Aβ peptide measurement method and reagent composition used in the method

Abstract

The present invention aims to provide a means for enabling a further improved measurement of Aβ peptide in blood. The above-mentioned problem is solved by a method for measuring Aβ peptide, which comprises a step of mixing a blood sample containing Aβ peptide and an antibody that specifically binds to Aβ peptide to form a complex of Aβ peptide and the antibody, a step of releasing Aβ peptide from the complex by an alkaline solution containing an organic solvent, and a step of measuring the freed Aβ peptide by mass spectrometry.

Description

[Technical field]

[0001] The present invention relates to a method for measuring amyloid β (Aβ) peptide and a reagent composition used in the measuring method. [Background technology]

[0002] It is known that Aβ peptide in biological samples collected from subjects is a biomarker for Alzheimer's disease. Among biological samples, cerebrospinal fluid (CSF) contains relatively more Aβ peptide, so a method for quantitatively measuring Aβ peptide in CSF has been established. For example, Non-Patent Document 1 describes measuring Aβ peptide in CSF by liquid chromatography-mass spectrometry (LC-MS).

[0003] [Prior art literature]

[0004]

Non-patent literature

[0005] [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)

[0006] [Summary of the Invention]

[0007] [Problems to be solved by the invention]

[0008] Since the collection of CSF is invasive and imposes a heavy burden on the subject, a method for measuring Aβ peptide using blood, which has a less burden on the collection, has been developed in recent years. However, since blood contains less Aβ peptide than CSF, a method for measuring Aβ peptide with high accuracy is required. The present invention aims to provide a means for making it possible to measure Aβ peptide in blood with high accuracy.

[0009]

Methods for solving the problem

[0010] The present invention provides a method for measuring Aβ peptide, comprising the steps of mixing a blood sample containing Aβ peptide and an antibody that specifically binds to Aβ peptide to form a complex of Aβ peptide and the antibody, releasing Aβ peptide from the complex using an alkaline solution containing an organic solvent, and measuring the released Aβ peptide by mass spectrometry. In addition, the present invention provides a reagent composition used in the above method, which is an alkaline solution containing an organic solvent.

[0011] [Effects of the Invention]

[0012] The present invention makes it possible to measure Aβ peptide in blood with high accuracy.

[0013] [Brief description of the attached drawings]

[0014]

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[0032] The method for measuring Aβ peptide in the present embodiment is a method for measuring Aβ peptide captured by an antibody by mass spectrometry (hereinafter also referred to as "measurement method"). This measurement method is characterized by releasing Aβ peptide from a complex of Aβ peptide and antibody by an alkaline solution containing an organic solvent, and measuring the free Aβ peptide by mass spectrometry.

[0033] In the past, an acidic solution was used to free the test substance from the test substance captured by the antibody (i.e., the complex of the antibody and the test substance). This is because acidic solutions are generally excellent in dissociating the binding of the test substance and the antibody. For example, in the Publication No. 2018-194374 of the Japanese Patent, it is recorded that Aβ peptide is freed from the complex by an acidic aqueous solution of pH 1 to 4 optionally containing an organic solvent. However, as shown in the examples described later, the inventors found that the acidic solution containing free Aβ peptide is not suitable for mass spectrometry combined with liquid chromatography. Specifically, when the acidic solution containing free Aβ peptide is used directly in liquid chromatography, a part of the Aβ peptide injected remains in the flow path and column of the liquid chromatography device, and the residual Aβ peptide is left in the measurement of the following sample (called carryover). When carryover occurs, the correct measurement of Aβ peptide cannot be performed. In order to reduce carryover, it is considered to free the Aβ peptide with an acidic solution and then replace the solvent with an alkaline solution. However, even if this method can reduce carryover, it is difficult to perform high-sensitivity measurements due to the loss of Aβ peptides caused by solvent replacement. In contrast, in the measurement method of this embodiment using an alkaline solution containing an organic solvent, the amount of Aβ peptide carried over is significantly reduced. Therefore, the correct measurement of Aβ peptide can be performed directly using an alkaline solution containing free Aβ peptide in liquid chromatography without the need for solvent replacement. As described above, since the content of Aβ peptide in blood samples is less than that in CSF, the measurement method of this embodiment is suitable for quantitative measurement of Aβ peptide in blood samples.

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

[0035] In this specification, the term "blood sample containing Aβ peptide" may also include blood samples suspected of containing Aβ peptide. Examples of blood samples include blood (whole blood), plasma, serum, etc. Among these, plasma and serum are also preferred. The blood sample may also be diluted with a suitable aqueous medium as needed. Such an aqueous medium is not particularly limited as long as it does not interfere with the determination described below, and examples include water, saline, buffer, etc. The buffer is preferably a buffer having a buffering effect at a pH near neutral (e.g., a pH of 6 to 8). Examples of such buffers include Good buffers such as HEPES, MES, PIPES, Tris-buffered saline (TBS), phosphate-buffered saline (PBS), etc.

[0036] The source of the blood sample is not particularly limited, and for example, it may be blood collected from a subject and plasma or serum prepared therefrom. In addition, commercially available mixed plasma, healthy plasma, etc. may also be used. If necessary, a labeled Aβ peptide as an internal standard substance may be added to the blood sample. The subject is not particularly limited, and examples thereof include healthy subjects, subjects with abnormal cognitive function, and subjects suspected of having abnormal cognitive function. Abnormal cognitive function may include, for example, mild cognitive impairment (MCI), Alzheimer's dementia, etc.

[0037] Aβ peptide is a polypeptide produced by the treatment of amyloid β precursor protein (APP) by β-secretase and γ-secretase. Aβ peptide includes polypeptides of any length unless otherwise specified, and is generally a polypeptide consisting of 39 to 43 amino acids. As Aβ peptide, Aβ40 (DAEFRHDSGY EVHHQKLVFF AEDVGSNKGAIIGLMVGGVV: SEQ ID NO: 1) consisting of 40 amino acid residues and Aβ42 (DAEFRHDSGY EVHHQKLVFFAEDVGSNKGA IIGLMVGGVV IA: SEQ ID NO: 2) consisting of 42 amino acid residues are preferred.

