Method for detecting test substance derived from amyloid-β precursor protein, method for reducing influence of chelating agent, buffer reagent, and reagent kit

Abstract

The present invention is such that a test substance derived from an amyloid-β precursor protein in a blood sample, a substance that binds to the test substance, and a buffer solution are brought into contact for 6 minutes or less. The buffer solution contains a buffering agent where the concentration at the time of contact is 25 mM or more, and is such that the pH is 3.5 to 9.0. A signal derived from the binding between the test substance and the substance binding to the test substance is measured.

Description

Method for detecting a test substance derived from amyloid-β precursor protein, method for reducing the influence of a chelating agent, buffer reagent, and reagent kit

[0001] The present disclosure relates to a method for detecting a test substance derived from amyloid-β precursor protein, a method for reducing the influence of a chelating agent, a buffer reagent, and a reagent kit.

[0002] In the brain of Alzheimer's disease, there are two pathological conditions: extracellular accumulation of amyloid-β (amyloid plaque) and intracellular accumulation of phosphorylated tau in neurons (neurofibrillary changes; neurofibrillary tangles, NFT). It is presumed that the presence of amyloid-β does not directly cause neuronal death, but induces tau lesions and causes NFT. Usually, amyloid PET (Positron Emission Tomography) examination images the deposition of amyloid-β in the brain. For the diagnosis of Alzheimer's disease, the amyloid-β deposition state by amyloid PET examination is used as a judgment material.

[0003] Amyloid-β is cleaved from amyloid-β precursor protein, a membrane protein present on the cell surface, and is also called amyloid-β peptide. Amyloid-β includes amyloid-β1-40 consisting of 40 amino acid residues, amyloid-β1-42 consisting of 42 amino acid residues, and the like. It is known that amyloid-β1-42 in cerebrospinal fluid decreases in patients with Alzheimer's disease, and it is known that the ratio of amyloid-β1-42 to amyloid-β1-40 in cerebrospinal fluid shows a strong correlation with the amyloid accumulation amount by amyloid PET examination. Therefore, the amyloid-β1-42 / amyloid-β1-40 ratio in cerebrospinal fluid is used as a biomarker for grasping the accumulation state of amyloid-β in the brain.

[0004] Patent Document 1 discloses a method for immunoassaying of amyloid-beta peptide in blood in the presence of an anionic polymer. The method disclosed in Patent Document 1 is said to be able to measure amyloid-beta peptide in blood with high sensitivity. Furthermore, Patent Document 2 discloses a method for diagnosing Alzheimer's disease based on the amyloid-beta degradation activity in the blood, by adding amyloid-beta peptide to the blood and measuring the amyloid-beta peptide over time, as it is difficult to measure amyloid-beta peptide in blood.

[0005] Patent Document 1: International Publication No. 2021 / 200940 Patent Document 2: International Publication No. 2007 / 119685

[0006] Incidentally, when measuring amyloid-beta peptides in the blood, chelating agents such as ethylenediaminetetraacetic acid (EDTA), which is an anticoagulant for blood, are used. However, the inventors have found that the presence of chelating agents reduces the sensitivity of immunoassays for amyloid-beta peptides. Therefore, in view of the above circumstances, this disclosure aims to provide a method for detecting a test substance derived from amyloid-beta precursor protein, a method for reducing the effect of chelating agents, a buffer reagent, and a reagent kit that prevent the reduction in sensitivity caused by chelating agents when performing immunoassays for amyloid-beta peptides.

[0007] To achieve the above-mentioned objectives, the present inventors conducted diligent studies and found that by setting the concentration, pH, and reaction time of the buffering agent within an appropriate range, the test substance derived from amyloid-beta precursor protein in a blood sample can be detected with high sensitivity, leading to the completion of this disclosure.

[0008] This disclosure includes: <1> A method for detecting a test substance derived from amyloid-beta precursor protein, comprising contacting a test substance derived from amyloid-beta precursor protein in a blood sample with a substance that binds to the test substance and a buffer containing an amount of buffering agent such that the concentration at contact is 25 mM (moles / l) or more and the pH is 3.5 or more and 9.0 or less for 6 minutes or less, and measuring a signal derived from the binding of the test substance to the substance that binds to the test substance. <2> The method according to <1>, wherein the test substance is amyloid-beta 1-40 or amyloid-beta 1-42. <3> The method according to <1> or <2>, wherein the test substance is amyloid-beta 1-40 and the pH of the buffer is 4.0 or more and 8.0 or less. <4> The method according to any one of <1> to <3>, wherein the test substance is amyloid-beta 1-42 and the pH of the buffer is 5.0 or more and 8.2 or less. <5> The method according to any one of <1> to <4>, wherein the test substance, a substance that binds to the test substance, and a buffer are in contact for 4 minutes or less. <6> The method according to any one of <1> to <5>, wherein the concentration of the buffer at the time of contact is 50 mM or more. <7> The method according to any one of <1> to <6>, wherein the blood sample is a plasma sample derived from a whole blood sample collected in a blood collection container that has been pre-filled with a chelating agent, which is an anticoagulant. <8> A method for reducing the influence of a chelating agent in the detection of a test substance derived from amyloid-beta precursor protein, comprising contacting the test substance derived from amyloid-beta precursor protein in a blood sample, a substance that binds to the test substance, and a buffer containing an amount of buffer that has a concentration of 25 mM (molecule / l) or more at the time of contact and has a pH of 3.5 or more and 9.0 or less for 6 minutes or less, and measuring the signal derived from the binding of the test substance and the substance that binds to the test substance. <9> A buffer reagent for detecting amyloid-beta 1-40 among the test substances derived from amyloid-beta precursor protein in a blood sample, with a buffer concentration of 50 mM or higher and a pH in the range of 4.0 to 8.0. <10> A buffer reagent for detecting amyloid-beta 1-42 among the test substances derived from amyloid-beta precursor protein in a blood sample, with a buffer concentration of 50 mM or higher and a pH in the range of 5.0 to 8.2.A reagent kit for detecting amyloid-beta 1-40 or amyloid-beta 1-42 among test substances derived from amyloid-beta precursor protein in a blood sample, comprising the buffer reagent described in <11> or <9>, or the buffer reagent described in <10>. <12> A reagent kit according to <11> comprising a first reagent containing a substance immobilized on a solid phase and binding to the test substance. <13> A reagent kit according to <12> in which the buffer reagent described in <9>, or the buffer reagent described in <10>, and the first reagent are the same reagent. <14> A reagent kit according to <11> comprising a test substance detection reagent containing a labeling substance that binds to the test substance. <15> A reagent kit according to <14> in which the buffer reagent described in <9>, or the buffer reagent described in <10>, and the test substance detection reagent are the same reagent. <16> A reagent kit according to any one of <11> to <15> used for the diagnosis of Alzheimer's disease.

