Immunoassay method for amyloid β

Abstract

Disclosed are an amyloid β measurement method and a reagent capable of more accurately measuring amyloid β and unaffected by molecular target drugs (antibody drugs) for amyloid β. Provided is an immunoassay method for amyloid β in a sample isolated from a living body including a pretreatment step for mixing a sample isolated from a living body, (1) an acidifying agent, and (2) at least one selected from the group consisting of cationic surfactants, zwitterionic surfactants, protein denaturants, and chelating agents.

Description

Amyloid beta immunoassay method

[0001] The present invention relates to a method for immunoassaying amyloid β in a sample and a kit therefor.

[0002] Amyloid beta (Aβ) is a peptide protein consisting of approximately 40 amino acids, produced by cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Aβ is biosynthesized in brain neurons and excreted into cerebrospinal fluid (CSF) or into the blood via the blood-brain barrier. Two major Aβ forms are known: Aβ1-40 (40 amino acids) and Aβ1-42 (42 amino acids). Aβ1-40 and Aβ1-42 differ only by two residues at their C-terminus. Of these, Aβ1-42 exhibits strong aggregating properties, and its aggregates accumulate in brain tissue to form senile plaques, which are thought to contribute to Alzheimer's disease. Decreased Aβ1-42 concentrations in CSF and blood have been shown to correlate with pathological findings in Alzheimer's disease, making Aβ1-42 a valuable diagnostic marker for Alzheimer's disease progression (Patent Document 1).

[0003] In recent years, progress has been made in the development of molecular targeted drugs (antibody drugs) that target Aβ as a treatment for Alzheimer's disease. Antibody drugs are expected to slow the progression of symptoms by binding to Aβ and inhibiting the aggregation reaction of Aβ molecules.

[0004] WO 00 / 68694

[0005] As mentioned above, the development of molecularly targeted drugs (antibody drugs) targeting Aβ is progressing. However, it is thought that antibody drugs and Aβ bind to each other in the samples of administered patients. When measuring Aβ in a sample by immunoassay, a problem arises in that when samples from patients administered antibody drugs are used, the measurement of Aβ can be inhibited depending on the recognition domain of the anti-Aβ antibody used for the measurement. Therefore, the present invention aims to provide a method and a measurement reagent for Aβ that can measure Aβ more accurately without being affected by molecularly targeted drugs (antibody drugs).

[0006] As a result of extensive research, the present inventors have discovered that by performing a pretreatment step of mixing the sample with (1) an acidifying agent and (2) at least one selected from the group consisting of a cationic surfactant, an amphoteric surfactant, a protein denaturant, and a chelating agent prior to immunoassay of Aβ, it is possible to measure the Aβ concentration in a sample relatively accurately, even if the sample is derived from a patient who has been administered an anti-Aβ antibody drug, and have completed the present invention.

[0007] That is, the present invention provides the following: (1) An immunoassay method for amyloid-β in a specimen isolated from a living body, comprising a pretreatment step of mixing the specimen isolated from a living body with (i) an acidifying agent and (ii) at least one selected from the group consisting of a cationic surfactant, a zwitterionic surfactant, a protein denaturant, and a chelating agent. (2) The immunoassay method according to (1), wherein the specimen is derived from a patient who has been administered an anti-amyloid-β antibody. (3) The immunoassay method according to (1) or (2), wherein the acidifying agent is an acid and the final concentration of the acid in the pretreatment step is 0.01N to 1.0N. (4) The immunoassay method according to (3), wherein the acid is at least one selected from the group consisting of hydrochloric acid, sulfuric acid, and acetic acid. (5) The immunoassay method according to any one of (1) to (4), wherein the cationic surfactant comprises a quaternary ammonium salt having an alkyl group bonded thereto. (6) The immunoassay method according to (5), wherein the cationic surfactant is at least one selected from the group consisting of hexadecyltrimethylammonium chloride, octyltrimethylammonium chloride, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, octyltrimethylammonium bromide, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide. (7) The immunoassay method according to any one of (1) to (6), wherein the zwitterionic surfactant comprises a quaternary ammonium salt having an alkyl group bonded thereto.(8) The immunoassay method according to any one of (1) to (8), wherein the zwitterionic surfactant is at least one selected from the group consisting of N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and N-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. (9) The immunoassay method according to any one of (1) to (8), wherein the protein denaturant is at least one selected from the group consisting of urea and guanidine hydrochloride. (10) The immunoassay method according to any one of (1) to (9), wherein the chelating agent is at least one selected from the group consisting of citric acid, ethylenediaminetetraacetic acid, glycoletherdiaminetetraacetic acid, and salts thereof. (11) A kit for immunoassay of amyloid beta in a sample isolated from a living body, comprising (i) an acidifying agent and (ii) at least one selected from the group consisting of a cationic surfactant, an amphoteric surfactant, a protein denaturant, and a chelating agent.