[0038] Aβ peptides can be in the form of monomers or polymers. Multimers are also called polymers, which are formed by physical or chemical polymerization or aggregation of multiple monomeric Aβ peptides. Multimers can contain multiple monomeric Aβ peptides, and can also contain other molecules. In multimers, monomeric Aβ peptides do not need to be strongly bound to each other by covalent bonds or the like. Multimers also contain aggregates in which multiple monomeric Aβ peptides are gathered due to slower binding. Examples of Aβ peptide multimers include Aβ oligomers.

[0039] In this specification, 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 antibodies, reduced IgG (rIgG), single chain antibodies (scFv), etc. The antibody that specifically binds to the Aβ peptide may be either a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

[0040] Monoclonal antibodies that can specifically bind to Aβ peptides are known per se, and examples thereof include an antibody of clone 82E1 that recognizes the 1st to 16th region as an epitope from the amino acid residues at the N-terminus of Aβ peptide (referred to as 82E1 antibody), an antibody of clone 6E10 that recognizes the 3rd to 8th region as an epitope (referred to as 6E10 antibody), an antibody of clone WO-2 that recognizes the 4th to 10th region as an epitope (referred to as WO-2 antibody), an antibody of clone 2H4 that recognizes the 1st to 8th region as an epitope (referred to as 2H4 antibody), and an antibody of clone 6E10 that recognizes the 3rd to 8th region as an epitope (referred to as 6E10 antibody). The antibody of clone H31L21 which recognizes the region of 33 to 42 as an epitope (referred to as H31L21 antibody), the antibody of clone G2-11 which recognizes the region of 33 to 42 as an epitope (referred to as G2-11 antibody), the antibody of clone 16C11 which recognizes the region of 33 to 42 as an epitope (referred to as 16C11 antibody), the antibody of clone 21F12 which recognizes the region of 34 to 42 as an epitope (referred to as 21F12 antibody), the antibody of clone 1A10 which recognizes the region of 35 to 40 as an epitope (referred to as 1A10 antibody), etc. These monoclonal antibodies are commercially available.

[0041] In the present embodiment, as an antibody that specifically binds to the Aβ peptide, an antibody that binds to one or both of Aβ40 and Aβ42 is preferably used. As an antibody that specifically binds to Aβ40, for example, an antibody of clone 1A10 can be cited. As an antibody that specifically binds to Aβ42, for example, antibodies of clones H31L21, G2-11, 16C11, and 21F12 can be cited. As an antibody that binds to both of Aβ40 and Aβ42, for example, antibodies of clones 82E1, 6E10, WO-2, and 2H4 can be cited. By using these antibodies, Aβ40 and / or Aβ42 can be selectively captured among the Aβ peptides in the blood sample. At this time, in the freeing step of the measurement method of the present embodiment, at least one of Aβ40 and Aβ42 can be freed, and in the measurement step, at least one of the measured values ​​of Aβ40 and Aβ42 can be obtained.

[0042] In a preferred embodiment, an antibody is used that binds to both Aβ40 and Aβ42. In this case, in the releasing step of the measuring method of this embodiment, Aβ40 and Aβ42 can be released, and in the measuring step, the measured value of Aβ40 and the measured value of Aβ42 can be obtained.

[0043] In the determination method of the present embodiment, in order to selectively obtain the Aβ peptide captured by the antibody, it is preferred to form a complex of the Aβ peptide and the antibody on a solid phase. A complex can be formed on the solid phase by bringing a solution containing the complex into contact with a solid phase on which the antibody can be fixed. Alternatively, an antibody that specifically binds to the Aβ peptide can be fixed to a solid phase in advance and used. By using an antibody fixed to a solid phase, a complex can be formed on the solid phase. Specifically, a complex is formed on the solid phase by mixing a solid phase on which an antibody that specifically binds to the Aβ peptide is fixed and a blood sample. Furthermore, by separating the unreacted components from the solid phase and recovering the solid phase, the complex can be selectively obtained.

[0044] The solid phase can be any insoluble carrier that can fix the antibody. The embodiment of fixing the antibody to the solid phase is not particularly limited. For example, the antibody and the solid phase can be directly bonded, or the antibody and the solid phase can be indirectly bound through another substance. As a direct bond, for example, physical adsorption can be mentioned. As an indirect bond, for example, a molecule that specifically binds to the antibody is immobilized on the solid phase, and the antibody is fixed to the solid phase through the binding of the molecule and the antibody. As molecules that specifically bind to the antibody, protein A or G, antibodies that specifically recognize antibodies (secondary antibodies), etc. can be mentioned. In addition, the antibody can also be fixed to the solid phase using a combination of substances between the antibody and the solid phase. As a combination of such substances, a combination of biotin and avidin, a hapten and an anti-hapten antibody, etc. can be mentioned. Biotin contains biotin, and biotin analogs such as desthiobiotin and oxybiotin. Avidin contains avidin, and avidin analogs such as streptavidin and Tamavidin (registered trademark). As a combination of a hapten and an anti-hapten antibody, a combination of a compound having a 2,4-dinitrophenyl (DNP) group and an anti-DNP antibody can be mentioned. For example, by using an antibody pre-modified with biotin (or a compound having a DNP group) and a solid phase pre-bound with avidins (or an anti-DNP antibody), the antibody can be fixed on the solid phase through the binding of biotin and avidins (or the binding of the DNP group and the anti-DNP antibody).