[0009] According to the method for detecting a test substance derived from amyloid-beta precursor protein, the method for reducing the effect of chelating agents, the buffer reagent, and the reagent kit disclosed herein, the test substance can be detected with high sensitivity even under conditions in which a chelating agent is present.

[0010] The embodiments of this disclosure are described below. The description is illustrative and does not limit the scope of this disclosure.

[0011] In the following embodiments, the components (including elemental steps, etc.) are not essential unless otherwise explicitly stated. The same applies to numerical values ​​and their ranges, and these do not limit the disclosure. For example, the disclosure allows for additions, omissions, substitutions, and changes to numbers, quantities, locations, ratios, materials, compositions, types, and sequences, etc., without departing from the intent of the disclosure.

[0012] In this disclosure, numerical ranges indicated using "~" include the numbers before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages in this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Also, in numerical ranges described in this disclosure, the upper or lower limit of that numerical range may be replaced with the values ​​shown in the examples. In this disclosure, each component may contain multiple types of the corresponding substance. If multiple types of the substance corresponding to each component exist in the composition, the content or amount of each component means the total content or amount of the multiple types of substances present in the composition, unless otherwise specified. In this disclosure, "pH" is the value at 25 ± 1°C and is measured using a pH meter.

[0013] The method for detecting a test substance derived from amyloid-beta precursor protein according to this disclosure (hereinafter referred to as the "detection method of this disclosure") includes contacting a test substance derived from amyloid-beta precursor protein in a blood sample with a substance that binds to the test substance and a buffer solution containing an amount of buffer solution having a concentration of 25 mM (moles / l; the same applies hereinafter) or more at the time of contact and a pH of 3.5 to 9.0 for 6 minutes or less, and measuring the signal derived from the binding of the test substance and the substance that binds to the test substance. In the detection method of this disclosure, by contacting the test substance and the substance that binds to the test substance under the above conditions, the test substance can be detected with excellent sensitivity even when a chelating agent is present in the blood sample. "At the time of contact" refers to the time when the test substance, the substance that binds to the test substance, and the buffer solution come into contact.

[0014] As described above, the detection method of this disclosure allows for the detection of a test substance with excellent sensitivity even when a chelating agent is present in the blood sample. In other words, the detection method of this disclosure is a method for reducing the influence of chelating agents in the detection of a test substance derived from amyloid-beta precursor protein, which includes contacting a test substance derived from amyloid-beta precursor protein in a blood sample with a substance that binds to the test substance and a buffer containing an amount of buffer that has a concentration of 25 mM or more at the time of contact and a pH of 3.5 to 9.0 for 6 minutes or less, and measuring the signal derived from the binding of the test substance and the substance that binds to the test substance.

[0015] [Blood Sample] The blood sample includes blood (e.g., peripheral blood or umbilical cord blood) collected from an animal, including a human, that is the subject of the test. The blood sample may be a whole blood sample, a plasma sample, or a serum sample, but it is preferably a whole blood sample or a plasma sample, and more preferably a plasma sample. If the blood sample is a whole blood sample or a plasma sample, it contains a chelating agent, which is an anticoagulant. In other words, the blood sample may contain a chelating agent. If the blood sample is a plasma sample, it can usually be obtained by centrifuging a whole blood sample containing a chelating agent, separating and removing the precipitated blood cell components, and then obtaining the supernatant.

[0016] Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and sodium citrate. Ethylenediaminetetraacetic acid (EDTA) is preferably used in the form of salts such as EDTA-2K, EDTA-3K, EDTA-4K, EDTA-2Na, EDTA-3Na, and EDTA-4Na. One type of EDTA salt may be used, or a mixture of multiple types may be used.

[0017] The chelating agent described above may be added to the whole blood sample to reach a predetermined concentration, or it may be pre-contained by, for example, coating the inner wall of the blood collection container (e.g., a vacuum blood collection tube) in which the whole blood sample will be placed, so that it dissolves when the whole blood sample placed in the container comes into contact with the inner wall. When the chelating agent is coated on the inner wall of the container, the concentration of the chelating agent in the blood sample depends on the amount of whole blood sample placed in the container. However, since a predetermined amount of whole blood sample is usually intended to be placed in the container, the concentration of the chelating agent will remain at a nearly constant value. If the amount of whole blood sample placed in the container is less than the predetermined amount, the chelating agent will be concentrated, resulting in a higher concentration.

