA. The vertical axis indicates the absorbance (Absorbance) at 450 nm / 650 nm. As a sample, standard included in ELISA Kit and Aβ1-40 of AnaSpec were used.
　以上の実施例１～４の結果から、分析対象ポリペプチドであるAPP669-711やAβ1-40に対して結合する抗体を添加することにより、それぞれに対応するサンドイッチELISAの反応性が向上する効果があることが判明した。 From the results of Examples 1 to 4 described above, by adding antibodies that bind to APP669-711 and Aβ1-40, which are polypeptides to be analyzed, the effect of improving the reactivity of the corresponding sandwich ELISA can be obtained. It turned out to be.
　以上の各実施例では、分析対象ポリペプチドがAPP669-711及びAβ1-40である例を示した。本発明は、これら以外のポリペプチドやタンパク質を分析対象とするサンドイッチELISAについても有用である。また、本発明は、ELISA法に限らず、他の標識を用いるサンドイッチ法についても同様に適用できる。 In each of the above examples, the analysis target polypeptides are APP669-711 and Aβ1-40. The present invention is also useful for sandwich ELISAs that analyze polypeptides and proteins other than these. Further, the present invention is not limited to the ELISA method, and can be similarly applied to a sandwich method using other labels.

Claims (10)

  1. 　試料中の標的ポリペプチドを認識可能な抗原結合部位を持つ第１の抗体、及び前記標的ポリペプチドを認識可能な抗原結合部位であって前記第１の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ第２の抗体を用いたポリペプチドのサンドイッチ型免疫測定法において、試料に標的ポリペプチドに結合可能な抗原親和性物質を添加することを含む、免疫測定法。 A first antibody having an antigen-binding site capable of recognizing a target polypeptide in the sample, and an antigen-binding site capable of recognizing the target polypeptide, which is different from the antigen-binding site of the first antibody A polypeptide sandwich-type immunoassay method using a second antibody having a site, comprising adding an antigen affinity substance capable of binding to a target polypeptide to a sample.
  2. 　前記標的ポリペプチドを含む試料に前記抗原親和性物質を添加し、その後、前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体と反応させるか、又は、
    　前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体と反応させている時に、反応系に前記抗原親和性物質を添加するか、又は、
    　前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体のいずれか一方と反応させ、その後、反応系に前記抗原親和性物質を添加し、その後、前記標的ポリペプチドを前記第１の抗体及び前記第２の抗体のいずれか他方と反応させる、請求項１に記載の免疫測定法。     Adding the antigen affinity substance to a sample containing the target polypeptide, and then reacting the target polypeptide with the first antibody and the second antibody, or
    When the target polypeptide is reacted with the first antibody and the second antibody, the antigen affinity substance is added to the reaction system, or
    The target polypeptide is reacted with one of the first antibody and the second antibody, and then the antigen affinity substance is added to a reaction system, and then the target polypeptide is converted into the first antibody. The immunoassay method according to claim 1, wherein the immunoassay is reacted with any one of the second antibody and the other.
  3. 　前記抗原親和性物質が、抗体、ペプチド、低分子化合物、及び核酸アプタマーからなる群から選ばれる、請求項１又は２に記載の免疫測定法。 The immunoassay method according to claim 1 or 2, wherein the antigen affinity substance is selected from the group consisting of antibodies, peptides, low molecular compounds, and nucleic acid aptamers.
  4. 　前記サンドイッチ型免疫測定法が、酵素免疫測定法（ＥＩＡ）、放射免疫測定法（ＲＩＡ）、化学発光免疫測定法（ＣＩＡ）、蛍光免疫測定法（ＦＩＡ）、電気化学発光免疫測定法（ＥＣＬＩＡ）、生物発光免疫測定法（ＢＬＩＡ）、イムノＰＣＲ、免疫比濁法（ＴＡＩ）、及びラテックス凝集比濁法（ＬＡ）からなる群から選ばれる、請求項１～３のうちのいずれかに記載の免疫測定法。 The sandwich immunoassay includes enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CIA), fluorescence immunoassay (FIA), and electrochemiluminescence immunoassay (ECLIA). The bioluminescent immunoassay (BLIA), immuno-PCR, immunoturbidimetric assay (TAI), and latex agglutination turbidimetric assay (LA) are selected from the group consisting of Immunoassay.
  5. 　前記第１の抗体が、前記標的ポリペプチドのＮ末端認識抗体であり、前記第２の抗体が、前記標的ポリペプチドのＣ末端認識抗体である、請求項１～４のうちのいずれかに記載の免疫測定法。 The first antibody is an N-terminal recognition antibody for the target polypeptide, and the second antibody is a C-terminal recognition antibody for the target polypeptide. Immunoassay.
  6. 　前記抗原親和性物質が、前記標的ポリペプチドのＮ末端付近には作用せず、且つ、前記標的ポリペプチドのＣ末端付近には作用しないものである、請求項５に記載の免疫測定法。 The immunoassay method according to claim 5, wherein the antigen affinity substance does not act near the N-terminus of the target polypeptide and does not act near the C-terminus of the target polypeptide.
  7. 　前記抗原親和性物質は、前記標的ポリペプチドのＮ末端から４残基部位からＣ末端から４残基部位までの中間部位に作用するものである、請求項５又は６に記載の免疫測定法。 The immunoassay method according to claim 5 or 6, wherein the antigen affinity substance acts on an intermediate site from the 4-residue site from the N-terminal to the 4-residue site from the C-terminal of the target polypeptide.
  8. 　前記標的ポリペプチドが、Ａβ及びＡβ関連ペプチドからなる群から選ばれる、請求項１～７に記載の免疫測定法。 The immunoassay method according to any one of claims 1 to 7, wherein the target polypeptide is selected from the group consisting of Aβ and an Aβ-related peptide.
  9. 　前記試料が、血液、脳脊髄液、尿、糞便、及び、体分泌液からなる群から選ばれる生体由来試料である、請求項１～８に記載の免疫測定法。 The immunoassay method according to any one of claims 1 to 8, wherein the sample is a biological sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, and body secretions.
  10. 　標的ポリペプチドを認識可能な抗原結合部位を持つ第１の抗体、
    　前記標的ポリペプチドを認識可能な抗原結合部位であって前記第１の抗体が持つ抗原結合部位とは異なる抗原結合部位を持つ第２の抗体、及び
    　前記標的ポリペプチドに結合可能な抗原親和性物質
    を含む、ポリペプチドのサンドイッチ型免疫測定用キット。     A first antibody having an antigen binding site capable of recognizing a target polypeptide;
    A second antibody having an antigen binding site capable of recognizing the target polypeptide and different from the antigen binding site of the first antibody; and an antigen affinity substance capable of binding to the target polypeptide A sandwich type immunoassay kit for polypeptides, comprising:
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