[0045] The raw material of the solid phase is not particularly limited, and can be, for example, selected from organic polymer compounds, inorganic compounds, biopolymers, etc. As organic polymer compounds, latex, polystyrene, polypropylene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylate copolymer, polyvinyl acetate acrylate, etc. can be cited. As inorganic compounds, magnetic bodies (iron oxide, chromium oxide, cobalt and ferrite, etc.), silicon oxide, aluminum oxide, glass, etc. can be cited. As biopolymers, insoluble agarose, insoluble dextran, gelatin, cellulose, etc. can be cited. It is also possible to use two or more of these in combination. The shape of the solid phase is not particularly limited, and can be, for example, particles, microplates, microtubes, test tubes, etc. Among them, particles are also preferred, and magnetic particles are particularly preferred. When particles are used as the solid phase, the formation process of the complex in the assay method of the present embodiment is equivalent to a general immunoprecipitation method.

[0046] Next, in the determination method of the present embodiment, the Aβ peptide is released from the complex by an alkaline solution containing an organic solvent (hereinafter, also referred to as a "freeing reagent"). The freeing reagent is believed to have the effect of dissociating the binding of the antibody and the Aβ peptide. In a preferred embodiment, a solution containing a complex formed on a solid phase and a freeing reagent are mixed. Thus, the Aβ peptide is released from the complex, and free Aβ peptide and a solid phase to which the antibody is fixed exist 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 centrifugal separation or magnetic separation.

[0047] The conditions for contacting the complex with the free reagent are not particularly limited. For example, the free reagent and the complex are contacted at a temperature of 4° C. to 42° C. and allowed to stand or stir for 1 minute to 10 minutes. The amount of the free reagent used is not particularly limited. For example, it can be appropriately determined in the range of 10 μL to 50 μL per sample.

[0048] The free reagent can be obtained by mixing an alkaline solution and an organic solvent, or by mixing water, an alkaline substance and an organic solvent. The alkaline solution can be obtained by mixing water and an alkaline substance. Commercially available alkaline solutions such as ammonia water can also be used. As the alkaline substance, for example, substances that provide ammonium ions can be mentioned. As the substance that provides ammonium ions, for example, ammonia, ammonium carbonate, ammonium bicarbonate, etc. can be mentioned. The alkaline substance in the free reagent can be one kind, or two or more kinds.

[0049] As the organic solvent, for example, acetonitrile, acetone, 1-propanol, 2-propanol, hexane, ethanol, dimethyl sulfoxide or methanol etc. can be mentioned. The organic solvent in the free reagent can be one kind, or can be more than two kinds. In the present embodiment, the free reagent preferably contains at least acetonitrile as an organic solvent, and more preferably contains only acetonitrile as an organic solvent.

[0050] The concentration of the organic solvent in the dissolution reagent is not particularly limited, but is preferably 20% or more, 30% or more, or 40% or more. In addition, it is preferably 65% ​​or less, 60% or less, or 55% or less. In this specification, the concentration "%" of the organic solvent is all volume / volume % (v / v%).

[0051] The pH of the free reagent is not particularly limited as long as it is a pH recognized as alkaline by a person skilled in the art. The pH of the free 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 free reagent is 11.4 or more. Thus, the Aβ peptide can be particularly effectively freed from the complex. In addition, the pH of the free 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.

[0052] The pH of the free reagent can be adjusted by the amount (or concentration) of the alkaline substance added. When ammonia or its salt is used as the alkaline substance, the molar concentration of ammonium ions in the free reagent is preferably 5.29mM or more, 10.57mM or more, 26.43mM or more, 52.85mM or more, 105.71mM or more, or 132.14mM or more. In addition, the molar concentration is preferably 1586mM or less, 1533mM or less, or 1480mM or less. For example, when the alkaline substance is ammonia, the concentration of ammonia in the free 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. Furthermore, in this specification, the concentration "%" of ammonia is all weight / weight % (w / w%).

[0053] The determination method of this embodiment may also include a washing step for removing unreacted free components that do not form a complex between the complex formation step and the free step. This washing step includes B / F (Bound / Free) separation. Unreacted free components refer to components that do not constitute a complex, and examples thereof include antibodies that do not bind to Aβ peptides. The washing method is not particularly limited. For example, when the complex is formed on a solid phase, when the solid phase is particles, the complex and the unreacted free components can be separated by recovering the particles by centrifugation or magnetic separation and removing the supernatant. When the solid phase is a container such as a microplate or microtube, the complex and the unreacted free components can be separated by removing the liquid containing the unreacted free components. After removing the unreacted free components, the solid phase that captures the complex may also be washed with a suitable aqueous medium such as PBS.

[0054] Furthermore, in the determination method of the present embodiment, the free Aβ peptide is determined by mass spectrometry. The mass spectrometry is not particularly limited as long as the free Aβ peptide can be determined, and a known ionization method that can measure the Aβ peptide can be used. As such an ionization method, for example, an electron spray ionization (ESI) method, an atmospheric pressure chemical ionization (APCI) method, or a matrix-supported laser desorption ionization (MALDI) method can be cited. Among these, the ESI method is particularly preferred.

[0055] The mass spectrometer used in mass spectrometry is not particularly limited and can be suitably selected from known mass spectrometers. For example, quadrupole (Q) type mass spectrometer, ion capture type (IT) type mass spectrometer, time of flight (TOF) type mass spectrometer, Fourier transform ion cyclotron resonance (FTICR) type mass spectrometer, IT-TOF type mass spectrometer, Q-TOF type mass spectrometer, and triple quadrupole (QqQ) type mass spectrometer etc. can be enumerated. Among these, it is preferably measured using a mass spectrometer that can carry out MS / MS measurement, and it is more preferably measured using a triple quadrupole type mass spectrometer.

[0056] Among mass spectrometry methods, liquid chromatography-mass spectrometry (LC-MS) combining a mass spectrometer and a liquid chromatograph is preferably used, and LC-MS / MS combining a mass spectrometer capable of liquid chromatography and MS / MS measurement is more preferably used.