[0018] In particular, in the detection method of this disclosure, the concentration of the chelating agent in the blood sample is not particularly limited, but may be 1.1 times, 1.25 times, 2.0 times, 2.5 times, or 3.0 times the specified concentration when a specified amount of whole blood sample is placed in a container. Typically, a blood sample contains, for example, 0.00% to 0.20% of the chelating agent, preferably 0.10% to 0.20%. In the detection method of this disclosure, the concentration of the chelating agent in the blood sample may be 1.1 times, 1.25 times, 2.0 times, 2.5 times, or 3.0 times the upper limit of these numerical ranges.

[0019] [Test Substance] In the detection method of this disclosure, the test substance is a peptide derived from amyloid-β precursor protein. Peptides derived from amyloid-β precursor protein are also called amyloid-β peptides and are cleaved from amyloid-β precursor protein, which is a membrane protein present on the cell surface. Known amyloid-β peptides include amyloid-β1-40, which consists of 40 amino acid residues from position 1 to 40 from the N-terminus of amyloid-β precursor protein, and amyloid-β1-42, which consists of 42 amino acid residues from position 1 to 42 from the N-terminus of amyloid-β precursor protein. Among these, it is preferable to use at least one selected from the group consisting of amyloid-β1-40 and amyloid-β1-42 as the test substance.

[0020] Furthermore, the detection method of this disclosure is not limited to amyloid β1-40 or amyloid β1-42, but may also use other amyloid β peptides. If we denote amyloid β peptides consisting of amino acids X to Y from the N-terminus of the amyloid β precursor protein as amyloid βX-Y, examples include amyloid β1-38, amyloid β1-39, amyloid β1-43, amyloid β5-42, amyloid β5-40, amyloid β11-42, amyloid β11-40, amyloid β16-42, amyloid β16-40, amyloid β17-42, amyloid β17-40, amyloid β20-42, amyloid β20-40, amyloid β21-42, amyloid β21-40, amyloid β35-42, amyloid β35-40, and the like.

[0021] [Substances that bind to the test substance] In the detection method of this disclosure, the substance that binds to the test substance is preferably a substance that specifically binds to any of the above-mentioned test substances. Examples of substances that bind to the test substance include proteins that bind to any of the above-mentioned test substances (e.g., antibodies or antibody fragments, peptides such as cyclic peptides), nucleic acids that bind to any of the above-mentioned test substances (e.g., aptamers), and substances that bind to the sugar chains of any of the above-mentioned test substances (e.g., lectins, anti-glycan antibodies). The antibody may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred. Polyclonal antibodies and monoclonal antibodies can be obtained by well-known production methods using the above-mentioned amyloid-beta peptide as an immunogen. The "antibody fragment" may be any antibody fragment as long as it maintains the binding ability (antigen-antibody reactivity) of the original antibody to the corresponding antigen. Specific examples include Fab, F(ab'). ２ Examples include, but are not limited to, Fab and F(ab'). ２As is well known, antibodies can be obtained by treating them with proteolytic enzymes such as papain or pepsin. Single-chain antibodies (scFv) can also be produced according to well-known methods. In this disclosure, the term "antibody" is used to include both full-length antibodies and antibody fragments. The substance that binds to the test substance may be one or two or more, but it is preferable to use only one. The substance that binds to the test substance may be a commercially available product or one that has been appropriately prepared by conventional methods.

[0022] For example, in the detection method of this disclosure, when detecting amyloid β1-40 and amyloid β1-42 as test substances, an antibody that specifically binds to amyloid β1-40 (anti-amyloid β1-40 antibody) and an antibody that specifically binds to amyloid β1-42 (anti-amyloid β1-42 antibody) are used. In this case, it is preferable to use an anti-amyloid β1-40 antibody that has lower cross-reactivity with amyloid β1-42. Similarly, it is preferable to use an anti-amyloid β1-42 antibody that has lower cross-reactivity with amyloid β1-40. In this case, it is preferable to use antibodies that have different C-terminal regions as epitopes in the antigens amyloid β1-40 and amyloid β1-42 as antitopes.

[0023] [Buffer Solution] In the detection method of this disclosure, the buffer solution means a buffering solution containing a buffering agent. As will be described in detail later, in the detection method of this disclosure, the above-mentioned test substance and a substance that binds to the above-mentioned test substance are brought into contact in a buffer solution. In the detection method of this disclosure, the concentration of the buffering agent in the reaction solution at the time of contact is 25 mM or higher. The concentration of the buffering agent at this time is particularly preferably 30 mM or higher, more preferably 50 mM or higher, and even more preferably 60 mM or higher. The concentration of the buffering agent in the buffer solution is defined as above, but it should be noted that this is the so-called final concentration and differs from the concentration of the buffering agent contained in the buffer solution before mixing with the blood sample, etc. The upper limit of the concentration of the buffering agent in the reaction solution is not particularly limited, but it is preferably 5000 mM or less, more preferably 2000 mM or less, and even more preferably 1000 mM or less.

[0024] A buffering agent is a compound that exhibits pH buffering properties when in solution, and is not particularly limited to any specific compound. Examples of buffering agents include weakly acidic compounds, combinations of weakly acidic compounds and their salts, weakly basic compounds, and combinations of weakly basic compounds and their salts. pH buffering properties refer to the ability to suppress rapid changes in pH and maintain the pH of a solution within a specific range, even when other acids or bases are added to a certain extent. Therefore, a solution containing a buffering agent can maintain its pH within a specific range even when acids or bases are added within a predetermined range.