[0008] According to the present invention, it has become possible to measure the Aβ concentration in a sample relatively accurately, even if the sample is derived from a patient who has been administered an anti-Aβ antibody drug.

[0009] The sample used in the method of the present invention is not particularly limited as long as it is a sample isolated from a living body, but body fluids such as blood (including whole blood, serum, and plasma) and cerebrospinal fluid (CSF) are preferred, with blood being more preferred. In particular, the present invention is effective in measuring Aβ in samples derived from patients receiving anti-Aβ antibody drugs, and therefore body fluids derived from patients receiving anti-Aβ antibody drugs are preferred. Examples of anti-Aβ antibody drugs include lecanemab, donanemab, aducanumab, and trontinemab.

[0010] The acidifying agent used in the pretreatment step in the immunoassay method of the present invention is preferably an acid. Preferred acids include, but are not limited to, hydrochloric acid, sulfuric acid, and acetic acid. The final concentration of the acid in the pretreatment step is preferably 0.01 M to 0.5 M (0.01 N to 1.0 N), and particularly preferably 0.05 M to 0.3 M (0.05 N to 0.6 N). The pH in the pretreatment step is preferably about 1.0 to 4.5, more preferably about 1.0 to 4.0, and even more preferably about 1.0 to 3.5.

[0011] In the pretreatment step, in addition to the above-mentioned acidifying agent, at least one selected from the group consisting of a cationic surfactant, an amphoteric surfactant, a protein denaturant, and a chelating agent is further mixed.

[0012] The cationic surfactant is not particularly limited, but is preferably a surfactant containing a quaternary ammonium salt to which an alkyl group is bonded. The alkyl group preferably has 8 to 18 carbon atoms, and the alkyl group may be branched but is preferably linear. Specific examples of such cationic surfactants include hexadecyltrimethylammonium chloride (C16TAC), octyltrimethylammonium chloride (C8TAC), decyltrimethylammonium chloride (C10TAC), dodecyltrimethylammonium chloride (C12TAC), tetradecyltrimethylammonium chloride (C14TAC), octadecyltrimethylammonium chloride (C18TAC), hexadecyltrimethylammonium bromide (C16TAB), octyltrimethylammonium bromide (C8TAB), decyltrimethylammonium bromide (C10TAB), dodecyltrimethylammonium bromide (C12TAB), tetradecyltrimethylammonium bromide (C14TAB), and octadecyltrimethylammonium bromide (C18TAB), but are not limited thereto. The final concentration of the cationic surfactant in the pretreatment step is usually about 0.005 w / v% to 0.4 w / v%, preferably about 0.005 w / v% to 0.2 w / v%, and more preferably about 0.005 w / v% to 0.15 w / v%.

