Antibody set for measuring Aβ peptide, method for measuring Aβ peptide and reagent kit

Abstract

To provide a means for enabling better measurement of Aβ peptide in blood.SOLUTION: An antibody set for measuring an Aβ peptide in a blood sample by an immunoassay includes a capture antibody and a detection antibody specifically binding to the Aβ peptide. The capture antibody binds to an epitope containing an N-terminal residue of the Aβ peptide. The detection antibody is an antibody that binds to an epitope different from the epitope to which the capture antibody binds. The Aβ peptide is at least one of Aβ40 and Aβ42.SELECTED DRAWING: Figure 10C

Description

[Technical field]

[0001] The present invention relates to an antibody set for measuring Aβ peptide.The present invention relates to a method for measuring Aβ peptide.The present invention relates to a reagent kit. [Background technology]

[0002] It is known that Aβ peptides in a biological sample collected from a subject can be a biomarker for Alzheimer's disease. Among biological samples, cerebrospinal fluid (CSF) contains a relatively large amount of Aβ peptides, and a method for quantitatively measuring Aβ peptides in CSF has been established. For example, Non-Patent Document 1 describes the measurement of Aβ peptides in CSF by liquid chromatography mass spectrometry (LC-MS). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Leinenbach A. et al., Mass Spectrometry-Based Candidate Reference Measurement Procedure for Quantification of Amyloid-β in Cerebrospinal Fluid. Clinical Chemistry 60:7 987-994 (2014) Summary of the Invention [Problem to be solved by the invention]

[0004] Since collection of CSF is invasive, it places a great burden on the subject. Therefore, in recent years, a method for measuring Aβ peptides using blood as a biological sample, which is less burdensome to collect, has been developed. However, since blood contains less Aβ peptides than CSF, a method for measuring Aβ peptides with high accuracy is required. The present invention aims to provide a means for enabling accurate measurement of Aβ peptides in blood. [Means for solving the problem]

[0005] The present invention provides an antibody set for measuring Aβ peptide in a blood sample by immunoassay, the antibody set comprising a capture antibody and a detection antibody that specifically bind to Aβ peptide, the capture antibody being an antibody that binds to an epitope that includes the N-terminal residue of Aβ peptide, the detection antibody being an antibody that binds to an epitope different from the epitope to which the capture antibody binds, and the Aβ peptide being at least one of Aβ40 and Aβ42. The present invention also provides a method for measuring Aβ peptide using an antibody set comprising a capture antibody and a detection antibody that specifically bind to Aβ peptide. The present invention also provides a reagent kit comprising a capture antibody and a detection antibody that specifically bind to Aβ peptide. Effect of the Invention

[0006] The present invention makes it possible to measure Aβ peptide in blood with high accuracy. [Brief description of the drawings]

[0007] [Figure 1A] FIG. 1 is a diagram showing an example of the appearance of a reagent kit according to the present embodiment. [Figure 1B] FIG. 1 is a diagram showing an example of the appearance of a reagent kit according to the present embodiment. [Figure 1C] FIG. 1 is a diagram showing an example of the appearance of a reagent kit according to the present embodiment. [Figure 1D] FIG. 1 is a diagram showing an example of the appearance of a reagent kit according to the present embodiment. [Diagram 2] FIG. 1 shows the results of measuring Aβ peptide using LC-MS / MS. [Figure 3A] FIG. 1 shows the concentration of Aβ40 peptide in plasma of each sample. [Figure 3B] FIG. 1 shows the concentration of Aβ42 peptide in the plasma of each sample. [Figure 4A] FIG. 1 shows a calibration curve prepared based on measured values ​​detected using Aβ40 peptide of known concentrations. [Figure 4B] FIG. 1 shows a calibration curve prepared based on measured values ​​detected using Aβ42 peptide of known concentrations. [Diagram 5] FIG. 13 shows the difference in elution efficiency of Aβ peptide due to differences in the composition of the release reagent. [Figure 6A] FIG. 13 shows the difference in elution efficiency of Aβ40 peptide depending on the organic solvent of the release reagent. [Figure 6B] FIG. 13 is a graph showing the difference in elution efficiency of Aβ42 peptide depending on the organic solvent of the release reagent. [Figure 7] FIG. 13 is a diagram showing the difference in carryover amount due to the difference in the composition of the free reagent. [Figure 8A] FIG. 1 shows the amount of Aβ40 peptide when a 190 pg / ml Aβ40 peptide solution was eluted with a basic solution. [Figure 8B] FIG. 1 shows the amount of Aβ40 peptide when a 190 pg / ml Aβ40 peptide solution was eluted with an acidic solution. [Figure 8C] FIG. 1 shows the amount of Aβ42 peptide when a 103 pg / ml Aβ42 peptide solution was eluted with a basic solution. [Figure 8D] FIG. 1 shows the amount of Aβ42 peptide when a 103 pg / ml Aβ42 peptide solution was eluted with an acidic solution. [Figure 9A] FIG. 1 shows the amount of Aβ40 peptide when a 50 pg / ml Aβ40 peptide solution was eluted with a basic solution. [Figure 9B] FIG. 1 shows the amount of Aβ40 peptide when a 50 pg / ml Aβ40 peptide solution was eluted with an acidic solution. [Figure 9C] FIG. 1 shows the amount of Aβ42 peptide when a 26 pg / ml Aβ42 peptide solution was eluted with a basic solution. [Figure 9D] FIG. 1 shows the amount of Aβ42 peptide when a 26 pg / ml Aβ42 peptide solution was eluted with an acidic solution. [Figure 10A]FIG. 13 shows the results of calculating the correlation coefficient by plotting the values ​​of Aβ40 peptide in plasma samples measured using immunoprecipitation and mass spectrometry against the values ​​of Aβ40 peptide in plasma samples measured using HISCL (registered trademark)-5000. [Figure 10B] FIG. 13 shows the results of calculating the correlation coefficient by plotting the values ​​measured by immunoprecipitation and mass spectrometry for Aβ40 peptide in peptide-spiked samples against the values ​​measured by HISCL (registered trademark)-5000 for Aβ40 peptide in peptide-spiked samples. [Figure 10C] FIG. 13 shows the results of calculating the correlation coefficient by plotting the values ​​measured for Aβ40 peptide in plasma samples and peptide-spiked samples using immunoprecipitation and mass spectrometry against the values ​​measured for Aβ40 peptide in plasma samples and peptide-spiked samples using HISCL (registered trademark)-5000. [Figure 11A] FIG. 13 shows the results of calculating a correlation coefficient by plotting the values ​​of Aβ42 peptide in plasma samples measured using immunoprecipitation and mass spectrometry against the values ​​of Aβ42 peptide in plasma samples measured using HISCL (registered trademark)-5000. [Figure 11B] FIG. 13 shows the results of calculating the correlation coefficient by plotting the values ​​of Aβ42 peptide in peptide-spiked samples measured using immunoprecipitation and mass spectrometry against the values ​​of Aβ42 peptide in peptide-spiked samples measured using HISCL (registered trademark)-5000. [Figure 11C] FIG. 13 shows the results of calculating the correlation coefficient by plotting the values ​​measured for Aβ42 peptide in plasma samples and peptide-spiked samples using immunoprecipitation and mass spectrometry against the values ​​measured for Aβ42 peptide in plasma samples and peptide-spiked samples using HISCL (registered trademark)-5000. [Figure 12] FIG. 13 shows the correlation coefficient obtained by plotting the values ​​measured by immunoprecipitation and mass spectrometry for Aβ42 peptide in plasma samples against the values ​​measured by HISCL®-5000 using 6E10 antibody as a capture antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] One of the present embodiments is an antibody set for measuring Aβ peptide in a blood sample by immunoassay. The antibody set refers to a combination of multiple antibodies including at least a capture antibody and a detection antibody used in the immunoassay. The type of immunoassay is not particularly limited, and can be appropriately selected from known immunoassay methods such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation / Western blot method, and immune complex transfer method (see Japanese Patent Application Laid-Open No. 1-254868). Among them, the ELISA method is preferable. The type of ELISA method may be any of a sandwich method, a competitive method, a direct method, an indirect method, and the like, but is preferably a sandwich method. The immunoassay using the antibody set of the present embodiment may be performed using a commercially available immunoassay device such as the HISCL (registered trademark) series (Sysmex Corporation).

[0009] In this specification, the term "blood sample" includes blood samples containing Aβ peptides and blood samples suspected of containing Aβ peptides. Examples of blood samples include blood (whole blood), plasma, serum, etc. Among these, plasma and serum are preferred. The blood sample may be diluted with an appropriate aqueous medium as necessary. Such an aqueous medium is not particularly limited as long as it does not interfere with the measurement described below, and examples thereof include water, saline, and buffer solutions. The buffer solution is preferably a buffer solution having a buffering effect at a pH near neutral (e.g., a pH of 6 to 8). Examples of such a buffer solution include Good's buffer solutions such as HEPES, MES, and PIPES, Tris-buffered saline (TBS), and phosphate-buffered saline (PBS).

[0010] The origin of the blood sample is not particularly limited, and may be, for example, blood collected from a subject and plasma or serum prepared therefrom. Commercially available pooled plasma, healthy subject plasma, etc. may also be used. If necessary, labeled Aβ peptide may be added to the blood sample as an internal standard. The subject is not particularly limited, and may be, for example, a healthy subject, a subject with abnormal cognitive function, or a subject suspected of having such abnormality. Examples of cognitive abnormalities include mild cognitive impairment (MCI), Alzheimer's dementia, etc.

[0011] Aβ peptide is a polypeptide produced by processing amyloid β precursor protein (APP) with β-secretase and γ-secretase. Unless otherwise specified, Aβ peptide includes polypeptides of any length, but is usually a polypeptide consisting of 39 to 43 amino acids. As Aβ peptide, Aβ40 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV: SEQ ID NO: 1) consisting of 40 amino acid residues and Aβ42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA: SEQ ID NO: 2) consisting of 42 amino acid residues are preferable.

[0012] The Aβ peptide may be in the form of a monomer or a multimer. A multimer is also called a polymer, and is formed by physical or chemical polymerization or aggregation of a plurality of monomeric Aβ peptides. A multimer may contain a plurality of monomeric Aβ peptides, and may contain other molecules. In a multimer, the monomeric Aβ peptides do not need to be strongly bonded to each other by a covalent bond or the like. Multimers also include aggregates in which a plurality of monomeric Aβ peptides are assembled by a looser bond. Examples of Aβ peptide multimers include Aβ oligomers.