[0025] Buffering agents include, but are not limited to, citrate, phosphate, succinate, cacodylate, 2-morpholinoethanesulfonic acid (referred to as MES), 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol, N-(2-acetamide)iminodiacetic acid, N-(2-acetamide)-2-aminoethanesulfonic acid, 1,4-piperazinedietanesulfonic acid, imidazole, hydroxypropanesulfonic acid, 3-morpholinopropanesulfonic acid (referred to as MOPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (referred to as TES), 4-(N-morpholino)butanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (referred to as TAPS), 2 -Hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (referred to as TAPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, piperazine-1,4-bis(2-hydroxypropanesulfonic acid), triethanolamine, 4-(2-hydroxyethyl)-1-piperazine-1-propanesulfonic acid, tris(hydroxymethyl)aminomethane (referred to as Tris), tricine, glycylglycine, bicine, 4-(2-hydroxy Examples include ethyl)piperazine-1-ethanesulfonic acid (referred to as HEPES), 2-amino-2-methyl-1,3-propanediol, 2-(hydroxyethyl)amino-1-propanesulfonic acid, boric acid, 2-cyclohexylaminoethanesulfonic acid (referred to as CHES), glycine, sodium 3-cyclohexylamino-2-hydroxypropanesulfonate, 3-cyclohexylaminopropanesulfonic acid, 4-(cyclohexylamino)-1-butanesulfonic acid, etc.

[0026] When using a weakly acidic compound or a combination of a weakly acidic compound and its salt as the buffer, the buffer solution containing these buffering agents is adjusted to a pH of 3.5 to 9.0 using an alkaline solution such as sodium hydroxide solution. Similarly, when using a weakly basic compound or a combination of a weakly basic compound and its salt as the buffer, the buffer solution is adjusted to a pH of 3.5 to 9.0 using an acidic solution such as hydrochloric acid. By maintaining the buffer solution's pH within this range, amyloid-beta peptides can be detected with excellent sensitivity even when chelating agents are present in the blood sample.

[0027] Furthermore, in the detection method of this disclosure, when the test substance is amyloid β1-40, it is more preferable to set the pH of the buffer solution to 4.0 or more and 8.0 or less, and particularly more preferable to set the pH to 4.5 or more and 8.0 or less. Also, when the test substance is amyloid β1-42, it is more preferable to set the pH of the buffer solution to 5.0 or more and 8.2 or less. By setting the pH of the buffer solution within this range, amyloid β1-40 and amyloid β1-42 can be detected with higher sensitivity, respectively.

[0028] In particular, when the test substance is amyloid-β1-40, a buffer solution with a buffer concentration of 50 mM or higher and a pH in the range of 4.0 to 8.0, preferably 4.5 to 8.0, can be used as a buffer reagent for detecting amyloid-β1-40 among the test substances derived from amyloid-β precursor protein in a blood sample. Furthermore, when the test substance is amyloid-β1-42, a buffer solution with a buffer concentration of 50 mM or higher and a pH in the range of 5.0 to 8.2 can be used as a buffer reagent for detecting amyloid-β1-42 among the test substances derived from amyloid-β precursor protein in a blood sample.

[0029] [Method for detecting the test substance] In the detection method of this disclosure, a blood sample is brought into contact with the substance that binds to the test substance as described above and the buffer solution described above, thereby binding the test substance contained in the blood sample with the substance that binds to the test substance. At this time, the concentration of the buffer solution contained in the reaction system is 25 mM or higher. In other words, in the detection method of this disclosure, the blood sample and the substance that binds to the test substance are bound in the presence of a buffer solution with a final concentration of 25 mM or higher. Furthermore, in the detection method of this disclosure, the reaction time for binding the test substance contained in the blood sample with the substance that binds to the test substance is 6 minutes or less, and it is particularly preferable that this reaction time be 4 minutes or less. In the detection method of this disclosure, the lower limit of this reaction time is not particularly limited, but it is preferably 1 minute or more, more preferably 2 minutes or more, even more preferably 2 minutes 30 seconds or more, and particularly preferably 3 minutes or more.

[0030] In the detection method disclosed herein, the signal derived from the binding of the test substance contained in the blood sample to a substance that binds to the test substance is then measured. According to the detection method disclosed herein, even if a chelating agent is included in the reaction system described above, a decrease in signal due to the chelating agent can be prevented, and the signal can be detected with high sensitivity.

[0031] In the detection method disclosed herein, the blood sample can be a plasma sample derived from a whole blood sample collected in a blood collection container that has been pre-filled with a chelating agent, which is an anticoagulant. In this case, the blood sample will contain the chelating agent. In particular, even if the collected whole blood sample is below a specified volume and the concentration of the chelating agent is higher than the expected value, the detection method disclosed herein can prevent a decrease in signal caused by the chelating agent and detect the signal with high sensitivity.

[0032] Typically, blood samples contain, for example, 0.00% to 0.20% of a chelating agent, preferably 0.10% to 0.20%. In the detection method of this disclosure, even if the concentration of the chelating agent in the blood sample is 1.1 times, 1.25 times, 2.0 times, 2.5 times, or 3.0 times the upper limit of these numerical ranges, a decrease in signal due to the chelating agent can be prevented, and the above signal can be detected with high sensitivity.

[0033] In the detection method disclosed herein, the method for measuring the signal derived from the binding of a test substance contained in a blood sample to a substance that binds to the test substance is not particularly limited, and any method may be applied. For example, the substance that binds to the test substance can be immobilized on a solid phase. As an example of such a method, as described above, an antibody can be used as the substance that binds to the test substance, and amyloid-beta peptide can be measured, for example, by the sandwich method. The sandwich method includes various techniques such as chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, electrochemiluminescence immunoassay (ECLIA), and fluorescence immunoassay (FIA), and any of these techniques can be used in the detection method disclosed herein. Furthermore, the sandwich method includes both one-step and two-step methods, both of which are applicable.