[0057] As a liquid chromatography apparatus, there is no particular limitation as long as it can be connected to a mass spectrometer, and a commercially available HPLC (High Performance Liquid Chromatography) apparatus can be used. The column connected to the liquid chromatography apparatus is not particularly limited, and a commercially available HPLC column can be used. In this embodiment, the Aβ peptide released into the free reagent can be supplied to the liquid chromatography apparatus. Therefore, any pre-measurement treatment such as solvent replacement is not required.

[0058] The column is not particularly limited, for example, a reverse phase column can be used. As the packing material of the reverse phase column, for example, a silicon oxide-based packing material, a polymer-based packing material, etc. can be cited. Among them, a silicon oxide-based packing material is also preferred. As a reverse phase column having a silicon oxide-based packing material, an alkali-resistant ODS column is more preferred.

[0059] As the solvent (mobile phase) used in liquid chromatography, a solution of the same composition as the free reagent or an alkaline solution used in the free reagent can be used. The concentration of the organic solvent in the mobile phase is not particularly limited and can be appropriately set corresponding 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 a gradient method using a combination of multiple mobile phases of different compositions.

[0060] The flow rate of the mobile phase in the determination can be suitably set corresponding to the properties of the material, pressure resistance, etc. of the device and the post. The flow rate of the mobile phase is preferably set to a flow rate that suitably performs mass spectrometry. In addition, the introduction amount of the sample of the column temperature or the determination device, etc., can also be adapted to the determination device and the post and suitably set.

[0061] In this embodiment, the measurement can be performed using a liquid chromatography device, and sample-related information can also be obtained. Examples of detectors for the measurement include UV detectors, fluorescence detectors, refractive index detectors, conductivity detectors, and electrochemical detectors.

[0062] The measurement of Aβ peptide by mass spectrometry can be appropriately set from known techniques according to the ionization method and mass spectrometer used. Among them, when using a triple quadrupole mass spectrometer, Aβ peptide is preferably measured by multiple reaction monitoring (MRM) measurement in which the measurement mode is set to positive ion measurement mode.

[0063] The triple quadrupole mass spectrometer is sandwiched between a collision cell (Q2) and has a first quadrupole (Q1) and a third quadrupole (Q3) before and after it. The object to be measured is ionized in the ion source to become precursor ions, and only ions with a specific mass-to-charge ratio (m / z) set by Q1 are introduced into Q2 through a filter. The precursor ions with a specific mass-to-charge ratio introduced into Q2 collide with an inactive gas and break to become product ions. The product ions transmitted from Q2 to Q3 pass through a filter in Q3 again, and only ions with a specific mass are transmitted from Q3 to the detector through the filter, and a signal is detected. Thus, specific product ions for specific precursor ions are detected by the detector. Among them, the combination of a specific mass-to-charge ratio set by Q1 and a specific mass-to-charge ratio set by Q3 is called an MRM conversion. By setting multiple MRM conversions in the MRM measurement, multiple product ions can be detected simultaneously. Thus, multiple substances contained in the object to be measured can be measured simultaneously. For example, by setting separate MRM transitions for samples containing Aβ40 and Aβ42, two types of Aβ peptides can be measured simultaneously to obtain respective measurement values.

[0064] In the MRM measurement, the measurement mode is not particularly limited, and it is preferred to use the mass spectrometer in the positive ion measurement mode. In addition, the cone voltage and collision energy in the MRM measurement are not particularly limited, and can be appropriately set to suitable conditions by those skilled in the art.

[0065] For example, the MRM transition for Aβ40 may be set to precursor ion / product ion = 1083 / 1953.6, and the MRM transition for Aβ42 may be set to precursor ion / product ion = 1129 / 1078.5. By setting such MRM transitions, Aβ peptide can be detected with high sensitivity.

[0066] In the measurement method of this embodiment, the blood sample and the labeled internal standard substance can also be mixed to obtain the measurement value of the Aβ peptide based on the measurement value of the internal standard substance. As an internal standard substance, it is not particularly limited as long as it is a substance that can quantify the Aβ peptide, and preferably it is an Aβ peptide labeled with a stable isotope. As an Aβ peptide labeled with a stable isotope, for example, Aβ40 and Aβ42 labeled with N15 stable isotope can be cited. 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 measuring device, and can be appropriately set according to the analytical capability of the measuring device.

[0067] Along with the MRM conversion for detecting Aβ peptide, the MRM conversion for Aβ peptide labeled with a stable isotope as an internal standard is also set and measured to obtain a measured value. The MRM conversion for Aβ40 labeled with N15 stable isotope can be set to precursor ion / product ion = 1096 / 1066.5, and the MRM conversion for Aβ42 labeled with N15 stable isotope can be set to precursor ion / product ion = 1142.5 / 1091.5.

[0068] A further embodiment is a reagent composition used in the determination method of this embodiment. This reagent composition is an alkaline solution containing an organic solvent. The alkaline solution containing an organic solvent is as described above. By using the reagent composition, the Aβ peptide can be efficiently released from the complex of the Aβ peptide and the antibody that specifically binds to the Aβ peptide.

[0069] The pH of the reagent composition is not particularly limited as long as it is a pH recognized as alkaline by a person skilled in the art. The pH of the reagent composition 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 reagent composition is 11.4 or more. Thus, the Aβ peptide can be particularly effectively released from the complex. In addition, the pH of the reagent composition 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.

[0070] The reagent composition may also contain additives such as stabilizers to the extent that it does not affect the release of the Aβ peptide from the complex and the determination of the Aβ peptide by mass spectrometry. As additives, monosaccharides such as bovine serum albumin (BSA), human albumin, ovalbumin, glucose, disaccharides such as maltose, sugar alcohols such as mannitol and sorbitol, or amino acids such as glycine can be cited. The additive may be one or more. It is preferred that the additive be contained in a range of 0 to 20 parts by weight relative to 100 parts by weight of an alkaline solution containing an organic solvent. In addition, the reagent composition may also be contained in a suitable container that does not affect the composition of the reagent composition. Figure 1 FIG. 1 is a diagram showing an example of a container 151 storing the reagent composition.