[0013] The amphoteric surfactant is not particularly limited, but is preferably one containing a quaternary ammonium salt to which an alkyl group is bonded. The alkyl group preferably has 8 to 16 carbon atoms, and the alkyl group may be branched or linear, preferably linear. Specific examples of such amphoteric surfactants include, but are not limited to, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C14APS), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS), N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C10APS), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C12APS), and N-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C8APS). The final concentration of the zwitterionic surfactant in the pretreatment step is usually about 0.005 w / v% to 5.0 w / v%, preferably about 0.005 w / v% to 3.0 w / v%, and more preferably about 0.05 w / v% to 3.0 w / v%.

[0014] Examples of protein denaturants include, but are not limited to, urea, guanidine hydrochloride, etc. The final concentration of the protein denaturant in the pretreatment step is usually about 0.05 M to 4 M, preferably about 0.2 M to 1.5 M.

[0015] Examples of chelating agents include, but are not limited to, citric acid, ethylenediaminetetraacetic acid, glycoletherdiaminetetraacetic acid, and salts thereof (sodium citrate, EDTA 2Na, EGTA 4Na, etc.). The final concentration of the chelating agent in the pretreatment step is usually about 0.5 mM to 200 mM, preferably about 10 mM to 80 mM, and more preferably about 25 mM to 80 mM.

[0016] The at least one selected from the group consisting of cationic surfactants, zwitterionic surfactants, protein denaturants, and chelating agents may be used in combination with a plurality of types, but even a single type will be effective. In this case, the combinations of an acidifier and a zwitterionic surfactant, an acidifier and a cationic surfactant, an acidifier and a chelating agent, and an acidifier and a protein denaturant are preferred in this order.

[0017] The pretreatment step can be carried out at, for example, 10°C to 50°C, preferably 20°C to 45°C, more preferably 30°C to 40°C, and even more preferably 37°C. The time for the pretreatment step is usually 1 minute or more, preferably 3 minutes or more. There is no particular upper limit to the time, but making it too long is meaningless, and the time is usually 20 minutes or less, preferably 10 minutes or less.

[0018] Following the pretreatment step described above, Aβ is measured by immunoassay. Aβ is produced by enzymatic cleavage of its precursor, and depending on the cleavage site of the cleavage enzyme, it can be divided into two types: amyloid β1-42 (Aβ), which consists of amino acids 1 to 42. １－４２ )), and those consisting of amino acids 1 to 40 (amyloid beta 1-40 (Aβ １－４０ The present invention is useful for measuring any of amyloid β, and any of these measurements is within the scope of the present invention, but it is particularly preferable to use the method for measuring amyloid β1-42 and amyloid β1-40.

[0019] The above pretreatment step is intended to dissociate Aβ from anti-Aβ antibodies, and is therefore clearly effective for any immunoassay method. Immunoassays include sandwich assays, competitive assays, agglutination assays, immunochromatography, and the like, and the present invention encompasses any of these immunoassays. These immunoassay methods are well known and do not need to be described in detail here, but each will be briefly described below.

[0020] Immunoassays include various techniques such as chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, electrochemiluminescence immunoassay (ECLIA), and fluorescence immunoassay (FIA), and the present invention encompasses all of these techniques. The immunoassay of the present invention is not particularly limited, and may be a sandwich method. Sandwich methods include, for example, a one-step sandwich method and a two-step sandwich method.

[0021] In the two-step sandwich method, for example, 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 first contacted with amyloid beta in a sample, allowing an antigen-antibody reaction between the solid-phase antibody and amyloid beta (primary 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 primary reaction. Then, B / F separation is performed. Next, the amyloid beta bound to the solid-phase antibody is contacted with an anti-amyloid beta antibody (labeled antibody) bound to a label for detection, allowing an antigen-antibody reaction between the amyloid beta and the labeled antibody (secondary reaction). Next, B / F separation is performed, and amyloid beta in the 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. Washing may be performed after B / F separation.