[0034] In the two-step sandwich method, for example, first, an anti-amyloid-beta antibody immobilized on a solid phase or an anti-amyloid-beta antibody immobilized on a solid phase (solid-phase antibody) is brought into contact with amyloid-beta peptide in a blood sample to perform an antigen-antibody reaction between the solid-phase antibody and the amyloid-beta peptide (first reaction). If the solid-phase antibody is an anti-amyloid-beta antibody immobilized on a solid phase, the solid-phase antibody is immobilized on the solid phase after the first reaction or simultaneously with the antigen-antibody reaction. Then, B / F separation is performed. Next, the amyloid-beta peptide bound to the solid-phase antibody is brought into contact with an anti-amyloid-beta antibody to which a labeling substance for detection is bound (labeled antibody) to perform an antigen-antibody reaction between the amyloid-beta peptide and the labeled antibody (second reaction). Then, B / F separation is performed, and the amyloid-beta peptide in the blood sample can be measured by detecting the signal derived from the label of the labeled antibody bound to the amyloid-beta bound to the solid phase.

[0035] In the one-step sandwich method, a solid-phase antibody is brought into contact with amyloid-beta peptide in a blood sample and a labeled antibody, allowing for an antigen-antibody reaction between the solid-phase antibody and amyloid-beta peptide, and an antigen-antibody reaction between the amyloid-beta peptide and the labeled antibody, all in a single step. If the solid-phase antibody is an anti-amyloid-beta antibody that is immobilized on a solid phase, the solid-phase antibody is immobilized on the solid phase after or simultaneously with the antigen-antibody reaction. Subsequently, B / F separation is performed, and the amyloid-beta peptide in the blood sample can be measured by detecting the signal derived from the label of the labeled antibody bound to the amyloid-beta peptide bound to the solid phase.

[0036] The label is not particularly limited, and an enzyme (e.g., alkaline phosphatase) may be used as the label, and a substrate that produces a chemiluminescent compound (e.g., AMPPD) may be used as the substrate. Enzyme immunoassay (EIA) is an immunoassay that uses an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc.) as the label. As the substrate for each enzyme, a compound that can be quantified by absorbance measurement, etc., is used. For example, in the case of peroxidase, 1,2-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB), etc. may be used in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP), etc. may be used in the case of β-galactosidase, MG: 4-methylumbelliferyl galactoside, NG: nitrophenyl galactoside, etc. may be used, and in the case of luciferase, luciferin, etc. may be used. Radioactive immunoassay (RIA) is a method that uses radioactive materials as labels, and examples of radioactive materials include radioactive elements such as 3H, 14C, 32P, 35S, and 125I. Fluorescent immunoassay (FIA) is a method that uses fluorescent substances or fluorescent proteins as labels, and examples of fluorescent substances or fluorescent proteins include fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, and red fluorescent protein.

[0037] More specifically, the detection method disclosed herein preferably employs chemiluminescent enzyme immunoassay (CLEIA). In this case, a solid-phase antibody, which is obtained by immobilizing an anti-amyloid-β peptide antibody on magnetic particles (solid phase), is mixed with a blood sample. At this time, the buffer solution with the pH described above is used, the concentration of the buffer in the reaction system is set to 25 mM or higher, and the reaction time is set to 6 minutes or less. This reaction binds the amyloid-β peptide to be detected (e.g., amyloid-β1-40 or amyloid-β1-42) contained in the blood sample to the solid-phase antibody. After recovering and washing the magnetic particles, the amyloid-β peptide bound to the solid-phase antibody is detected using a peroxidase-labeled anti-amyloid-β peptide antibody and a peroxidase substrate. The peroxidase substrate is luminol or 8-amino-5-corolo-2,3-dihydro-7-phenylpyrido[3,4-d]pyridazine sodium salt (L-012). Furthermore, hydrogen peroxide catalyzes the reaction between peroxidase and the substrate. That is, by measuring the fluorescence derived from luminol or L-012, amyloid-beta peptides bound to solid-phase antibodies can be detected.

[0038] [Reagent Kit] The reagent kit of this disclosure is a reagent kit used in the [Method for Detecting a Test Substance] described above, and in particular is a reagent kit for detecting amyloid β1-40 or amyloid β1-42 among test substances derived from amyloid β precursor protein in a blood sample. The reagent kit of this disclosure includes, as a buffer reagent for detecting amyloid β1-40 among test substances derived from amyloid β precursor protein in a blood sample, a buffer with a buffer concentration of 50 mM or more and a pH in the range of 4.0 to 8.0, or as a buffer reagent for detecting amyloid β1-42 among test substances derived from amyloid β precursor protein in a blood sample, a buffer with a buffer concentration of 50 mM or more and a pH in the range of 5.0 to 8.2. The reagent kit of this disclosure may include both a buffer reagent for detecting amyloid β1-40 and a buffer reagent for detecting amyloid β1-42.

[0039] Furthermore, as described in [Method for detecting a test substance], the detection method of this disclosure can use a solid phase on which a substance that binds to the test substance is immobilized, such as a solid-phase antibody. Therefore, the reagent kit of this disclosure can include a first reagent containing a substance that is immobilized on a solid phase and binds to the test substance. Here, the solid phase is not particularly limited, but the magnetic particles mentioned above are preferred. Other examples of solid phases include polystyrene, polyethylene, Sepharose, latex, dextran, agarose, gelatin, and polyacrylamide. In particular, the solid phase used is preferably one on which antibodies can be easily immobilized on its surface and on which the immunocomplexes formed during measurement and unreacted components can be easily separated. The immobilization of antibodies on these solid phases can be carried out by conventional methods well known to those skilled in the art. The immobilization of antibodies on the solid phase may be carried out by physical adsorption or by covalent bonding. Antibody immobilization to a solid phase may also be achieved by immobilizing one affinity substance, such as biotin or streptoavidin, to the solid phase, binding the other to the antibody, and mixing them, thereby immobilizing the antibody via the affinity substance.