[0071] Hereinafter, the present invention will be described in more detail by way of examples.

[0072] [Example]

[0073] [Example 1: Measurement of plasma Aβ peptide using a combination of immunoprecipitation using an alkaline solution containing an organic solvent and mass spectrometry]

[0074] Aβ peptide is released from a complex of Aβ peptide and an antibody that specifically binds to Aβ peptide using an alkaline solution containing an organic solvent, and Aβ peptide is measured using mass spectrometry.

[0075] (1) Capture and release of Aβ peptide using immunoprecipitation

[0076] (1.1) Blood sample

[0077] As blood samples containing Aβ peptide, five types of commercially available plasma samples from different batches (ProMedeX) were used.

[0078] (1.2) Antibodies that specifically bind to Aβ peptide

[0079] As an antibody that specifically binds to Aβ peptide, 6E10 antibody (BioLegend), which is a commercially available mouse monoclonal anti-Aβ antibody, was used. 6E10 antibody was immobilized on magnetic particles (M-270 Epoxy-activated Dynabeads: Thermo Fisher Scientific) by a conventional method.

[0080] (1.3) Aβ peptide

[0081] For the preparation of the calibration curve, Aβ40 peptide and Aβ42 peptide were purchased from AnaSpec. As the internal standard substance, the stable isotope 15 N-labeled Aβ40 peptide and Aβ42 peptide 15N-Aβ40 and 15N-Aβ42 (rPeptide). Aβ40 peptide was suspended in a PBS solution containing 3% BSA at a final concentration 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 peptide was suspended in a PBS solution containing 3% BSA at a final concentration 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 a PBS solution containing 3% BSA at a concentration of 500 pg / ml each.

[0082] (1.4) Preparation of alkaline solution containing organic solvent

[0083] As an alkaline solution containing an organic solvent (free reagent), 1.2 ml of 28% concentrated ammonia water (NACALAITESQUE) and 6.0 ml of acetonitrile (Kanto Chemical Co., Ltd.) were added to 12.8 ml of pure water and mixed to prepare a 1.68% ammonia / 30% acetonitrile solution.

[0084] (1.5) Immune Sedimentation

[0085] Add 250 μl of plasma sample or each Aβ40 peptide solution or Aβ42 peptide solution prepared in (1.3) above to a 1.5 ml sample tube (Eppendorf). Add 250 μl of the solution containing 15N-Aβ40 and 15N-Aβ42 prepared in (1.3) above to each sample tube containing the above solution, and let it stand at room temperature for 30 minutes. After standing, add 40 μl of the suspension of magnetic particles (4 μg antibody / 0.4 mg magnetic particles) immobilized with the 6E10 antibody prepared in (1.2) above to each sample solution, and mix by inversion at room temperature for 1 hour using a rotator to form a complex of Aβ peptide and antibody. Collect these solutions using a magnetic stand and remove the supernatant.

[0086] (1.6) Cleaning process

[0087] After removing the supernatant, add 1 mL of PBS solution containing 3% BSA to the magnetic particles remaining in the sample tube and mix, then collect the magnet and remove the supernatant. This operation is repeated twice in 1 mL of PBS solution containing 3% BSA, twice in 1 mL of 50 mM ammonium acetate solution, and once in 1 mL of ultrapure water to wash the magnetic particles.

[0088] Freeing of Aβ peptide from (1.7) complex

[0089] After washing the magnetic particles in (1.6), 25 μL of the freeing reagent prepared in (1.4) was added to the remaining magnetic particles after the washing solution was removed, mixed, and allowed to stand for 1 minute. The magnetic particles were re-magnetized and the supernatant was recovered as the eluate.

[0090] (2) Mass spectrometry

[0091] The eluate prepared in (1.7) was subjected to LC-MS / MS and subjected to MRM measurement to measure Aβ peptide. ACQUITY(registered trademark) UPLC(registered trademark) H-type Biosystem (Waters: hereinafter also referred to as UPLC) was used in the liquid chromatography part of LC-MS / MS. As a column, ACQUITY(registered trademark) UPLC(registered trademark) Peptide BEH C was used as a reverse phase column. 18Column (Waters). As a mass spectrometer, a Xevo (registered trademark) TQ-XS triple quadrupole mass spectrometer (Waters: hereinafter also referred to as TQ-XS mass spectrometer) was used.

[0092] Each eluate was placed in an automatic sampler of UPLC, and 10 μl of the eluate was introduced into UPLC to perform fractionation with a gradient. The gradient conditions were as follows.

[0093]

Table 1

[0094]

[0095] The eluate eluted from the column was supplied to a TQ-XS mass spectrometer directly connected to the UPLC. The TQ-XS mass spectrometer uses an electrospray ionization method and measures in a positive ion mode. The conditions for MRM measurement are set as shown in Table 2 below.

[0096]

Table 2

[0097] Precursor ion (m / z) · 4+ Product ion (m / z) ·4+ Cone voltage (V) Collision Energy(eV) Aβ40 1083.4 1054.0 32 22 Aβ42 1129.5 1078.8 28 25 15N-Aβ40 1096 1066.5 32 22 15N-Aβ42 1142.6 1091.5 28 25

[0098] (3) Measurement results

[0099] The results of the LC-MS / MS measurements are shown in Figure 2 , Figure 3A and Figure 3B .exist Figure 2 The results of MRM measurements for Aβ40 peptide, Aβ42 peptide, 15N-Aβ40 and 15N-Aβ42 are shown. Figure 3A and Figure 3B The measured Aβ40 peptide and Aβ42 peptide concentrations in the plasma test subjects are shown. These results show that Aβ peptide can be released from the complex using an alkaline solution containing an organic solvent and measured using mass spectrometry.