[0013] As used herein, the term "antibody" includes full-length antibodies and fragments thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, light chain, heavy chain variable region (VHH) of heavy chain antibody, reduced IgG (rIgG), single chain antibody (scFv), etc. The antibody that specifically binds to Aβ peptide may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.

[0014] The capture antibody is an antibody that specifically binds to the test substance and is fixed to a solid phase. The test substance is fixed on the solid phase by binding between the test substance and the capture antibody. The detection antibody is an antibody that specifically binds to the test substance. The detection antibody is preferably labeled with a labeling substance. The labeling substance is as described below. The detection antibody is usually not fixed to a solid phase, but the labeling substance itself may be a solid phase carrier such as a particle. The antibody set of this embodiment includes a capture antibody and a detection antibody that specifically bind to Aβ peptide. The capture antibody that specifically binds to Aβ peptide is an antibody that binds to an epitope containing the N-terminal residue of Aβ peptide. The detection antibody that specifically binds to Aβ peptide is an antibody that binds to an epitope different from the epitope to which the capture antibody binds. In the antibody set of this embodiment, the Aβ peptide is at least one of Aβ40 and Aβ42.

[0015] In this embodiment, the epitope containing the N-terminal residue of the Aβ peptide to which the capture antibody binds refers to a region containing the N-terminal residue of the Aβ peptide and consisting of a part of the amino acid sequence of the Aβ peptide. A preferred epitope of the capture antibody is contained in a region containing the N-terminal residue of the Aβ peptide and consisting of the 1st to 16th amino acid residues counting from the N-terminus of the Aβ peptide. Examples of such epitopes include a region consisting of the 1st to 5th, 1st to 6th, 1st to 7th, 1st to 8th, 1st to 9th, 1st to 10th, 1st to 11th, 1st to 12th, 1st to 13th, 1st to 14th, 1st to 15th, or 1st to 16th amino acid residues counting from the N-terminus of the Aβ peptide. Examples of antibodies that bind to such epitopes include the antibody of clone 82E1 (referred to as the 82E1 antibody), which recognizes the region of 1st to 16th amino acid residues counting from the N-terminus of Aβ peptide as an epitope, and the antibody of clone 2H4 (referred to as the 2H4 antibody), which recognizes the region of 1st to 8th amino acid residues counting from the N-terminus of Aβ peptide as an epitope. Among them, the 82E1 antibody is particularly preferred. These monoclonal antibodies are commercially available.

[0016] In this embodiment, the epitope to which the detection antibody binds is different from the epitope to which the capture antibody binds. In other words, the capture antibody and the detection antibody do not substantially compete for binding to the antigen Aβ peptide. The epitope of the detection antibody preferably includes the C-terminal residue of the Aβ peptide. The epitope including the C-terminal residue of the Aβ peptide to which the detection antibody binds refers to a region including the C-terminal residue of the Aβ peptide and consisting of a part of the amino acid sequence of the Aβ peptide. A preferred epitope of the detection antibody is included in a region including the C-terminal residue of the Aβ peptide and consisting of the 35th to 40th amino acid residues counting from the N-terminus of Aβ40 or the 36th to 42nd amino acid residues counting from the N-terminus of Aβ42. Examples of such epitopes include a region consisting of the 35th to 40th amino acid residues counting from the N-terminus of Aβ40 and a region consisting of the 36th to 40th amino acid residues counting from the N-terminus of Aβ42. Examples of antibodies that bind to an epitope containing the C-terminal residue of Aβ peptide include the antibody of clone H31L21 (referred to as H31L21 antibody), which recognizes the region of 36th to 42nd residues counting from the N-terminus of Aβ42 as an epitope, and the antibody of clone 1A10 (referred to as 1A10 antibody), which recognizes the region of 35th to 40th residues counting from the N-terminus of Aβ40 as an epitope. These monoclonal antibodies are commercially available.

[0017] A measurement method using an antibody set of a different combination from the antibody set of this embodiment is described in WO2007 / 022015A. In the method described in this document, 1A10 antibody is used as a capture antibody, and 82E1 antibody is used as a detection antibody. This document describes that the use of 82E1 antibody as a capture antibody reduces the sensitivity of Aβ detection. However, in this embodiment, high sensitivity detection of Aβ peptide is possible by using an antibody that binds to an epitope containing the N-terminal residue of Aβ peptide, such as 82E1 antibody, as a capture antibody in an immunoassay.

[0018] The capture antibody and the detection antibody included in the antibody set may each be either a monoclonal antibody or a polyclonal antibody, but are preferably monoclonal antibodies. In a preferred embodiment, both the capture antibody and the detection antibody are monoclonal antibodies.

[0019] In this embodiment, it is preferable that the capture antibody is immobilized on a solid phase in advance. The solid phase may be an insoluble carrier capable of immobilizing the antibody. The manner of immobilization of the antibody on the solid phase is not particularly limited. For example, the antibody may be directly bound to the solid phase, or the antibody may be indirectly bound to the solid phase via another substance. An example of direct binding is physical adsorption. An example of indirect binding is immobilizing a molecule that specifically binds to the antibody on the solid phase, and immobilizing the antibody on the solid phase via the binding between the molecule and the antibody. Examples of molecules that specifically bind to the antibody include protein A or G, and an antibody that specifically recognizes the antibody (secondary antibody). The antibody can also be immobilized on the solid phase using a combination of substances that are interposed between the antibody and the solid phase. Examples of such combinations of substances include combinations of biotins and avidins, haptens and anti-hapten antibodies, etc. Biotins include biotin and biotin analogues such as desthiobiotin and oxybiotin. The avidins include avidin and avidin analogues such as streptavidin and tamavidin (registered trademark). An example of a combination of a hapten and an anti-hapten antibody is a combination of a compound having a 2,4-dinitrophenyl (DNP) group and an anti-DNP antibody. For example, by using an antibody modified in advance with biotins (or a compound having a DNP group) and a solid phase to which avidins (or an anti-DNP antibody) are bound in advance, the antibody can be immobilized on the solid phase via the bond between the biotins and the avidins (or the bond between the DNP group and the anti-DNP antibody).

[0020] The material of the solid phase is not particularly limited, and can be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, etc. Examples of organic polymer compounds include latex, polystyrene, polypropylene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylic acid ester copolymer, polyvinyl acetate acrylate, etc. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, cobalt, ferrite, etc.), silica, alumina, glass, etc. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, cellulose, etc. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples include particles, microplates, microtubes, test tubes, etc. Among them, particles are preferred, and magnetic particles are particularly preferred.

[0021] The detection antibody is preferably labeled with a labeling substance. The labeling substance is not particularly limited, and may be, for example, a substance that generates a signal by itself (hereinafter, also referred to as a "signal generating substance"), or a substance that catalyzes the reaction of another substance to generate a signal. Examples of signal generating substances include fluorescent substances and radioisotopes. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioisotopes include: 125 I, 14 C. 32 Examples of the labeling substance that catalyzes the reaction of other substances to generate a detectable signal include enzymes. The labeling substance is preferably an enzyme. Examples of the enzyme include alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase, and luciferase. Among these, alkaline phosphatase is particularly preferable.

[0022] The method for detecting a signal is known in the art. In this embodiment, a measurement method may be appropriately selected according to the type of signal derived from the labeling substance. For example, when the labeling substance is an enzyme, a signal such as light or color generated by reacting with a substrate for the enzyme can be measured using a known device such as a spectrophotometer.

[0023] The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when alkaline phosphatase is used as the enzyme, the substrate can be a chemiluminescent substrate such as CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), or a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, or p-nitrophenyl phosphate. When peroxidase is used as the enzyme, examples of the substrate include chemiluminescent substrates such as luminol and its derivatives, and color-developing substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB).

[0024] When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined depending on the type of the fluorescent substance used.

[0025] As an example of immunoassay, the case of measuring Aβ peptides in a blood sample by sandwich ELISA using the antibody set of this embodiment will be described below. First, a blood sample is mixed with a capture antibody to form a complex between the Aβ peptides in the blood sample and the capture antibody. Then, a solution containing the complex is contacted with a solid phase capable of immobilizing the capture antibody to form a complex on the solid phase. Alternatively, a solid phase on which the capture antibody is immobilized in advance may be used. That is, a complex between the Aβ peptides in the blood sample and the capture antibody is formed on the solid phase by contacting the solid phase on which the capture antibody is immobilized with the blood sample. Next, a sandwich complex between the Aβ peptides, the capture antibody, and the detection antibody is formed on the solid phase by contacting the complex formed on the solid phase with a detection antibody.

[0026] The complex formed on the solid phase is then detected by a method known in the art, thereby obtaining the measured value of the Aβ peptide contained in the blood sample. For example, when an antibody labeled with a labeling substance is used as the detection antibody, the measured value of the Aβ peptide in the blood sample can be obtained by detecting the signal generated by the labeling substance. Alternatively, when a labeled secondary antibody for the detection antibody is used, the measured value of the Aβ peptide can be obtained in the same manner. The labeling substance is as described above.

[0027] As used herein, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection refers to indicating the signal intensity in stages, such as "no signal generated," "weak," "medium," and "strong." In this embodiment, it is preferable to quantitatively detect the signal intensity.

[0028] The detection result of the signal can be used as the measurement result of Aβ peptide in the blood sample. For example, when quantifying the signal intensity, the measured value of the signal intensity itself or a value obtained from the measured value can be used as the measurement result of Aβ peptide. Examples of the value obtained from the measured value of the signal intensity include a value obtained by subtracting the measured value of the negative control or the background value from the measured value. The negative control can be appropriately selected, and is, for example, a solution not containing Aβ peptide (e.g., a buffer solution).

[0029] In this embodiment, the amount or concentration of Aβ peptide may be determined by applying the measured value of the signal intensity to the calibration curve. The calibration curve can be created from the measured values ​​obtained by measuring a calibrator containing Aβ peptide at a known concentration by the immunoassay of this embodiment in the same manner as a blood sample. Specifically, the measured values ​​of Aβ peptide obtained from a plurality of calibrators are plotted on an XY plane in which the concentration of Aβ peptide in the calibrator is taken on the X-axis and the measured value (e.g., signal intensity) is taken on the Y-axis, and a calibration curve can be created by obtaining a straight line or curve by a known method such as the least squares method. A calibrator containing Aβ peptide at a known concentration can be prepared, for example, by adding synthetic Aβ peptide at an arbitrary concentration to a buffer solution not containing Aβ peptide.

[0030] In the immunoassay using the antibody set of this embodiment, B / F separation for removing unreacted free components that have not formed a complex may be performed between the formation and detection of the complex. The B / F separation will be described later.