[0040] Furthermore, in the reagent kit of this disclosure, the buffer reagent described above may be the same reagent as the first reagent. That is, in the reagent kit of this disclosure, the buffer reagent for detecting amyloid β1-40 or the buffer reagent for detecting amyloid β1-42 may include a solid phase on which a substance that binds to the test substance is immobilized, such as a solid-phase antibody. Specifically, the buffer reagent for detecting amyloid β1-40 may include a buffer with a buffer concentration of 50 mM or more and a pH in the range of 4.0 to 8.0, and a solid phase on which a substance that binds to the test substance is immobilized. Similarly, the buffer reagent for detecting amyloid β1-42 may include a buffer with a buffer concentration of 50 mM or more and a pH in the range of 5.0 to 8.2, and a solid phase on which a substance that binds to the test substance is immobilized.

[0041] Furthermore, as described in [Method for detecting a test substance], the detection method of this disclosure can use a labeled substance that binds to the test substance, such as a labeled antibody. Therefore, the reagent kit of this disclosure can include a test substance detection reagent containing a labeled substance that binds to the test substance. Also, in the reagent kit of this disclosure, the buffer reagent described above can be the same reagent as the test substance detection reagent. That is, in the reagent kit of this disclosure, the buffer reagent for detecting amyloid β1-40 or the buffer reagent for detecting amyloid β1-42 may contain a labeled substance that binds to the test substance, such as a labeled antibody. Specifically, the buffer reagent for detecting amyloid β1-40 may include a buffer with a buffer concentration of 50 mM or more and a pH in the range of 4.0 to 8.0, and a labeled substance that binds to the test substance, such as a labeled antibody. Furthermore, a buffer reagent for detecting amyloid β1-42 may include a buffer with a buffer concentration of 50 mM or higher and a pH in the range of 5.0 to 8.2, and a labeling substance that binds to the test substance, such as a labeled antibody.

[0042] Furthermore, the reagent kit of this disclosure may also include a solid phase on which a substance that binds to the test substance is immobilized, such as a solid-phase antibody, and a labeled substance that binds to the test substance, such as a labeled antibody. That is, in the reagent kit of this disclosure, a buffer reagent for detecting amyloid β1-40 or a buffer reagent for detecting amyloid β1-42 may include a solid phase on which a substance that binds to the test substance is immobilized and a labeled substance that binds to the test substance. Specifically, a buffer reagent for detecting amyloid β1-40 may include a buffer with a buffer concentration of 50 mM or more and a pH in the range of 4.0 to 8.0, a solid phase on which a substance that binds to the test substance is immobilized, and a labeled substance that binds to the test substance. Furthermore, a buffer reagent for detecting amyloid β1-42 may include a buffer solution with a buffer concentration of 50 mM or higher and a pH in the range of 5.0 to 8.2, a solid phase on which a substance that binds to the test substance is immobilized, and a labeled substance that binds to the test substance.

[0043] According to the reagent kit of the present disclosure configured as described above, even when a chelating agent is contained in a blood sample, reduction of the signal can be prevented and amyloid-β peptide can be detected with excellent sensitivity. In particular, the result of detecting amyloid-β peptide using the reagent kit of the present disclosure can be used for the diagnosis of Alzheimer's disease. That is, the reagent kit of the present disclosure can be used for the diagnosis of Alzheimer's disease.

[0044] Specifically, as an example, amyloid-β1-40 and amyloid-β1-42 in a blood sample are measured using the reagent kit of the present disclosure. Then, the ratio of these, that is, amyloid-β1-42 / amyloid-β1-4 can be used for the diagnosis of Alzheimer's disease. Specifically, when this ratio is smaller than that of a healthy person, it can be determined that there is a high possibility of Alzheimer's disease.

[0045] Hereinafter, the present disclosure will be described in more detail by way of examples, but the technical scope of the present disclosure is not limited to the following examples.

[0046] [Experimental Example 1] <Examination using EDTA-2K plasma specimens> Using EDTA-2K plasma specimens, reaction conditions having an effect of suppressing signal reduction due to addition of a high concentration of EDTA-2K were examined. In this example, amyloid-β1-40 and amyloid-β1-42 contained in the specimens were used as test substances.

[0047] Preparation of Reagents 1-1 The first to fifth reagents and other reagents necessary for measurement were prepared using the following reagent raw materials. Reagent raw materials: glycine (manufactured by Fujifilm Wako Pure Chemical Corporation), citric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), MES (2-morpholinoethanesulfonic acid, manufactured by Dojindo Laboratories), MOPS (3-morpholinopropane-1-sulfonic acid, manufactured by Dojindo Laboratories), TES (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, manufactured by Dojindo Laboratories), HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, manufactured by Dojindo Laboratories), TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid, manufactured by Dojindo Laboratories), TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, manufactured by Dojindo Laboratories), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation), BSA (manufactured by Sigma-Aldrich Japan LLC), boric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), L-012 (manufactured by Fujifilm Wako Pure Chemical Corporation), phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Corporation), capture antibody: anti-amyloid β monoclonal antibody (manufactured by Fujifilm Wako Pure Chemical Corporation, recognizing the N-terminal side), detection antibodies: anti-amyloid β40 monoclonal antibody, anti-amyloid β42 monoclonal antibody (both manufactured by Fujifilm Wako Pure Chemical Corporation)