[0100] Figure 4A and Figure 4B The results of preparing a calibration curve based on the measured values ​​of the Aβ peptide with a known concentration prepared in (1.3) above are recorded. Figure 4A and Figure 4B Similarly, in either Aβ40 peptide or Aβ42 peptide, R 2 The calibration curve was 0.999 or higher. In addition, Aβ peptides at low concentrations of 100 pg / ml or less could be detected. Therefore, it was shown that the above-mentioned measurement method combining an alkaline solution containing an organic solvent and mass spectrometry can measure Aβ peptides with high sensitivity and has excellent quantitative properties.

[0101] [Example 2: Comparison of dissolution efficiency]

[0102] The composition of the free reagent was changed, and the dissolution efficiency of the Aβ peptide from the complex due to the difference in the composition of the free reagent was calculated based on the following calculation formula and compared.

[0103] [Dissolution efficiency (%)] = [Aβ peptide concentration of sample C] / ([Aβ peptide concentration of sample A] - [Aβ peptide concentration of sample B]) × 100

[0104] (1) Comparison of alkaline substances in free reagents

[0105] (1.1) Preparation of free reagent

[0106] DDM (n-dodecyl-β-D-maltoside, Sigma-Aldrich), acetonitrile and / or 28% concentrated ammonia water were appropriately selected and mixed in pure water to form the composition of the following Table 3, and various free reagents were modulated. Among them, the solution without alkaline substances or organic solvents was referred to as free reagent. After modulation, the pH of the solution containing alkaline substances was measured using a pH meter (Horiba Co., Ltd.).

[0107]

Table 3

[0108] Free reagent composition pH Comparative reagent 1 DDM, 70% acetonitrile Comparative reagent 2 DDM, 0.056% ammonia 11.013 Comparative reagent 3 DDM, 0.56% ammonia 11.582 Reagent 1 50% acetonitrile, 0.028% ammonia 10.854 Reagent 2 50% acetonitrile, 0.07% ammonia 11.059 Reagent 3 50% acetonitrile, 0.14% ammonia 11.219 Reagent 4 50% acetonitrile, 0.28% ammonia 11.448 Reagent 5 50% acetonitrile, 0.56% ammonia 11.613 Reagent 6 50% acetonitrile, 1.12% ammonia 11.801 Reagent 7 50% acetonitrile, 1.68% ammonia 11.973 Reagent 8 50% acetonitrile, 2.24% ammonia 11.974 Reagent 9 50% acetonitrile, 2.80% ammonia 12.038

[0109] (1.2) Sample preparation

[0110] 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 of sample A corresponds to the initial concentration of the Aβ40 peptide.

[0111] (1.3) Immune sedimentation

[0112] Sample A prepared in (1.2) above was subjected to immunoprecipitation in the same manner as in (1.5) of Example 1 to recover magnetic particles. At this time, the supernatant after magnetic collection was recovered and stored as sample B. The Aβ peptide concentration of sample B corresponds to the concentration of Aβ peptide not captured by the antibody that specifically binds to the Aβ peptide.

[0113] (1.4) Cleaning-Dissolution

[0114] For the magnetic particles recovered in (1.3) above, the washing operation is performed in the same manner as in (1.6) of Example 1, and the washing solution is removed. 15 μl of each freeing reagent prepared in (1.1) above is added to the washed magnetic particles, and the mixture is allowed to stand for 1 minute to release the Aβ peptide. After standing, the magnetic particles are magnetized and the supernatant is recovered. The recovered supernatant is 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 of sample C corresponds to the concentration of the free Aβ peptide after capture by immunoprecipitation.

[0115] (2) Determination by immunoassay

[0116] For the above-mentioned samples A, B and C, the Aβ peptide concentration in each sample was measured by immunoassay using the fully automatic immunoassay apparatus HISCL (registered trademark)-5000 (Sysmex Corporation). The 82E1 antibody was labeled with biotin by a conventional method and dissolved in a buffer solution containing 1% BSA, 0.1M Tris-HCl, 0.15M NaCl, and 0.1% NaN3 at pH 7.5 to prepare R1 reagent (capture antibody reagent). As R2 reagent (solid phase), HISCL (registered trademark) R2 reagent (Sysmex Corporation) containing streptavidin-bound magnetic particles was used. The 1A10 antibody was labeled with alkaline phosphatase (ALP) by a conventional method and dissolved in a buffer solution containing 1% BSA, 0.1M Tris-HCl, 0.15M NaCl, and 0.1% NaN3 at pH 7.5 to prepare R3 reagent (detection antibody reagent). As the R4 reagent (measurement buffer), HISCL (registered trademark) R4 reagent (Sysmex Corporation) was used. As the R5 reagent (ALP substrate solution), HISCL (registered trademark) R5 reagent (Sysmex Corporation) was used.

[0117] The measurement procedure of HISCL (registered trademark)-5000 is as follows. Sample A, B or C (30 μL) and R1 reagent (110 μL) are mixed and reacted at 42°C for 4 minutes. After the reaction, R2 reagent (30 μL) is added and reacted at 42°C for 3 minutes. The magnetic particles in the obtained mixed solution are collected and the supernatant is removed, and HISCL (registered trademark) cleaning solution (300 μL) is added to wash the magnetic particles. After the supernatant is removed, R3 reagent (100 μL) is added to the magnetic particles and mixed, and reacted at 42°C for 5 minutes. The magnetic particles in the obtained mixed solution are collected and the supernatant is removed, and HISCL (registered trademark) cleaning solution (300 μL) is added to wash the magnetic particles. After the supernatant is removed, R4 reagent (50 μL) and R5 reagent (100 μL) are added to the magnetic particles, and the chemiluminescence intensity is measured. As a calibrant (antigen for preparing a calibration curve), Aβ40 peptide was suspended in a solution containing 0.1% BSA, 0.14M triethanolamine, 0.15M NaCl, 0.1% NaN3 at pH 7.0 to prepare a calibration curve by similar measurement. The chemiluminescence intensity obtained in the measurement was applied to the calibration curve to determine the concentration of Aβ40 peptide.