[0031] Immunoassays using the antibody set of this embodiment may be performed using a commercially available fully automated immunoassay device. A fully automated immunoassay device is a device that, when a user sets a biological sample such as a blood sample and inputs an instruction to start measurement, automatically prepares a measurement sample and performs an immunoassay on the sample, and outputs the measurement results of the test substance. Examples of such fully automated immunoassay devices include the HISCL (registered trademark) series, such as HISCL (registered trademark)-5000 and HISCL (registered trademark)-800 (Sysmex Corporation), and HI-1000 (Sysmex Corporation).

[0032] On the other hand, Aβ peptides can be measured by a method in which Aβ peptides captured by an antibody are measured by mass spectrometry (hereinafter also referred to as the "measurement method"). This measurement method is characterized in that the Aβ peptides are liberated from the complex between the Aβ peptide and the antibody by a basic solution containing an organic solvent, and the liberated Aβ peptides are measured by mass spectrometry.

[0033] Conventionally, when a test substance is released from a test substance captured by an antibody (i.e., a complex of an antibody and a test substance), an acidic solution is used. This is because an acidic solution is generally excellent at dissociating the bond between the test substance and the antibody. For example, JP 2018-194374 A describes that Aβ peptides are released from the complex using an acidic aqueous solution of pH 1 to 4 that may contain an organic solvent. However, as shown in the examples described later, the present inventors have found that an acidic solution containing released Aβ peptides is not suitable for mass spectrometry combined with liquid chromatography. Specifically, when the acidic solution containing released Aβ peptides is used directly for liquid chromatography, a part of the input Aβ peptides remains in the flow path and column of the liquid chromatography device, and the remaining Aβ peptides are carried over to the measurement of the next sample (called carryover). When carryover occurs, accurate measurement of Aβ peptides is not possible. In order to reduce carryover, it is possible to release Aβ peptides with an acidic solution and then exchange the solvent with a basic solution. However, even if the carryover can be reduced by this method, Aβ peptides are lost due to solvent exchange, so it may be difficult to measure with high sensitivity. In contrast, the measurement method of the present embodiment using a basic solution containing an organic solvent significantly reduces the amount of Aβ peptides carried over. Therefore, even if the basic solution containing the free Aβ peptides is used directly in liquid chromatography, accurate measurement of Aβ peptides is possible, and solvent exchange is not required.

[0034] In the above measurement method, first, a blood sample is mixed with an antibody that specifically binds to Aβ peptide to form a complex between the Aβ peptide and the antibody. The complex between the Aβ peptide and the antibody can be formed by mixing the blood sample with the antibody that specifically binds to Aβ peptide.

[0035] In the above measurement method, the antibody is preferably a monoclonal antibody capable of specifically binding to Aβ peptide. Examples of such antibodies include the 82E1 antibody, the antibody of clone 6E10 (referred to as the 6E10 antibody) which recognizes the region of 3rd to 8th amino acid residues counting from the N-terminus of Aβ peptide as an epitope, the antibody of clone WO-2 (referred to as the WO-2 antibody) which recognizes the region of 4th to 10th amino acid residues counting from the N-terminus of Aβ peptide as an epitope, the 2H4 antibody, the H31L21 antibody, the antibody of clone G2-11 (referred to as the G2-11 antibody) which recognizes the region of 33rd to 42nd amino acid residues counting from the N-terminus of Aβ42 as an epitope, the antibody of clone 16C11 (referred to as the 16C11 antibody) which recognizes the region of 33rd to 42nd amino acid residues counting from the N-terminus of Aβ42 as an epitope, the antibody of clone 21F12 (referred to as the 21F12 antibody) which recognizes the region of 34th to 42nd amino acid residues counting from the N-terminus of Aβ42 as an epitope, and the 1A10 antibody. These monoclonal antibodies are commercially available.

[0036] In the above measurement method, it is preferable to use an antibody that binds to one or both of Aβ40 and Aβ42 as the antibody that specifically binds to Aβ peptide. An example of an antibody that specifically binds to Aβ40 is the antibody of clone 1A10. An example of an antibody that specifically binds to Aβ42 is the antibody of clone H31L21, G2-11, 16C11, and 21F12. An example of an antibody that binds to both Aβ40 and Aβ42 is the antibody of clone 82E1, 6E10, WO-2, and 2H4. By using these antibodies, Aβ40 and / or Aβ42 can be selectively captured among the Aβ peptides in the blood sample. In this case, at least one of Aβ40 and Aβ42 can be released in the release step of the measurement method of this embodiment, and at least one measured value of Aβ40 and Aβ42 can be obtained in the measurement step.

[0037] In the above measurement method, it is preferable to use an antibody that binds to both Aβ40 and Aβ42. In this case, Aβ40 and Aβ42 can be released in the release step of the above measurement method, and the measured values ​​of Aβ40 and Aβ42 can be obtained in the measurement step.

[0038] In the above measurement method, in order to selectively obtain the Aβ peptide captured by the antibody, it is preferable to form a complex between the Aβ peptide and the antibody on a solid phase. The complex can be formed on the solid phase by contacting a solution containing the complex with a solid phase capable of immobilizing the antibody. Alternatively, an antibody that specifically binds to the Aβ peptide may be immobilized on the solid phase in advance. The complex can be formed on the solid phase by using an antibody immobilized on the solid phase. Specifically, the complex is formed on the solid phase by mixing a solid phase on which an antibody that specifically binds to the Aβ peptide is immobilized with a blood sample. Then, the unreacted components and the solid phase are separated, and the solid phase is collected, whereby the complex can be selectively obtained. When particles are used as the solid phase, the complex formation process in the above measurement method corresponds to a general immunoprecipitation method.

[0039] Next, in the above measurement method, the Aβ peptide is released from the complex by a basic solution containing an organic solvent (hereinafter, also referred to as a "release reagent"). The release reagent is considered to have the effect of dissociating the bond between the antibody and the Aβ peptide. In a preferred embodiment, a solution containing the complex formed on the solid phase is mixed with the release reagent. As a result, the Aβ peptide is released from the complex, and the released Aβ peptide and the solid phase to which the antibody is fixed are present in the mixed solution. For example, when the solid phase is a magnetic particle, the solution containing the Aβ peptide can be recovered by separating the solid phase to which the antibody is fixed and the solution containing the Aβ peptide by centrifugation or magnetic separation.

[0040] The conditions for contacting the complex with the releasing reagent are not particularly limited, and for example, the complex may be contacted with the releasing reagent at a temperature of 4° C. to 42° C. and left to stand or stirred for 1 minute to 10 minutes. The amount of the releasing reagent used is not particularly limited, and can be appropriately determined, for example, from the range of 10 μL to 50 μL per sample.

[0041] The liberating reagent can be obtained by mixing a basic solution with an organic solvent, or by mixing water, a basic substance, and an organic solvent. The basic solution can be obtained by mixing water with a basic substance. A commercially available basic solution such as ammonia water may be used. Examples of the basic substance include substances that donate ammonium ions. Examples of the substance that donates ammonium ions include ammonia, ammonium carbonate, and ammonium bicarbonate. The basic substance in the liberating reagent may be one type, or two or more types.

[0042] Examples of the organic solvent include acetonitrile, acetone, 1-propanol, 2-propanol, hexane, ethanol, dimethyl sulfoxide, and methanol. The organic solvent in the release reagent may be one type or two or more types. In this embodiment, the release reagent preferably contains at least acetonitrile as an organic solvent, and more preferably contains only acetonitrile as an organic solvent.

[0043] The concentration of the organic solvent in the elution reagent is not particularly limited, but is preferably 20% or more, 30% or more, or 40% or more. Also, it is preferably 65% ​​or less, 60% or less, or 55% or less. In this specification, the concentration "%" of the organic solvent is always volume / volume % (v / v%).

[0044] The pH of the release reagent is not particularly limited as long as it is a pH that is recognized as basic by those skilled in the art. The pH of the release reagent is preferably 10.85 or more, 10.9 or more, 10.95 or more, 11.0 or more, 11.05 or more, 11.1 or more, 11.15 or more, 11.2 or more, 11.25 or more, 11.3 or more, 11.35 or more, or 11.4 or more. Most preferably, the pH of the release reagent is 11.4 or more. This allows the Aβ peptide to be particularly efficiently released from the complex. In addition, the pH of the release reagent is preferably 14.0 or less, 13.5 or less, 13.0 or less, 12.5 or less, 12.4 or less, 12.35 or less, 12.3 or less, 12.25 or less, 12.2 or less, 12.15 or less, 12.1 or less, 12.05 or less, or 12.0 or less.

[0045] The pH of the release reagent can be adjusted by the amount (or concentration) of the basic substance added. When ammonia or a salt thereof is used as the basic substance, the molar concentration of ammonium ions in the release reagent is preferably 5.29 mM or more, 10.57 mM or more, 26.43 mM or more, 52.85 mM or more, 105.71 mM or more, or 132.14 mM or more. In addition, the molar concentration is preferably 1586 mM or less, 1533 mM or less, or 1480 mM or less. For example, when the basic substance is ammonia, the concentration of ammonia in the release reagent is preferably 0.01% or more, 0.02% or more, 0.05% or more, 0.1% or more, 0.2% or more, or 0.25% or more. In addition, the concentration is preferably 3% or less, 2.9% or less, or 2.8% or less. In this specification, the concentration of ammonia "%" is all weight / weight % (w / w%).

[0046] The release reagent may contain additives such as stabilizers to the extent that they do not affect the release of Aβ peptide from the complex and the measurement of Aβ peptide by mass spectrometry. Examples of additives include bovine serum albumin (BSA), human albumin, ovalbumin, monosaccharides such as glucose, disaccharides such as maltose, sugar alcohols such as mannitol and sorbitol, and amino acids such as glycine. The additives may be one type or two or more types.

[0047] The above measurement method may include a washing step between the complex formation step and the release step to remove unreacted free components that do not form a complex. This washing step includes B / F (Bound / Free) separation. The unreacted free components are components that do not form a complex, and examples of such components include antibodies that have not bound to Aβ peptides. The washing method is not particularly limited, but for example, when a complex is formed on a solid phase, if the solid phase is particles, the particles can be collected by centrifugation or magnetic separation, and the supernatant can be removed to separate the complex from the unreacted free components. If the solid phase is a container such as a microplate or a microtube, the liquid containing the unreacted free components can be removed to separate the complex from the unreacted free components. After removing the unreacted free components, the solid phase that has captured the complex may be washed with an appropriate aqueous medium such as PBS.