[0048] (1) First Reagent (Antibody-Solid Magnetic Particle Reagent) Magnetic particles (magnetic silica particles, trade name: Magrapid, manufactured by Sanyo Chemical Industries, Ltd.) were sequentially reacted with 3-aminopropyltriethoxysilane (manufactured by Tokyo Chemical Industries, Ltd.) and succinic anhydride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The supernatant was removed while the magnetic particles were magnetized with a neodymium magnet to obtain magnetic particles with carboxyl groups introduced on their surface. Subsequently, anti-amyloid β monoclonal antibody was reacted with N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide, manufactured by Dojin Chemical Laboratories) at 25°C for 12 to 16 hours. The supernatant was removed while the magnetic particles were magnetized with a neodymium magnet to prepare magnetic particles with immobilized anti-amyloid β monoclonal antibody (antibody-solid magnetic particles). The first reagent was prepared to have the following composition. • 0.50 mg / mL antibody-solid magnetic particles • 50 mM MES (pH 5.5) • 500 mM sodium chloride

[0049] (2) Second reagent (reaction buffer) A buffer solution consisting of the following components was prepared: • 100 mM Tris (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), pH = 7.2 • 150 mM sodium chloride

[0050] (3) Third Reagent (Antibody Reagent for Detection) A third reagent containing a peroxidase-labeled anti-amyloid-β monoclonal antibody, which is a labeled antibody for measuring the amount of antigen in an EDTA-2K plasma sample, was prepared with the following composition: • 50 mM MES (pH = 6.5) • 150 mM sodium chloride • 2.0% BSA • 20 nM peroxidase-labeled anti-amyloid-β40 monoclonal antibody or peroxidase-labeled anti-amyloid-β42 monoclonal antibody

[0051] (4) Reagent 4 was prepared, consisting of the following components: 50 mM TAPSO, 0.9% boric acid, 0.50 mM L-012, and 0.2 mM thiazolylphenol.

[0052] (5) Reagent 5 was prepared, consisting of the following components: 0.0068% phosphoric acid and 0.0201% hydrogen peroxide (30%)

[0053] (6) AccuraSeed B / F Separation Solution (manufactured by Fujifilm Wako Pure Chemical Industries Ltd.) was used as the other reagent washing solution.

[0054] 1-2 Measurement and Results of Luminescence (Signal) (1) Preparation of EDTA-2K Plasma Sample The EDTA-2K plasma sample (blood sample) used in this example was prepared as follows. First, a specified amount of whole blood sample was collected in a blood collection container containing EDTA-2K, and the whole blood sample containing EDTA-2K was subjected to centrifugation to separate and remove the precipitated blood cell components. The supernatant portion was used as the EDTA-2K plasma sample. In addition, 0.5 (w / v)% of EDTA-2K (manufactured by Dojin Chemical Laboratories Co., Ltd.) was added to the EDTA-2K plasma sample prepared as described above to prepare a high-concentration EDTA-2K sample.

[0055] (2) Effect of buffer concentration and reaction time The supernatant was removed from 50 μL of the first reagent added to the reaction cuvette while the antibody solid magnetic particles were magnetized using a neodymium magnet. Subsequently, 50 μL of the second reagent (final concentration of Tris 25 mM) and 25 μL of the measurement sample (EDTA-2K plasma sample or EDTA-2K high-concentration sample) were added and mixed, and the mixture was heated at 37°C for reaction times of 3 minutes, 5 minutes, 8 minutes, or 11 minutes (first reaction). The first reaction was also performed in the same manner by adjusting the amount of the second reagent to achieve a final concentration of Tris of 33.3 mM or 66.7 mM.

[0056] After the completion of the first reaction, the antibody-solid magnetic particles were magnetized using a neodymium magnet to remove reagents other than the antibody-solid magnetic particles, and the mixture was washed three times with the washing solution. Subsequently, 50 μL of the third reagent was added and the mixture was heated at 37°C for 3 minutes (second reaction). After heating, the antibody-solid magnetic particles were magnetized using a neodymium magnet to remove reagents other than the antibody-solid magnetic particles, and the mixture was washed three times with the washing solution. After washing, 100 μL of the fourth reagent and 100 μL of the fifth reagent were added, and the mixture was reacted at 37°C for 20 seconds, after which the luminescence was measured. The luminescence was measured using an automated chemiluminescent enzyme immunoassay analyzer, Accuraseed (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0057] The luminescence obtained from high-concentration EDTA-2K samples was calculated as a percentage of the luminescence obtained from EDTA-2K plasma samples. The results are shown in Table 1.

[0058]

[0059] As shown in Table 1, under the conditions of the example, where the buffer concentration in the first reaction was 25 mM or higher and the second reagent with a pH of 3.5 to 9.0 was contacted for 6 minutes or less, it was found that the signal reduction due to high concentrations of EDTA (chelating agent) could be largely prevented. Furthermore, it was found that the shorter the first reaction time, the more the impact on signal reduction was suppressed. From these results, it became clear that when the chelating agent is at a high concentration, shortening the reaction time prevents signal reduction due to the chelating agent and enables highly sensitive detection of amyloid-β peptide.

[0060] [Experimental Example 2] In this example, the following buffer was used as the second reagent, and the luminescence was measured for EDTA-2K plasma samples and EDTA-2K high-concentration samples in the same manner as in Experimental Example 1. The ratio of the luminescence obtained from the EDTA-2K high-concentration sample to the luminescence obtained from the EDTA-2K plasma sample was calculated as a percentage. The results measured for amyloid β1-40 are shown in Table 2, and the results measured for amyloid β1-42 are shown in Table 3. In this example, the reaction time for the first reaction was set to 3 minutes.

