WO2019167128A1 - Immunoessai en sandwich - Google Patents

Immunoessai en sandwich 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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English (en)
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/fr
Priority to CN201980015355.9A priority patent/CN111770935A/zh
Priority to PCT/JP2019/006778 priority patent/WO2019167830A1/fr
Priority to EP19760318.6A priority patent/EP3760640A4/fr
Priority to US16/975,559 priority patent/US20210155680A1/en
Priority to JP2020503468A priority patent/JP7434144B2/ja
Publication of WO2019167128A1 publication Critical patent/WO2019167128A1/fr
Priority to JP2021190264A priority patent/JP2022031783A/ja
Priority to JP2024023850A priority patent/JP2024056971A/ja

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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

L'invention concerne un immunoessai en sandwich dans lequel un polypeptide à analyser peut être détecté avec une sensibilité élevée et quantifié. L'invention concerne un immunoessai en sandwich pour un polypeptide, qui utilise un premier anticorps ayant un site de liaison à l'antigène capable de reconnaître un polypeptide cible dans un échantillon, et un deuxième anticorps ayant un site de liaison à l'antigène qui est différent du site de liaison à l'antigène du premier anticorps et qui est capable de reconnaître le polypeptide cible. L'immunoessai comprend l'ajout à l'échantillon d'une substance d'affinité à l'antigène capable de se lier au polypeptide cible. Le polypeptide cible n'est pas limité, et il est choisi dans le groupe constitué de peptides Aβ et de peptides apparentés à Aβ, par exemple.
PCT/JP2018/007295 2018-02-27 2018-02-27 Immunoessai en sandwich WO2019167128A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
PCT/JP2018/007295 WO2019167128A1 (fr) 2018-02-27 2018-02-27 Immunoessai en sandwich
CN201980015355.9A CN111770935A (zh) 2018-02-27 2019-02-22 特异性识别APP669-x的N末端的抗体及免疫测定法
PCT/JP2019/006778 WO2019167830A1 (fr) 2018-02-27 2019-02-22 ANTICORPS RECONNAISSANT DE MANIÈRE SPÉCIFIQUE L'EXTRÉMITÉ N-TERMINALE DE APP669-x, ET PROCÉDÉ DE DOSAGE IMMUNOLOGIQUE
EP19760318.6A EP3760640A4 (fr) 2018-02-27 2019-02-22 Anticorps reconnaissant de manière spécifique l'extrémité n-terminale de app669-x, et procédé de dosage immunologique
US16/975,559 US20210155680A1 (en) 2018-02-27 2019-02-22 ANTIBODY THAT SPECIFICALLY RECOGNIZES N TERMINUS OF APP669-x, AND IMMUNOASSAY METHOD
JP2020503468A JP7434144B2 (ja) 2018-02-27 2019-02-22 ＡＰＰ６６９－ｘのＮ末端を特異的に認識する抗体、及び免疫測定法
JP2021190264A JP2022031783A (ja) 2018-02-27 2021-11-24 ＡＰＰ６６９－ｘのＮ末端を特異的に認識する抗体、及び免疫測定法
JP2024023850A JP2024056971A (ja) 2018-02-27 2024-02-20 ＡＰＰ６６９－ｘのＮ末端を特異的に認識する抗体、及び免疫測定法

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WO2021005857A1 (fr) * 2019-07-05 2021-01-14 株式会社 島津製作所 Anticorps monoclonal dirigé contre l'amyloïde bêta, et procédé de mesure d'un peptide apparenté à l'amyloïde bêta utilisant ledit anticorps
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