[0048] In the above measurement method, the released Aβ peptide is measured by mass spectrometry. The mass spectrometry is not particularly limited as long as it can measure the released Aβ peptide, and any known ionization method capable of measuring Aβ peptide can be used. Examples of such ionization methods include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization (MALDI). Among these, the ESI method is particularly preferred.

[0049] The mass spectrometer used in mass spectrometry is not particularly limited and can be appropriately selected from known mass spectrometers. For example, quadrupole (Q) mass spectrometer, ion trap (IT) mass spectrometer, time of flight (TOF) mass spectrometer, Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, IT-TOF mass spectrometer, Q-TOF mass spectrometer, and triple quadrupole (QqQ) mass spectrometer can be mentioned. Among these, it is preferable to use a mass spectrometer capable of MS / MS measurement for measurement, and it is more preferable to use a triple quadrupole mass spectrometer for measurement.

[0050] In mass spectrometry, it is preferable to use liquid chromatography-mass spectrometry (LC-MS), which combines a mass spectrometer with a liquid chromatography device, and it is more preferable to use LC-MS / MS, which combines liquid chromatography with a mass spectrometer capable of MS / MS measurement.

[0051] The liquid chromatography device is not particularly limited as long as it can be connected to a mass spectrometer, and a commercially available HPLC (High Performance Liquid Chromatography) device can be used. The column connected to the liquid chromatography device is not particularly limited, and a commercially available HPLC column can be used. In this embodiment, the Aβ peptide liberated in the liberating reagent can be provided to the liquid chromatography device. Therefore, pre-measurement processing such as solvent exchange is not required.

[0052] The column is not particularly limited, but for example, a reversed-phase column can be used. Examples of the packing material of the reversed-phase column include silica-based packing materials and polymer-based packing materials. Among them, silica-based packing materials are preferred. As the reversed-phase column having a silica-based packing material, a base-resistant ODS column is more preferred.

[0053] The solvent (mobile phase) used in liquid chromatography can be a solution of the same composition as the free reagent, or a basic solution used in the free reagent. The concentration of the organic solvent in the mobile phase is not particularly limited and can be appropriately set according to the measurement conditions. Liquid chromatography can be performed using an isocratic method using a mobile phase of a single concentration, or a stepwise method or gradient method using a combination of multiple mobile phases with different compositions.

[0054] The flow rate of the mobile phase in the measurement can be appropriately set according to the properties such as the material and pressure resistance of the device and column. The flow rate of the mobile phase is preferably set to a flow rate at which mass spectrometry can be performed appropriately. In addition, the column temperature and the amount of sample introduced into the measurement device can also be appropriately set according to the measurement device and column.

[0055] In the above measurement method, the measurement may also be performed using a liquid chromatography device to obtain information about the sample. Examples of detectors used for the measurement include a UV detector, a fluorescence detector, a differential refractive index detector, an electrical conductivity detector, and an electrochemical detector.

[0056] The measurement of Aβ peptides by mass spectrometry can be appropriately set from known techniques according to the ionization method and mass spectrometer used. Among these, when a triple quadrupole mass spectrometer is used, it is preferable that Aβ peptides are measured by multiple reaction monitoring (MRM) measurement in a positive ion measurement mode.

[0057] A triple quadrupole mass spectrometer is equipped with a first quadrupole (Q1) and a third quadrupole (Q3) on either side of a collision cell (Q2). The object to be measured is ionized in the ion source to become precursor ions, and only ions having a specific mass-to-charge ratio (m / z) set in Q1 pass through a filter and are introduced into Q2. The precursor ions having a specific mass-to-charge ratio introduced into Q2 collide with an inert gas and are fragmented to become product ions. The product ions sent from Q2 to Q3 are filtered again in Q3, and only ions having a specific mass pass through the filter and are sent from Q3 to the detector, where a signal is detected. As a result, a specific product ion for a specific precursor ion is detected by the detector. Here, the combination of a specific mass-to-charge ratio set in Q1 and a specific mass-to-charge ratio set in Q3 is called an MRM transition. In MRM measurement, by setting multiple MRM transitions, multiple product ions can be detected simultaneously. This allows multiple substances contained in the object to be measured to be measured simultaneously. For example, for a sample containing Aβ40 and Aβ42, by setting the respective MRM transitions, two types of Aβ peptides can be measured simultaneously and respective measurement values ​​can be obtained.

[0058] In the MRM measurement, the measurement mode is not particularly limited, but it is preferable to use the mass spectrometer in a positive ion measurement mode. In addition, the cone voltage and collision energy in the MRM measurement are also not particularly limited, and can be appropriately set to appropriate conditions by a person skilled in the art.

[0059] The MRM transition for Aβ40 can be, for example, precursor ion / product ion=1083 / 1953.6, and the MRM transition for Aβ42 can be, for example, precursor ion / product ion=1129 / 1078.5. By setting the MRM transitions in this way, Aβ peptides can be detected with high sensitivity.

[0060] In the above measurement method, the blood sample may be mixed with a labeled internal standard substance, and the measurement value of Aβ peptide may be obtained based on the measurement value of the internal standard substance. The internal standard substance is not particularly limited as long as it is a substance capable of quantifying Aβ peptide, but is preferably an Aβ peptide labeled with a stable isotope. Examples of Aβ peptides labeled with a stable isotope include Aβ40 and Aβ42 labeled with a stable N15 isotope. The amount of the internal standard substance added is not particularly limited as long as it is a concentration that can be measured by the measurement device, and can be appropriately set according to the analytical capacity of the measurement device.

[0061] In addition to the MRM transitions for detecting Aβ peptides, MRM transitions can also be set for Aβ peptides labeled with stable isotopes as internal standards, and measurements can be obtained. The MRM transition for Aβ40 labeled with N15 stable isotope can be precursor ion / product ion=1096 / 1066.5, and the MRM transition for Aβ42 labeled with N15 stable isotope can be precursor ion / product ion=1142.5 / 1091.5.

[0062] When Aβ peptides in a blood sample are measured using the antibody set of this embodiment with a fully automated immunoassay device, the Aβ peptides can be measured with the same quantitative accuracy as when the blood sample is measured with the above-mentioned measurement method. Specifically, the correlation coefficient r calculated by linear regression analysis of the measured value X of Aβ peptides obtained by measurement with a fully automated immunoassay device using the capture antibody and detection antibody of the antibody set of this embodiment and the measured value Y of Aβ peptides obtained by measurement by mass spectrometry is 0.8 or more, preferably 0.85 or more, and more preferably 0.9 or more. The procedure for calculating the correlation coefficient r will be described below.

[0063] Measurement by a fully automated immunoassay device includes measuring Aβ peptides in a blood sample using a capture antibody and a detection antibody labeled with alkaline phosphatase (ALP). In general, in a measurement by a fully automated immunoassay device, a reagent containing a capture antibody, a reagent containing magnetic particles as a solid phase, a reagent containing an ALP-labeled detection antibody, and a reagent containing an ALP substrate solution are used. In a preferred embodiment, the capture antibody is labeled with biotins in advance, and avidins are fixed to the magnetic particles in advance. Alternatively, a reagent containing a capture antibody fixed to magnetic particles, a reagent containing an ALP-labeled detection antibody, and a reagent containing an ALP substrate solution are used. In a preferred embodiment, a reagent containing a buffer for diluting the blood sample and a buffer for promoting the reaction between ALP and the substrate (hereinafter also referred to as a measurement buffer) may be further used. The buffer for diluting the blood sample is the same as that described in the measurement method of this embodiment above. The measurement buffer is a buffer containing metal ions necessary for the enzymatic reaction of ALP and having a pH and salt concentration suitable for the reaction, such as R4 reagent (Sysmex Corporation). These reagents are placed in a fully automated immunoassay device, a blood sample containing Aβ peptide is placed in the device, and the measurement is started, whereby the Aβ peptide in the blood sample is measured by the above-mentioned sandwich ELISA method. The blood sample may be one or more.

[0064] In the measurement by mass spectrometry, the same blood sample as that in the measurement by the fully automated immunoassay device is measured. The measurement by mass spectrometry includes immunoprecipitation of Aβ peptides in the blood sample with the 6E10 antibody, which is an anti-Aβ monoclonal antibody, liberating the Aβ peptides from the complex of the immunoprecipitated Aβ peptides and the antibody with a basic solution containing an organic solvent, separating the solution containing the liberated Aβ peptides by liquid chromatography, and ionizing the separated Aβ peptides and measuring them with a triple quadrupole mass spectrometer. The details of the measurement by mass spectrometry are the same as those described in the measurement method of this embodiment.

[0065] In a preferred embodiment, a measured value X is obtained using HISCL (registered trademark)-5000 (Sysmex Corporation), and a measured value Y is obtained by a process using a predetermined release reagent, LC separation using a predetermined column, and measurement using a predetermined mass spectrometer as a mass spectrometry method. That is, the correlation coefficient r calculated by linear regression analysis of the measured value X of Aβ peptide obtained by measurement using HISCL (registered trademark)-5000 using the capture antibody and detection antibody of the antibody set of this embodiment and the measured value Y of Aβ peptide obtained by measurement using mass spectrometry is 0.8 or more, preferably 0.85 or more, and more preferably 0.9 or more. Here, the measurement using HISCL (registered trademark)-5000 includes measuring Aβ peptide in a blood sample using a capture antibody and a detection antibody labeled with alkaline phosphatase. Specifically, it is as follows. In the measurement by HISCL (registered trademark)-5000, the R1 reagent containing a biotin-labeled capture antibody, the R2 reagent containing streptavidin-immobilized magnetic particles, the R3 reagent containing an ALP-labeled detection antibody, the R4 reagent containing a measurement buffer, and the R5 reagent containing an ALP substrate solution are used. When these reagents are prepared in the HISCL (registered trademark)-5000 and a blood sample containing Aβ peptide is set and measurement is started, the blood sample and the R1 reagent are mixed to form a complex between the Aβ peptide and the biotin-labeled capture antibody. The R2 reagent is added to this, and the complex is formed on the magnetic particles through the binding between biotin and streptavidin. Here, the magnetic particles are magnetically collected to remove the liquid, and the magnetic particles are washed with the HISCL (registered trademark) washing solution (B / F separation). The R3 reagent is added to the magnetic particles to form a complex between the Aβ peptide, the biotin-labeled capture antibody, and the ALP-labeled detection antibody on the magnetic particles. Here, B / F separation is carried out in the same manner as above, R4 reagent and R5 reagent are added to the magnetic particles, the chemiluminescence intensity is measured, and a measured value X is obtained.