[0118] (3) Measurement results

[0119] The results of the measurement of the dissolution efficiency of the comparative reagents 1 to 3 and reagents 1 to 9 in Table 3 are shown in Figure 5 .like Figure 5 As shown in the results, the dissolution efficiency is low when using only an organic solvent or only an alkaline solution as a free reagent for Aβ peptide, but a high dissolution efficiency is obtained when using a free reagent containing an organic solvent and an alkaline substance. In particular, it is shown that a reagent with a pH of 11.4 or more and 12.0 or less has a high dissolution efficiency exceeding 70%.

[0120] (4) Comparison of organic solvents for free reagents

[0121] In the above (1.1), the free reagents in Table 4 below were used instead of the free reagents in Table 3, and the dissolution efficiency was measured. In comparative reagent 5 and samples 17 to 23, Aβ42 peptide was used as a sample instead of Aβ40 peptide. When Aβ42 peptide was used, H31L21 antibody was used as a detection antibody instead of 1A10 antibody, and as a calibrator, 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 was used. Except for the above, the same operation as (1) and (2) was performed to measure the dissolution efficiency for each free reagent.

[0122]

Table 4

[0123] Free reagent composition Free reagent composition Comparative Reagent 4 0.56% Ammonia Comparative Reagent 5 0.56% Ammonia Reagent 10 40% acetonitrile, 0.56% ammonia Reagent 17 40% acetonitrile, 0.56% ammonia Reagent 11 40% acetone, 0.56% ammonia Reagent 18 40% acetone, 0.56% ammonia Reagent 12 40% 2-propanol, 0.56% ammonia Reagent 19 40% 2-propanol, 0.56% ammonia Reagent 13 40% hexane, 0.56% ammonia Reagent 20 40% hexane, 0.56% ammonia Reagent 14 40% 1-propanol, 0.56% ammonia Reagent 21 40% 1-propanol, 0.56% ammonia Reagent 15 40% ethanol, 0.56% ammonia Reagent 22 40% ethanol, 0.56% ammonia Reagent 16 40% DMSO, 0.56% ammonia Reagent 23 40% DMSO, 0.56% ammonia

[0124] The measurement results of Comparative Reagent 4 and Samples 10 to 16 are shown in Fig. 6A The measurement results for comparative reagent 5 and samples 17 to 23 are shown in Figure 6B .from Fig. 6A and Figure 6B It was found that samples 10 to 16 and 17 to 23 showed superior dissolution efficiency compared to comparative reagents 4 and 5 not containing an organic solvent, and that superior dissolution efficiency was obtained by the free reagent containing an alkaline substance and an organic solvent.

[0125] [Example 3: Carryover Determination]

[0126] Conventionally, an acidic solution is used to release Aβ peptide from a complex of Aβ peptide captured by an antibody and an antibody, and the carryover of Aβ peptide to a liquid chromatography apparatus under acidic-alkaline conditions of a free reagent for eluting the complex is measured.

[0127] (1) Sample preparation

[0128] As an acidic free reagent, formic acid (Fuji Film Wako Pure Chemical Industries, Ltd.) and acetonitrile were mixed with pure water to prepare an acidic free reagent (wherein the acidic solution is also referred to as a free reagent) having a composition of 0.1% formic acid and 50% acetonitrile. In addition, a free reagent (1.68% ammonia, 50% acetonitrile) was prepared in the same manner as in (1.4) of Example 1. To these free reagents, the same Aβ40 peptide as in (1.3) of Example 1 was added (a solution containing Aβ40 peptide) in such a manner that the final concentration became 100 fmol / μl. In addition, for each of the acidic free reagent and the alkaline free reagent, a solution not containing Aβ40 peptide (a solution not containing Aβ40 peptide) was also prepared.

[0129] (2) Introduction of sample into liquid chromatography

[0130] The four solutions prepared in (1) above were subjected to LC-MS / MS. The LC-MS / MS apparatus and the columns used were the mass spectrometry method described in (2) of Example 1. Each solution was placed in an automatic sampler of UPLC, and 10 μl of each solution was introduced into UPLC for fractional separation. The analysis conditions of UPLC were as shown in Table 5 below. The conditions for MRM measurement in mass spectrometry were as shown in Table 2 of Example 1.

[0131]

Table 5

[0132]

[0133] The signal intensities obtained by supplying the two solutions containing Aβ40 peptide prepared in (1) above and the corresponding solutions not containing Aβ40 to UPLC in the order of a solution not containing Aβ40 peptide (background), a solution containing Aβ40 peptide, and a solution not containing Aβ40 peptide (carryover measurement) are shown in the table below. Figure 7 .from Figure 7 It was found that when an acidic free reagent was used, a large amount of carryover occurred compared to when an alkaline free reagent was used. On the other hand, when an alkaline free reagent was used as a free reagent for Aβ peptide, carryover was significantly suppressed. This indicates that the use of an alkaline free reagent is suitable for continuous analysis of Aβ peptide using mass spectrometry.

[0134] [Example 4: Comparison of acidity and alkalinity of free reagents]

[0135] When Aβ peptide released by using an acidic releasing agent was measured by LC-MS / MS as in Example 3, carryover occurred. When Aβ peptide was released from the complex, the difference in the amount of Aβ peptide detected when using an alkaline releasing agent and when using an acidic releasing agent during release but then replacing it with an alkaline releasing agent and subjecting to LC-MS / MS was compared.