[0061] Cushioning material used: 100 mM Glycine (pH = 3.0, 3.5, 9.0, 9.5, or 10.0); Citric acid (pH = 4.0, 4.5, or 5.0); MES (pH = 5.5, 6.0, 6.5, or 7.0); MOPS (pH = 6.5, 7.0, or 7.5); Phosphoric acid (pH = 6.2, 7.2, or 8.2); TES (pH = 7.0, 7.5, or 8.0); HEPES (pH = 7.0, 7.5, or 8.0); TAPSO (pH = 7.0, 7.5, or 8.0); Tris (pH = 7.0, 8.0, or 9.0); TAPS (pH = 8.0, 8.5, or 9.0); Boric acid (pH = 9.0, 10.0, or 11.0); or CHES (pH = 9.0, 9.5, or 10.0)

[0062]

[0063]

[0064] As can be seen from Tables 2 and 3, in both amyloid β1-40 measurement and amyloid β1-42 measurement, using a buffer with a pH of 3.5 to 9.0 for the first reaction, as in the example, suppressed the decrease in luminescence due to the addition of high concentrations of EDTA to within 20%.

[0065] Furthermore, as can be seen from Table 2, in the measurement of amyloid β1-40, using a buffer solution with a pH of 4.0 to 8.0 for the first reaction suppressed the decrease in luminescence due to the addition of high concentrations of EDTA to within 5%.

[0066] Furthermore, as can be seen from Table 3, in the measurement of amyloid β1-42, using a buffer solution with a pH of 5.0 to 8.2 for the first reaction suppressed the decrease in luminescence due to the addition of high concentrations of EDTA to within 10%. Furthermore, as can be seen from Table 3, in the measurement of amyloid β1-42, using a buffer solution with a pH of 6.0 to 8.2 for the first reaction further suppressed the decrease in luminescence due to the addition of high concentrations of EDTA to within 5%.

[0067] While not bound by theory, blood samples may contain numerous substances that affect the detection of amyloid-beta. The above experimental example showed that the presence of chelating agents in the measurement system, particularly at high concentrations, significantly increased the impact of these substances on signal reduction. This is presumed to be because the chelating agent causes fluctuations in the pH of the reaction system, or the chelating agent itself acts as a buffer, suppressing the effects on amyloid-beta within the specified range.

[0068] The disclosure of Japanese Patent Application No. 2024-171219, filed on 30 September 2024, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A method for detecting a test substance derived from amyloid-beta precursor protein, comprising: contacting a test substance derived from amyloid-beta precursor protein in a blood sample with a substance that binds to the test substance and a buffer solution containing an amount of buffering agent such that the concentration at the time of contact is 25 mM or more, and having a pH of 3.5 to 9.0 for 6 minutes or less; and measuring a signal derived from the binding of the test substance to the substance that binds to the test substance.

2. The method according to claim 1, wherein the test substance is amyloid β1-40 or amyloid β1-42.

3. The method according to claim 1, wherein the test substance is amyloid β1-40 and the pH of the buffer solution is 4.0 or higher and 8.0 or lower.

4. The method according to claim 1, wherein the test substance is amyloid β1-42 and the pH of the buffer solution is 5.0 or higher and 8.2 or lower.

5. The method according to claim 1, wherein the substance to be tested, the substance that binds to the substance to be tested, and the buffer solution are brought into contact for four minutes or less.

6. The method according to claim 1, wherein the concentration of the buffering agent at the time of contact is 50 mM or more.

7. The method according to claim 1, wherein the blood sample is a plasma sample derived from a whole blood sample collected in a blood collection container that has been pre-filled with a chelating agent, which is an anticoagulant.

8. A method for reducing the influence of chelating agents in the detection of amyloid-beta precursor protein-derived test substance, comprising: contacting a test substance derived from amyloid-beta precursor protein in a blood sample with a substance that binds to the test substance and a buffer containing an amount of buffering agent such that the concentration at the time of contact is 25 mM or more, and having a pH of 3.5 to 9.0 for 6 minutes or less; and measuring a signal derived from the binding of the test substance to the substance that binds to the test substance.

9. A buffer reagent for detecting amyloid-beta 1-40, a test substance derived from amyloid-beta precursor protein in a blood sample, with a buffer concentration of 50 mM or higher and a pH in the range of 4.0 to 8.

0.

10. A buffer reagent for detecting amyloid-beta 1-42, a test substance derived from amyloid-beta precursor protein in a blood sample, with a buffer concentration of 50 mM or higher and a pH in the range of 5.0 to 8.

2.

11. A reagent kit comprising the buffer reagent described in claim 9 or the buffer reagent described in claim 10, for detecting amyloid-beta 1-40 or amyloid-beta 1-42 among test substances derived from amyloid-beta precursor protein in a blood sample.

12. The reagent kit according to claim 11, comprising a first reagent which is immobilized on a solid phase and contains a substance that binds to the test substance.

13. The reagent kit according to claim 12, wherein the buffer reagent according to claim 9, or the buffer reagent according to claim 10 and the first reagent are the same reagent.

14. The reagent kit according to claim 11, comprising a reagent for detecting a test substance containing a labeling substance that binds to the test substance.

15. The reagent kit according to claim 14, wherein the buffer reagent according to claim 9, or the buffer reagent according to claim 10 and the reagent for detecting the test substance are the same reagent.

16. The reagent kit according to claim 11, used for the diagnosis of Alzheimer's disease.

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