[0066] In the measurement by mass spectrometry, the same blood sample as that in the measurement by the fully automated immunoassay device is measured. In a preferred embodiment, the measurement by mass spectrometry includes immunoprecipitating Aβ peptides in a blood sample with 6E10 antibody, which is an anti-Aβ monoclonal antibody, liberating the Aβ peptides from the complex between the immunoprecipitated Aβ peptides and the antibody with a solution containing 0.56% ammonia and 40% acetonitrile, and subjecting the solution containing the liberated Aβ peptides to an ACQUITY® UPLC® Peptide BEH C 18 The method includes separating the Aβ peptides by liquid chromatography using an ACQUITY® UPLC® H-Class Bio System with a column, and measuring the separated Aβ peptides using a Xevo® TQ-XS triple quadrupole mass spectrometer with electrospray ionization. Here, the measurement by the mass spectrometer is a multiple reaction monitoring (MRM) measurement. In this MRM measurement, the mass spectrometer is used in a positive ion measurement mode, and the MRM transitions are precursor ion / product ion=1083.4 / 1054 for Aβ40 peptide and precursor ion / product ion=1129.5 / 1078.8 for Aβ42 peptide. A blood sample is measured by such mass spectrometry to obtain a measured value Y.

[0067] The measured value X by the fully automated immunoassay device and the measured value Y by mass spectrometry may be either signal intensity or the amount or concentration of Aβ peptide obtained from a calibration curve or the like. In calculating the correlation coefficient r, the measured values ​​of Aβ peptide obtained from a blood sample by each measurement method may be plotted on an XY plane with the measured value X by the fully automated immunoassay device on the X axis and the measured value Y by mass spectrometry on the Y axis to obtain a regression line. The correlation coefficient r can be calculated by known linear regression analysis. An example of the linear regression analysis is the least square method. In this embodiment, the correlation coefficient r is preferably 0.85 or more, and more preferably 0.9 or more. The calculation of the correlation coefficient itself can be performed by software such as Excel (registered trademark) (Microsoft Corporation).

[0068] A further embodiment is a method for measuring Aβ peptide in vitro by immunoassay using an antibody set including a capture antibody and a detection antibody that specifically bind to Aβ peptide. This immunoassay is characterized in that an antibody that binds to an epitope including the N-terminal residue of Aβ peptide is used as the capture antibody that specifically binds to Aβ peptide, and an antibody that binds to an epitope different from the epitope to which the capture antibody binds is used as the detection antibody that specifically binds to Aβ peptide. In this embodiment, the Aβ peptide is at least one of Aβ40 and Aβ42. In this embodiment, it is preferable to use the above antibody set.

[0069] By using the above-mentioned capture antibody and detection antibody, the immunoassay can measure Aβ peptides with the same quantitative accuracy as the measurement method of Aβ peptides of the present embodiment using mass spectrometry. Specifically, the antibody set used in the immunoassay is an antibody set that calculates a correlation coefficient r of 0.8 or more when a linear regression analysis is performed between the measurement value X of Aβ peptide obtained by measurement using a fully automated immunoassay device using a capture antibody and a detection antibody and the measurement value Y of Aβ peptide obtained by measurement using mass spectrometry. In a more preferred embodiment, the antibody set used in the immunoassay is an antibody set with a correlation coefficient r of 0.85 or more, and an even more preferred embodiment is an antibody set with a correlation coefficient r of 0.9 or more. Details of the measurement using the fully automated immunoassay device and mass spectrometry are the same as those described for the antibody set of the present embodiment.

[0070] In a further embodiment, the antibody set used in the immunoassay is preferably an antibody set that, when linear regression analysis is performed between the measured value X of Aβ peptide obtained by measurement with HISCL (registered trademark)-5000 using a capture antibody and a detection antibody and the measured value Y of Aβ peptide obtained by treatment with a specified release reagent, LC separation using a specified column, and measurement by mass spectrometry using a specified mass spectrometer, a correlation coefficient r of 0.8 or more is calculated. In a more preferred embodiment, the antibody set used in the immunoassay is an antibody set that has a correlation coefficient r of 0.85 or more, and an even more preferred embodiment is an antibody set that has a correlation coefficient r of 0.9 or more. Details of the measurement by HISCL (registered trademark)-5000 and mass spectrometry are the same as those described for the antibody set of this embodiment.

[0071] A further embodiment is a reagent kit for measuring Aβ peptide. That is, a reagent kit for measuring Aβ peptide (hereinafter also referred to as a "reagent kit") is provided, which includes a capture antibody and a detection antibody of the antibody set of the present embodiment. The capture antibody and the detection antibody are as described above. The reagent kit of the present embodiment includes one or more reagents.

[0072] The reagent kit of this embodiment may contain a basic solution containing an organic solvent for releasing the Aβ peptide from the complex formed by binding the Aβ peptide to the capture antibody. The basic solution containing an organic solvent is as described above.

[0073] The detection antibody may be labeled with a labeling substance. The labeling substance is not particularly limited, but is preferably an enzyme. Alternatively, the reagent kit may further include a labeling substance for labeling the detection antibody. In addition, when the labeling substance is an enzyme, the reagent kit may further include a substrate for the enzyme. The labeling substance and the substrate are as described above. The form of the capture antibody, the detection antibody, the labeling substance, and the substrate is not particularly limited, and may be a solid (e.g., a powder, a crystal, a lyophilized product, etc.) or a liquid (e.g., a solution, a suspension, an emulsion, etc.).

[0074] In this embodiment, the containers containing the reagents may be packed in a box and provided to the user. A package insert may be included in the box. The package insert may describe the configuration of the reagent kit, how to use it, and the relationship between the measurement results of the fully automated immunoassay device obtained using the reagent kit and the measurement results of the mass spectrometer. Examples of such reagent kits are shown in several figures below. This embodiment is not limited to these examples.

[0075] An example of the reagent kit of this embodiment is shown in Fig. 1A. In Fig. 1A, 10 indicates the reagent kit, 11 indicates a first container containing a reagent including a capture antibody for Aβ peptide, 12 indicates a second container containing a reagent including a detection antibody for Aβ peptide, 13 indicates a packaging box, and 14 indicates an attached document. In this example, the reagent kit may further include a solid phase for immobilizing the capture antibody. The solid phase is as described above.

[0076] An example of a reagent kit according to a further embodiment is shown in FIG. 1B. In FIG. 1B, 20 indicates a reagent kit, 21 indicates a first container containing a reagent including a capture antibody for Aβ peptide, 22 indicates a second container containing a reagent including a detection antibody for Aβ40, 23 indicates a third container containing a reagent including a detection antibody for Aβ42, 24 indicates a packaging box, and 25 indicates an attached document. In this example, the reagent kit may further include a solid phase for immobilizing the capture antibody. Details of the solid phase are as described above.

[0077] An example of a reagent kit of a further embodiment is shown in FIG. 1C. In FIG. 1C, 30 indicates a reagent kit, 31 indicates a first container containing a reagent including a capture antibody of Aβ40 peptide, 32 indicates a second container containing a reagent including a detection antibody of Aβ40, 33 indicates a third container containing a reagent including a detection antibody of Aβ42, 34 indicates a fourth container containing a reagent including a basic solution including an organic solvent, 35 indicates a packaging box, and 36 indicates an attached document. In this example, the reagent kit may further include a solid phase for immobilizing the capture antibody. The solid phase is as described above.

[0078] An example of a reagent kit according to a further embodiment is shown in Fig. 1D. In Fig. 1D, 40 indicates a reagent kit, 41 indicates a first container containing a reagent including a capture antibody for Aβ peptide, 42 indicates a second container containing a reagent including an enzyme-labeled detection antibody for Aβ40, 43 indicates a third container containing a reagent including an enzyme-labeled detection antibody for Aβ42, 44 indicates a fourth container containing a reagent including magnetic particles, 45 indicates a fifth container containing a cleaning reagent, 46 indicates a sixth reagent containing an enzyme substrate, 47 indicates a packaging box, and 48 indicates an attached document.

[0079] The reagent kit of this embodiment may include a calibrator for Aβ peptide. Examples of the calibrator include a calibrator for Aβ40 quantification and a calibrator for Aβ42 quantification. The calibrator for Aβ40 quantification may include, for example, a buffer solution not containing Aβ40 (negative control) and a buffer solution containing Aβ40 at a known concentration. Another example of the calibrator includes a buffer solution not containing either Aβ40 or Aβ42 (negative control), a buffer solution containing Aβ40 at a known concentration, and a buffer solution containing Aβ42 at a known concentration. Another example of the calibrator includes a buffer solution not containing either Aβ40 or Aβ42 (negative control), and a buffer solution containing Aβ40 and Aβ42 at known concentrations.

[0080] The present invention will now be described in more detail with reference to examples. EXAMPLES

[0081] Example 1: Measurement of plasma Aβ peptides using immunoprecipitation with a basic solution containing an organic solvent in combination with mass spectrometry The Aβ peptide was released from the complex between the Aβ peptide and the antibody that specifically binds to the Aβ peptide using a basic solution containing an organic solvent, and the Aβ peptide was measured using mass spectrometry.

[0082] (1) Capture and release of Aβ peptides using immunoprecipitation (1.1) Blood samples As blood samples containing Aβ peptides, five different lots of commercially available plasma samples (ProMedeX) were used.

[0083] (1.2) Antibody that specifically binds to Aβ peptide The antibody that specifically binds to Aβ peptide was a commercially available mouse monoclonal anti-Aβ antibody, 6E10 antibody (BioLegend). The 6E10 antibody was immobilized on magnetic particles (M-270 Epoxy-activated Dynabeads: Thermo Fisher Scientific) by a conventional method.

[0084] (1.3) Aβ peptide To prepare the calibration curve, Aβ40 peptide and Aβ42 peptide were purchased from AnaSpec. 15 N-labeled Aβ40 and Aβ42 peptides, 15N-Aβ40 and 15N-Aβ42 (rPeptide), were used. Aβ40 peptides were suspended in PBS solution containing 3% BSA to final concentrations of 10.8 pg / ml, 21.7 pg / ml, 43.3 pg / ml, 86.6 pg / ml, 173.2 pg / ml, 346.4 pg / ml, and 692.8 pg / ml, respectively. Aβ42 peptides were suspended in PBS solution containing 3% BSA to final concentrations of 2.8 pg / ml, 5.6 pg / ml, 11.3 pg / ml, 22.6 pg / ml, 45.2 pg / ml, 90.3 pg / ml, and 180.6 pg / mL, respectively. 15N-Aβ40 and 15N-Aβ42 were suspended in the same solution containing 3% BSA in PBS to a concentration of 500 pg / ml.