[0136] (1) Sample preparation

[0137] As Aβ peptide samples, the same Aβ40 peptide and Aβ42 peptide as in Example 1 (1.3) were prepared, and solutions prepared in a 3% BSA solution such that the Aβ40 peptide was 50 pg / ml or 190 pg / ml, or solutions prepared in a 3% BSA solution such that the Aβ42 peptide was 26 pg / ml or 103 pg / ml were used. As an acidic freeing agent, trifluoroacetic acid (TFA, Fuji Film Co., Ltd. Wako Pure Chemical Industries, Ltd.) and acetonitrile were mixed with pure water to prepare an acidic solution having a composition of 0.1% TFA and 30% acetonitrile. As an alkaline freeing agent, a 1.68% ammonia and 50% acetonitrile solution was prepared in the same manner as in Example 1 (1.4).

[0138] (2) Determination

[0139] (2.1) Immune sedimentation

[0140] 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 on which the 6E10 antibody was immobilized were added to form a complex.

[0141] (2.2) Dissolution of Aβ peptide

[0142] After the same washing step as in (1.6) of Example 1 was performed on each complex prepared in (2.1), 25 μL of the acidic or alkaline dissociation reagent prepared in (1.1) was added and mixed, and allowed to stand for 1 minute. The magnetic particles were remagnetized and the supernatant was recovered as an eluate.

[0143] (2.3) Solvent replacement

[0144] Among the eluates recovered in (2.2) above, the eluate obtained using the acidic free reagent was dried under reduced pressure for 1 hour using a spin dryer standard product VC-96R (TAITEC Co., Ltd.) in the SpinDryer mode at a speed of 1500 r / min and a temperature of 55° C. to volatilize the solvent. To the residue after the reduced pressure drying, 25 μL of the alkaline free reagent prepared in (1) above was added to resuspend the residue.

[0145] (2.4) Mass spectrometry

[0146] The eluate obtained by using each alkaline free reagent recovered in (2.2) above (referred to as solution X) and the solution resuspended in (2.3) above (referred to as solution Y) were subjected to LC-MS / MS in the same manner as in (2) of Example 1, and MRM measurement was performed.

[0147] (3) Measurement results

[0148] The measurement results of solution A and solution B are shown in Figures 8A to 9D . Fig. 8A The solution X using 190 pg / ml of Aβ peptide 40 is shown. Figure 8B The results show that solution Y using 190 pg / ml of Aβ40 peptide, Figure 8C Solution X using 103 pg / ml of Aβ42 peptide, Fig.8D Solution Y using 103 pg / ml of Aβ42 peptide, Fig.9A The solution X using 50 pg / ml of Aβ40 peptide is shown. Fig. 9B The results show that solution Y using 50 pg / ml of Aβ40 peptide, Fig. 9C Solution X using 26 pg / ml of Aβ42 peptide is shown. Fig.9D The measurement results of solution Y using 26 pg / ml Aβ42 are shown. These results show that, in the case of all concentrations of Aβ peptide, the square area value is larger when solution X is used than when solution Y is used. In the sample treatment, when the same process as (2.3) above is used, it is suggested that Aβ peptide loss occurs.

[0149]

Explanation of symbols

[0150] 151: Reagent container Sequence Listing <110> SYSMEX CORPORATION <120> Aβ peptide measurement method and reagent composition used in the method <130> 20-003JP <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 40 <212> PRT <213> Homo sapiens <400> 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val 35 40 <210> 2 <211> 42 <212> PRT <213> Homo sapiens <400> 2 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40

Claims

1. A method for determining Aβ peptide, comprising: a step of mixing a blood sample containing Aβ peptide and an antibody that specifically binds to the Aβ peptide to form a complex of the Aβ peptide and the antibody; a step of releasing the Aβ peptide from the antibody in the complex using an alkaline solution containing an organic solvent, and a step of measuring the free Aβ peptide by mass spectrometry, in, The organic solvent is at least one selected from the group consisting of acetonitrile, acetone, 1-propanol, 2-propanol, hexane, ethanol, dimethyl sulfoxide and methanol. The concentration of the organic solvent is 30% or more and 50% or less, and The pH of the alkaline solution is 10.85 or more and 14.0 or less.

2. The method of claim 1, wherein the pH of the alkaline solution is above 11.

4.

3. The method of claim 1, wherein the alkaline solution contains ammonium ions.

4. The method of claim 1, wherein The antibody that specifically binds to Aβ peptide is an antibody that binds to one or both of Aβ40 and Aβ42, In the releasing step, the Aβ peptide released from the complex is at least one of Aβ40 and Aβ42, In the measurement step, a measurement value of at least one of Aβ40 and Aβ42 is acquired.

5. The method of claim 4, wherein The antibody that binds to Aβ peptide is an antibody that binds to both Aβ40 and Aβ42. In the measurement step, the measurement values ​​of Aβ40 and Aβ42 are obtained. The method of claim 1 , wherein the antibody that specifically binds to the Aβ peptide is immobilized on a solid phase.

7. The method of claim 1, wherein the mass spectrometry is liquid chromatography mass spectrometry.

8. The method of claim 7, wherein a reverse phase column is used in the liquid chromatography mass spectrometry.

9. The method according to any one of claims 1 to 8, wherein The blood sample is mixed with a labeled internal standard substance. In the measurement step, the measurement value of the Aβ peptide is acquired based on the measurement value of the internal standard substance.

10. The method of claim 9, wherein the internal standard substance is Aβ peptide labeled with a stable isotope.

11. Use of an alkaline solution containing an organic solvent in the manufacture of a reagent composition for measuring Aβ peptide, wherein The organic solvent is at least one selected from the group consisting of acetonitrile, acetone, 1-propanol, 2-propanol, hexane, ethanol, dimethyl sulfoxide and methanol. The concentration of the organic solvent is 30% or more and 50% or less, and The pH of the alkaline solution is 10.85 or more and 14.0 or less.

12. The use according to claim 11, wherein the pH of the alkaline solution is above 11.

4.

13. The use according to claim 11 or 12, wherein the alkaline solution contains ammonium ions.

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