[0085] (1.4) Preparation of a basic solution containing an organic solvent As a basic solution containing an organic solvent (free reagent), 1.2 ml of 28% concentrated aqueous ammonia (Nacalai Tesque) and 6.0 ml of acetonitrile (Kanto Chemical) were added to 12.8 ml of pure water and mixed to prepare a 1.68% ammonia / 30% acetonitrile solution.

[0086] (1.5) Immunoprecipitation 250 μl of plasma sample or each Aβ40 peptide solution or Aβ42 peptide solution prepared in (1.3) above was added to a 1.5 ml sample tube (Eppendorf). 250 μl of the solution containing 15N-Aβ40 and 15N-Aβ42 prepared in (1.3) above was added to each sample tube containing the above solution, and the mixture was left to stand at room temperature for 30 minutes. After standing, 40 μl of the suspension of magnetic particles (4 μg antibody / 0.4 mg magnetic particles) with 6E10 antibody immobilized thereon prepared in (1.2) above was added to each sample solution, and the mixture was mixed by inversion at room temperature for 1 hour using a rotator to form a complex between Aβ peptide and antibody. These solutions were collected using a magnetic stand to remove the supernatant.

[0087] (1.6) Cleaning process After removing the supernatant, 1 mL of PBS solution containing 3% BSA was added to the magnetic particles remaining in the sample tube, mixed, and then magnetically collected again to remove the supernatant. This procedure was repeated twice with 1 mL of PBS solution containing 3% BSA, twice with 1 mL of 50 mM ammonium acetate solution, and once with 1 mL of ultrapure water to wash the magnetic particles.

[0088] (1.7) Release of Aβ peptide from the complex After washing the magnetic particles in (1.6) above, the washing solution was removed, and 25 μL of the release reagent prepared in (1.4) above was added to the remaining magnetic particles, mixed, and allowed to stand for 1 minute. The magnetic particles were again collected, and the supernatant was collected as the eluate.

[0089] (2) Mass spectrometry The eluate prepared in (1.7) above was subjected to LC-MS / MS for MRM measurement to measure Aβ peptides. The liquid chromatography portion of the LC-MS / MS was an ACQUITY (registered trademark) UPLC (registered trademark) H-Class Biosystem (Waters Corporation: hereinafter also referred to as UPLC). The column used was a reverse-phase column, ACQUITY (registered trademark) UPLC (registered trademark) Peptide BEH C 18 A column (Waters) was used. As a mass spectrometer, a Xevo (registered trademark) TQ-XS triple quadrupole mass spectrometer (Waters: hereinafter also referred to as a TQ-XS mass spectrometer) was used.

[0090] Each eluate was placed on the autosampler of the UPLC, and 10 μl of the eluate was introduced into the UPLC and fractionated by gradient. The gradient conditions were as follows:

[0091] [Table 1]

[0092] The eluate eluted from the column after being subjected to the gradient was directly fed to a TQ-XS mass spectrometer connected to a UPLC. The TQ-XS mass spectrometer was used for measurement in positive ion mode using electrospray ionization. The MRM measurement conditions were set as shown in Table 2 below.

[0093] [Table 2]

[0094] (3) Measurement results The results of the measurement using LC-MS / MS are shown in Figure 2, Figure 3A, and Figure 3B. Figure 2 shows the results of MRM measurement for Aβ40 peptide, Aβ42 peptide, 15N-Aβ40, and 15N-Aβ42, and Figures 3A and 3B show the concentrations of Aβ40 peptide and Aβ42 peptide in the measured plasma samples. These results demonstrated that Aβ peptides can be released from the complex using a basic solution containing an organic solvent, and then measured using mass spectrometry.

[0095] Figures 4A and 4B show the results of creating a calibration curve based on the measured values ​​detected using the Aβ peptide of known concentration prepared in (1.3) above. As shown in Figures 4A and 4B, for both the Aβ40 peptide and the Aβ42 peptide, R 2 A calibration curve with a value of 0.999 or more could be created. In addition, Aβ peptides at low concentrations of 100 pg / ml or less could be detected. This shows that the above measurement method, which combines a basic solution containing an organic solvent with mass spectrometry, is capable of measuring Aβ peptides with high sensitivity and has excellent quantitative properties.

[0096] Example 2: Comparison of elution efficiency The composition of the release reagent was changed, and the elution efficiency of Aβ peptide from the complex due to the difference in the composition of the release reagent was calculated based on the following formula and compared.

[0097] [Elution efficiency (%)] = [Aβ peptide concentration of sample C] / ([Aβ peptide concentration of sample A] - [Aβ peptide concentration of sample B]) × 100

[0098] (1) Comparison of basic substances in free reagents (1.1) Preparation of the release reagent Various free reagents were prepared by mixing DDM (n-dodecyl-β-D-maltoside, Sigma-Aldrich), acetonitrile, and / or 28% concentrated aqueous ammonia with pure water to obtain the compositions shown in Table 3 below. For convenience, solutions that do not contain basic substances or organic solvents are also referred to as free reagents. After preparation, the pH of the solutions containing basic substances was measured using a pH meter (HORIBA, Ltd.).

[0099] [Table 3]

[0100] (1.2) Sample preparation The Aβ40 peptide used in (1.3) of Example 1 was suspended in a PBS solution containing 3% BSA to a concentration of 1000 pg / ml to prepare sample A. The Aβ peptide concentration in sample A corresponds to the initial concentration of the Aβ40 peptide.

[0101] (1.3) Immunoprecipitation Sample A prepared in (1.2) above was subjected to immunoprecipitation in the same manner as in (1.5) of Example 1 to recover the magnetic particles. In this case, the supernatant after magnetic collection was recovered and stored as sample B. The Aβ peptide concentration in sample B corresponds to the concentration of Aβ peptide that was not captured by the antibody that specifically binds to Aβ peptide.

[0102] (1.4) Washing and elution The magnetic particles collected in (1.3) above were washed in the same manner as in (1.6) of Example 1, and the washing solution was removed. 15 μl of each release reagent prepared in (1.1) above was added to the washed magnetic particles, and the mixture was allowed to stand for 1 minute to release the Aβ peptide. After standing, the magnetic particles were collected and the supernatant was collected. The collected supernatant was mixed with a pH neutralizing solution (pH 7.4) containing 300 mM Tris and 300 mM NaCl to obtain sample C. The Aβ peptide concentration in sample C corresponds to the concentration of the Aβ peptide released after capture by immunoprecipitation.

[0103] (2) Measurement by immunoassay The Aβ peptide concentration in each sample was measured for the above samples A, B, and C by immunoassay using a fully automated immunoassay device HISCL (registered trademark)-5000 (Sysmex Corporation). The R1 reagent (capture antibody reagent) was prepared by labeling the 82E1 antibody with biotin by a conventional method and dissolving it in a buffer containing 1% BSA, 0.1 M Tris-HCl, 0.15 M NaCl, and 0.1% NaN3 at pH 7.5. The R2 reagent (solid phase) was HISCL (registered trademark) R2 reagent (Sysmex Corporation) containing streptavidin-bound magnetic particles. The R3 reagent (detection antibody reagent) was prepared by labeling the 1A10 antibody with alkaline phosphatase (ALP) by a conventional method and dissolving it in a buffer containing 1% BSA, 0.1 M Tris-HCl, 0.15 M NaCl, and 0.1% NaN3 at pH 7.5. HISCL® R4 reagent (Sysmex Corporation) was used as the R4 reagent (measurement buffer solution). HISCL® R5 reagent (Sysmex Corporation) was used as the R5 reagent (ALP substrate solution).

[0104] The measurement procedure using HISCL (registered trademark)-5000 was as follows. Sample A, B, or C (30 μL) was mixed with R1 reagent (110 μL) and reacted at 42° C. for 4 minutes. After the reaction, R2 reagent (30 μL) was added and reacted at 42° C. for 3 minutes. The magnetic particles in the resulting mixture were collected, the supernatant was removed, and HISCL (registered trademark) washing solution (300 μL) was added to wash the magnetic particles. The supernatant was removed, and R3 reagent (100 μL) was added to the magnetic particles and mixed, and reacted at 42° C. for 5 minutes. The magnetic particles in the resulting mixture were collected, the supernatant was removed, and HISCL (registered trademark) washing solution (300 μL) was added again to wash the magnetic particles. The supernatant was removed, and R4 reagent (50 μL) and R5 reagent (100 μL) were added to the magnetic particles, and the chemiluminescence intensity was measured. As a calibrator (antigen for creating a calibration curve), Aβ40 peptide was suspended in a solution containing 0.1% BSA, 0.14M triethanolamine, 0.15M NaCl, and 0.1% NaN3 at pH 7.0 to obtain concentrations of 0 pg / ml, 8.6 pg / ml, 33.3 pg / ml, 99.2 pg / ml, 319.1 pg / ml, and 1188.1 pg / ml, respectively, and a calibration curve was created by measuring the concentrations in the same manner. The chemiluminescence intensity obtained by the measurement was applied to the calibration curve to determine the concentration of Aβ40 peptide.

[0105] (3) Measurement results The measurement results of the elution efficiency for Comparative Reagents 1 to 3 and Reagents 1 to 9 in Table 3 are shown in Figure 5. As shown in Figure 5, the elution efficiency was low when the release reagent was only an organic solvent or only a basic solution, and it was shown that a high elution efficiency could be obtained by using a release reagent containing an organic solvent and a basic substance as the Aβ peptide release reagent. In particular, it was shown that a high elution efficiency of more than 70% could be obtained with the reagent having a pH of 11.4 or more and 12.0 or less.

[0106] (4) Comparison of organic solvents of free reagents In the above (1.1), the release reagent in Table 4 below was used instead of the release reagent in Table 3 to measure the elution efficiency. In Comparative Reagent 5 and Samples 17 to 23, Aβ42 peptide was used instead of Aβ40 peptide as the sample. When Aβ42 peptide was used, H31L21 antibody was used instead of 1A10 antibody as the detection antibody, and each solution prepared by suspending Aβ42 peptide in a solution containing 0.1% BSA, 0.14M triethanolamine, 0.15M NaCl, and 0.1% NaN3 at pH 7.0 to give concentrations of 0 pg / ml, 0.5 pg / ml, 6.1 pg / ml, 65.2 pg / ml, and 804.5 pg / ml was used as the calibrator. The same operations as in (1) and (2) were performed except for the above, and the elution efficiency for each release reagent was measured.

[0107] [Table 4]

[0108] The measurement results for Comparative Reagent 4 and Samples 10 to 16 are shown in Fig. 6A, and the measurement results for Comparative Reagent 5 and Samples 17 to 23 are shown in Fig. 6B. Figs. 6A and 6B show that Samples 10 to 16 and 17 to 23 exhibit superior elution efficiency compared to Comparative Reagents 4 and 5, which do not contain an organic solvent. It was shown that superior elution efficiency can be obtained by the free reagent containing a basic substance and an organic solvent.

[0109] Example 3: Carry-over measurements Conventionally, an acidic solution is used to release Aβ peptides from the complex between the Aβ peptide and the antibody captured by the antibody. Here, we measured the carryover of Aβ peptides to a liquid chromatography device depending on the acidic or basic condition of the release reagent used to elute the complex.

[0110] (1) Sample preparation As an acidic release reagent, pure water was mixed with formic acid (FUJIFILM Wako Pure Chemical Industries, Ltd.) and acetonitrile to prepare an acidic release reagent having a composition of 0.1% formic acid and 50% acetonitrile (here, for convenience, the acidic solution is also referred to as the release reagent). In addition, a release reagent (1.68% ammonia, 50% acetonitrile) was prepared in the same manner as in (1.4) of Example 1. The same Aβ40 peptide as in (1.3) of Example 1 was added to these release reagents to a final concentration of 100 fmol / ul (Aβ40 peptide-containing solution). In addition, a solution not containing Aβ40 peptide (Aβ40 peptide-free solution) was also prepared for each of the acidic release reagent and the basic release reagent.

[0111] (2) Introducing the sample into liquid chromatography The four types of solutions prepared in (1) above were subjected to LC-MS / MS. The LC-MS / MS device and column used were those described in the mass spectrometry method in (2) of Example 1. Each solution was placed in the autosampler of the UPLC, and 10 μl of each solution was introduced into the UPLC for fractionation. The UPLC analysis conditions were as shown in Table 5 below. The conditions for MRM measurement in mass spectrometry were as shown in Table 2 of Example 1.

[0112] [Table 5]

[0113] The two types of Aβ40 peptide-containing solutions prepared in (1) above and the corresponding Aβ40-free solutions were fed to the UPLC in the order of Aβ40 peptide-free solution (background), Aβ40 peptide-containing solution, and Aβ40 peptide-free solution (carry-over measurement), and the results of comparing the signal intensities obtained at that time are shown in Figure 7. From Figure 7, it was found that when an acidic releasing reagent was used, a larger amount of carry-over occurred than when a basic releasing reagent was used. On the other hand, when a basic releasing reagent was used as the releasing reagent for Aβ peptide, the carry-over could be significantly suppressed. Therefore, it was shown that the use of a basic releasing reagent is appropriate for the continuous analysis of Aβ peptides using mass spectrometry.

[0114] Reference example: Comparison of acidity and base of free reagents As in Example 3 above, carryover occurred when Aβ peptides released using an acidic releasing reagent were measured by LC-MS / MS. In this reference example, the difference in the amount of Aβ peptide detected was compared between a case in which a basic releasing reagent was used to release Aβ peptides from a complex, and a case in which an acidic releasing reagent was used during release, but then replaced with a basic releasing reagent and subjected to LC-MS / MS.

[0115] (1) Sample preparation As the Aβ peptide sample, the same Aβ40 peptide and Aβ42 peptide as those in (1.3) of Example 1 were used, and a solution in which the Aβ40 peptide was adjusted to 50 pg / ml or 190 pg / ml in a 3% BSA solution, or a solution in which the Aβ42 peptide was adjusted to 26 pg / ml or 103 pg / ml in a 3% BSA solution, was prepared and used. As an acidic release reagent, an acidic solution having a composition of 0.1% TFA and 30% acetonitrile was prepared by mixing trifluoroacetic acid (TFA, Fujifilm Wako Pure Chemical Industries, Ltd.) and acetonitrile with pure water. As a basic release reagent, a 1.68% ammonia, 50% acetonitrile solution was prepared in the same manner as in (1.4) of Example 1.

[0116] (2) Measurement (2.1) Immunoprecipitation To each sample prepared in (1) above, an internal standard was added in the same manner as in (1.5) of Example 1, and magnetic particles having 6E10 antibody immobilized thereon were added to form a complex.

[0117] (2.2) Elution of Aβ peptides After carrying out the same washing step as in (1.6) of Example 1 for each complex prepared in (2.1) above, 25 μL of the acidic or basic release reagent prepared in (1) above was added and mixed, and the mixture was allowed to stand for 1 minute. The magnetic particles were collected again, and the supernatant was collected as an eluate.

[0118] (2.3) Solvent exchange Of the eluates recovered in (2.2) above, the eluate obtained using the acidic release reagent was dried under reduced pressure for 1 hour using a Spin Dryer Standard VC-96R (Taitec Corporation) in SpinDryer mode at a rotation speed of 1500 r / min and a temperature of 55°C to volatilize the solvent. 25 μL of the basic release reagent prepared in (1) above was added to the residue after the reduced pressure drying to resuspend the residue.

[0119] (2.4) Mass spectrometry The eluate (referred to as solution X) obtained using each basic releasing reagent recovered in (2.2) above and the resuspended solution (referred to as solution Y) in (2.3) above were subjected to LC-MS / MS in the same manner as in (2) of Example 1, and MRM measurement was performed.

[0120] (3) Measurement results The measurement results of solution A and solution B are shown in Figures 8A to 9D, respectively. Figure 8A shows solution X using 190 pg / ml Aβ peptide 40, Figure 8B shows solution Y using 190 pg / ml Aβ40 peptide, Figure 8C shows solution X using 103 pg / ml Aβ42 peptide, Figure 8D shows solution Y using 103 pg / ml Aβ42 peptide, Figure 9A shows solution X using 50 pg / ml Aβ40 peptide, Figure 9B shows solution Y using 50 pg / ml Aβ40 peptide, Figure 9C shows solution X using 26 pg / ml Aβ42 peptide, and Figure 9D shows solution Y using 26 pg / ml Aβ42. From these results, in the example of Aβ peptide at all concentrations, the area value was larger when solution X was used than when solution Y was used. It was suggested that the loss of Aβ peptide occurs when the process such as (2.3) above is used in sample processing.

[0121] Example 4: Comparison of immunoassay measurements with mass spectrometry measurements Aβ peptide was released from the complex using a basic solution containing an organic solvent as a release reagent, and the resulting eluate was subjected to immunoassay or mass spectrometry, respectively, and the obtained measured values ​​were compared.

[0122] (1) Reagent preparation (1.1) Plasma samples Eighteen plasma samples from healthy individuals were purchased and used.

[0123] (1.2) Aβ peptide The Aβ40 peptide and Aβ42 peptide described in Example 1 (1.3) were used.

[0124] (1.3) Peptide spiked sample Among the plasma samples (1.1) above, five specimens were mixed to prepare a mixed sample. This mixed sample was divided into eight portions, and the Aβ40 peptide and Aβ42 peptide (1.2) above were added to each portion in amounts that would increase the Aβ40 peptide concentration by 48.5 pg / mL, 57.7 pg / mL, 56.8 pg / mL, 112.0 pg / mL, 120.3 pg / mL, 127.8 pg / mL, 248.0 pg / mL, and 513.7 pg / mL, and the Aβ42 peptide concentration by 5.7 pg / mL, 6.6 pg / mL, 7.5 pg / mL, 13.4 pg / mL, 13.7 pg / mL, 14.7 pg / mL, 29.3 pg / mL, and 54.2 pg / mL, respectively, to prepare peptide spike samples.

[0125] (1.4) Antibody that specifically binds to Aβ peptide The 6E10 antibody described in (1.2) of Example 1 was used. In addition, 15N-Aβ40 and 15N-Aβ42 described in (1.2) of Example 1 were used as internal standard substances. 15N-Aβ40 and 15N-Aβ42 were each suspended in a PBS solution containing 3% BSA to a concentration of 500 pg / ml.

[0126] (1.5) Samples for preparing calibration curves in mass spectrometry The Aβ40 peptide was suspended in PBS containing 3% BSA at 10.8 pg / ml, 21.7 pg / ml, 43.3 pg / ml, 86.6 pg / ml, 173.2 pg / ml, 346.4 pg / ml, and 692.8 pg / ml, respectively. The Aβ42 peptide was suspended in PBS containing 3% BSA at 2.8 pg / ml, 5.6 pg / ml, 11.3 pg / ml, 22.6 pg / ml, 45.2 pg / ml, 90.3 pg / ml, and 180.6 pg / ml, respectively, and prepared as samples for creating a calibration curve.

[0127] (1.6) Calibrator for HISCL (registered trademark)-5000 measurement As calibrators for HISCL®-5000 measurements, Aβ40 peptide was suspended in a solution containing 0.1% BSA, 0.14 M triethanolamine, 0.15 M NaCl, 0.1% NaN3, pH 7.0 to give concentrations of 0 pg / ml, 8.6 pg / ml, 33.3 pg / ml, 99.2 pg / ml, 319.1 pg / ml, and 1188.1 pg / ml, respectively.

[0128] (1.7) Preparation of basic solutions containing organic solvents As a basic solution containing an organic solvent (free reagent), a 1.68% ammonia, 30% acetonitrile solution was prepared as described in (1.4) of Example 1.

[0129] (2) Measurement using mass spectrometry (2.1) Immunoprecipitation 250 μl of the plasma sample (1.1), the peptide spike sample (1.3), or each Aβ40 peptide solution or Aβ42 peptide solution prepared in (1.5) above was added to a 1.5 ml sample tube (Eppendorf). 250 μl of the solution containing 15N-Aβ40 and 15N-Aβ42 prepared in (1.4) above was added to each sample tube containing the above solution, and the tube was left to stand at room temperature for 30 minutes. After standing, 40 μl of the suspension of magnetic particles (4 mg antibody / 0.4 mg magnetic particles) with the 6E10 antibody immobilized thereon prepared in (1.4) above was added to each sample solution, and the mixture was mixed by inversion at room temperature for 1 hour using a rotator. These solutions were collected using a magnetic stand, and the supernatant was removed.

[0130] (2.2) Cleaning process After removing the supernatant, 1 mL of PBS solution containing 3% BSA was added to the magnetic particles remaining in the sample tube, mixed, and then magnetically collected again to remove the supernatant. This procedure was repeated twice with 1 mL of PBS solution containing 3% BSA, twice with 1 mL of 50 mM ammonium acetate solution, and once with 1 mL of ultrapure water to wash the magnetic particles.

[0131] (2.3) Release of Aβ peptides After washing the magnetic particles in (2.2) above, the washing solution was removed, and 25 μL of the release reagent prepared in (1.7) above was added to the remaining magnetic particles, mixed, and allowed to stand for 1 minute. The magnetic particles were again collected, and the supernatant was collected as the eluate.

[0132] (2.4) Mass spectrometry The eluate prepared in (2.3) above was subjected to LC-MS / MS to carry out MRM measurement. The LC-MS / MS conditions were the same as those in (2) of Example 1.

[0133] (3) Measurement using immunoassay Apart from the above (2), the Aβ peptide concentration was measured by immunoassay using HISCL (registered trademark)-5000 for the plasma sample (1.1) and the peptide-spiked sample (1.3). The R1 reagent (capture antibody reagent) was prepared by labeling the 82E1 antibody with biotin by a conventional method and dissolving it in a buffer containing 1% BSA, 0.1 M Tris-HCl, 0.15 M NaCl, and 0.1% NaN3 at pH 7.5. The R2 reagent (solid phase) was HISCL (registered trademark) R2 reagent (Sysmex Corporation) containing streptavidin-bound magnetic particles. The R3 reagent (detection antibody reagent) was prepared by labeling the 1A10 antibody with alkaline phosphatase (ALP) by a conventional method and dissolving it in a buffer containing 1% BSA, 0.1 M Tris-HCl, 0.15 M NaCl, and 0.1% NaN3 at pH 7.5. HISCL® R4 reagent (Sysmex Corporation) was used as the R4 reagent (measurement buffer solution). HISCL® R5 reagent (Sysmex Corporation) was used as the R5 reagent (ALP substrate solution).

[0134] The measurement procedure using HISCL (registered trademark)-5000 was carried out in the same manner as in Example 2 (2), except that the plasma sample described in (1.1) above or the peptide-spiked sample described in (1.3) above was used.

[0135] (4) Calculation of correlation coefficient The results of the measurement of Aβ40 peptide and Aβ42 peptide measured in (2) and (3) above are plotted on the horizontal axis using mass spectrometry and on the vertical axis using HISCL (registered trademark)-5000, and the results of calculating the correlation coefficient r using each plotted data are shown in Figures 10 and 11. Figure 10A is a graph showing the measurement results of Aβ40 peptide in a plasma sample, Figure 10B is a graph showing the measurement results of Aβ40 peptide in a peptide-spiked sample, and Figure 10C is a graph including both the measurement results of Figures 10A and 10B. Figure 11A is a graph showing the measurement results of Aβ42 peptide in a plasma sample, Figure 11B is a graph showing the measurement results of Aβ42 peptide in a peptide-spiked sample, and Figure 11C is a graph including both the measurement results of Figures 11A and 11B.

[0136] (5) When measuring the correlation coefficient by changing the capture antibody As a comparative example, the same experiment was performed in (3) above, except that the capture antibody for Aβ42 peptide was replaced with 6E10 instead of 82E1. The 6E10 antibody is an antibody that recognizes the 3rd to 8th region epitope counting from the amino acid residues at the N-terminus of the Aβ peptide. The measurement results and the measurement results for the Aβ42 peptide measured in (2) above were plotted in the same manner as in (4) above, and are shown in FIG. 12. FIG. 12 shows that when the capture antibody is the 6E10 antibody, the results of the immunoassay do not correlate with the results of the mass spectrometry measurement. The results in FIG. 12 and the results in FIG. 10 to FIG. 11 show that there is a high correlation between the results of the measurement using mass spectrometry and the measurement using immunoassay by using the 82E1 antibody as the capture antibody. [Explanation of symbols]

[0137] 10, 20, 30, 40: Reagent kit 11, 21, 31, 41: 1st container 12, 22, 32, 42: 2nd container 23, 33, 43: 3rd container 34, 44: 4th container 45: 5th container 46: 6th container 13, 24, 35, 47: Packing box 14, 25, 36, 48: Attached document

Claims

1. A set of antibodies for measuring Aβ peptide in a blood sample by immunoassay, comprising: the antibody set comprises a capture antibody and a detection antibody that specifically bind to the Aβ peptide; the capture antibody is a monoclonal antibody of clone 82E1, the detection antibody is a monoclonal antibody of clone H31L21, and the Aβ peptide is Aβ42; a correlation coefficient r calculated by linear regression analysis of a measured value X of Aβ peptide obtained by measurement using a fully automated immunoassay device using the capture antibody and the detection antibody and a measured value Y of Aβ peptide obtained by measurement using mass spectrometry is 0.8 or more; the measurement by the fully automated immunoassay device includes measuring Aβ peptide in the blood sample using the capture antibody and the detection antibody labeled with alkaline phosphatase; the measurement by mass spectrometry comprises immunoprecipitating Aβ peptides in the blood sample with anti-Aβ monoclonal antibody 6E10, liberating the Aβ peptides from a complex between the immunoprecipitated Aβ peptides and the antibody with a basic solution containing an organic solvent and ammonium ions, separating a solution containing the liberated Aβ peptides by liquid chromatography, and ionizing the separated Aβ peptides and measuring them with a triple quadrupole mass spectrometer.

2. The measurement using the fully automated immunoassay device is a measurement using HISCL (registered trademark)-5000, the measurement by the HISCL®-5000 includes measuring Aβ peptide in the blood sample using the capture antibody and the detection antibody labeled with alkaline phosphatase; The measurement by mass spectrometry includes immunoprecipitating Aβ peptides in the blood sample with anti-Aβ monoclonal antibody 6E10, liberating the Aβ peptides from a complex between the immunoprecipitated Aβ peptides and the antibody with a solution containing 1.68% ammonia and 30% acetonitrile as the basic solution, and subjecting the solution containing the liberated Aβ peptides to an ACQUITY® UPLC® Peptide BEH C 18 and measuring the separated Aβ peptides using a Xevo TQ-XS triple quadrupole mass spectrometer with electrospray ionization; The measurement by the mass spectrometer is a multiple reaction monitoring (MRM) measurement, and in the MRM measurement, the mass spectrometer is used in a positive ion measurement mode, and the MRM transition for Aβ42 peptide is precursor ion / product ion=1129.5 / 1078.

8. The antibody set described in claim 1.

3. An antibody set as described in claim 1 or 2, wherein the capture antibody and the blood sample are mixed during measurement by the fully automated immunoassay device.

4. An antibody set described in any one of claims 1 to 3, wherein the correlation coefficient r is 0.9 or more.

5. An antibody set described in any one of claims 1 to 4, wherein the pH of the basic solution is 11.4 or higher.

6. An antibody set described in any one of claims 1 to 5, wherein the capture antibody is fixed to a solid phase.

7. The antibody set described in claim 6, wherein the solid phase is a magnetic particle.

8. An antibody set described in any one of claims 1 to 7, wherein the detection antibody is labeled with a labeling substance.

9. The antibody set described in claim 8, wherein the labeling substance is an enzyme.

10. The antibody set described in claim 9, wherein the enzyme is at least one selected from alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase and luciferase.

11. 1. A method for measuring Aβ peptide in a blood sample in vitro, comprising: measuring the Aβ peptide by immunoassay using an antibody set including a capture antibody and a detection antibody that specifically bind to the Aβ peptide; the capture antibody is a monoclonal antibody of clone 82E1, the detection antibody is a monoclonal antibody of clone H31L21, and the Aβ peptide is Aβ42; the antibody set is an antibody set that, when a measured value X of an Aβ peptide obtained by measurement using a fully automated immunoassay device using the capture antibody and the detection antibody is subjected to linear regression analysis with a measured value Y of an Aβ peptide obtained by measurement using mass spectrometry, gives a correlation coefficient r of 0.8 or more; the measurement by the fully automated immunoassay device includes measuring Aβ peptide in the blood sample using the capture antibody and the detection antibody labeled with alkaline phosphatase; The method for measuring Aβ peptides, wherein the measurement by mass spectrometry includes immunoprecipitating Aβ peptides in the blood sample with anti-Aβ monoclonal antibody 6E10, liberating the Aβ peptides from a complex between the immunoprecipitated Aβ peptides and the antibody with a basic solution containing an organic solvent and ammonium ions, separating a solution containing the liberated Aβ peptides by liquid chromatography, and ionizing the separated Aβ peptides and measuring them with a triple quadrupole mass spectrometer.

12. The measurement using the fully automated immunoassay device is a measurement using HISCL (registered trademark)-5000, The measurement using the HISCL®-5000 includes measuring Aβ peptide in the blood sample using the capture antibody and the detection antibody labeled with alkaline phosphatase; The measurement by mass spectrometry includes immunoprecipitating Aβ peptides in the blood sample with anti-Aβ monoclonal antibody 6E10, liberating the Aβ peptides from a complex between the immunoprecipitated Aβ peptides and the antibody with a solution containing 1.68% ammonia and 30% acetonitrile as the basic solution, and subjecting the solution containing the liberated Aβ peptides to an ACQUITY® UPLC® Peptide BEH C chromatography. 18 and measuring the separated Aβ peptides using a Xevo TQ-XS triple quadrupole mass spectrometer with electrospray ionization; The measurement by the mass spectrometer is a multiple reaction monitoring (MRM) measurement, and in the MRM measurement, the mass spectrometer is used in a positive ion measurement mode, and the MRM transition for Aβ42 peptide is precursor ion / product ion=1129.5 / 1078.

8. The method of claim 11.

13. The method described in claim 11 or 12, wherein the capture antibody and the blood sample are mixed during measurement by the fully automated immunoassay device.

14. The method according to any one of claims 11 to 13, wherein the correlation coefficient r is 0.9 or more.

15. The method according to any one of claims 11 to 14, wherein the pH of the basic solution is 11.4 or higher.

16. The method according to any one of claims 11 to 15, wherein the capture antibody is immobilized on a solid phase.

17. The method of claim 16 , wherein the solid phase is a magnetic particle.

18. The method according to any one of claims 11 to 17, wherein the detection antibody is labeled with a labeling substance.

19. The method of claim 18 , wherein the labeling substance is an enzyme.

20. 20. The method of claim 19, wherein the enzyme is at least one selected from alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase and luciferase.

21. A reagent kit comprising the capture antibody according to claim 1 or 2 and a detection antibody.

22. 22. The reagent kit of claim 21, wherein the detection antibody is labeled with an enzyme and further comprises a substrate for the enzyme.

Images