[0022] In the one-step sandwich method, for example, a solid-phase antibody is contacted with amyloid beta in a sample and a labeled antibody, and an antigen-antibody reaction between the solid-phase antibody and amyloid beta and an antigen-antibody reaction between amyloid beta and the labeled antibody are carried out in a single step. 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 or simultaneously with the antigen-antibody reaction. Then, B / F separation is performed, and amyloid beta in the sample can be measured by detecting a signal derived from the label of the labeled antibody bound to the amyloid beta bound to the solid phase. Washing may be performed after B / F separation.

[0023] The direct competitive assay involves immobilizing an antibody against the target antigen (in this case, amyloid beta) to a solid phase (immobilization), blocking the solid phase to prevent nonspecific adsorption (treating the solid phase with a protein solution such as serum albumin), reacting the antibody with a test sample (e.g., a specimen) containing the target antigen, and a certain amount of labeled antigen. After washing, the amount of label bound to the solid phase is quantified. Because the antigen in the test sample and the labeled antigen bind competitively to the antibody, the greater the amount of antigen in the test sample, the less label binds to the solid phase. Various antigen standard solutions with known concentrations are prepared, and the amount of label immobilized on the solid phase (absorbance, luminescence intensity, fluorescence intensity, etc., depending on the nature of the label; the same applies hereinafter) is measured. A calibration curve is then created, with antigen concentration on the horizontal axis and label amount on the vertical axis. The amount of label is measured for a test sample with an unknown amount of antigen, and the amount of antigen in the unknown test sample can be determined by applying the measured amount of label to the calibration curve. The direct competition method itself is well known in the art and is described, for example, in US20150166678A.

[0024] In the indirect competitive method, for example, a target antigen (amyloid beta in this invention) is immobilized. Then, after blocking the solid phase, a sample containing the target antigen is mixed with a certain amount of anti-target antigen antibody and allowed to react with the immobilized antigen. After washing, the amount of anti-target antigen antibody bound to the solid phase is quantified. This can be achieved by reacting a labeled secondary antibody against the anti-target antigen antibody, washing, and then measuring the amount of label. Various antigen standard solutions with known concentrations are prepared, and the amount of label immobilized on the solid phase for each is measured to create a calibration curve. The amount of label for an unknown sample can be measured and applied to the calibration curve to determine the amount of antigen in the unknown sample. It is also possible to use a labeled primary antibody without a labeled secondary antibody. The indirect competitive method itself is well known in the art and is described, for example, in the aforementioned US20150166678A.

[0025] Among the various immunoassays mentioned above, chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescent immunoassay (FIA) are immunoassays classified based on the type of label used when performing the above-mentioned direct competitive method, indirect competitive method, sandwich method, etc. Chemiluminescent enzyme immunoassay (CLEIA) is an immunoassay that uses an enzyme (e.g., alkaline phosphatase) as a label and a substrate (e.g., AMPPD) that generates a chemiluminescent compound as a substrate. Enzyme immunoassay (EIA) is an immunoassay that uses an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc.) as a label. A compound that can be quantified by absorbance measurement or the like is used as the substrate for each enzyme. For example, in the case of peroxidase, 1,2-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB) or the like is used, in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP), 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) or the like is used, in the case of β-galactosidase, 4-methylumbelliferyl galactoside (MG), nitrophenyl galactoside (NG) or the like is used, and in the case of luciferase, luciferin or the like is used. Radioimmunoassay (RIA) is a method using a radioactive substance as a label, and examples of the radioactive substance include: ３ H. １４ C. ３２ P. ３５ S. １２５ Examples of such labels include radioactive elements such as I. Fluorescence immunoassay (FIA) is a method that uses a fluorescent substance or a fluorescent protein as a label, and examples of such fluorescent substances or fluorescent proteins include fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, and red fluorescent protein. Immunoassays using these labels are well known in the art and are described, for example, in US8039223B and US20150309016A1.

[0026] Turbidimetry (TIA) is an immunoassay that utilizes the phenomenon of increased turbidity due to the antigen-antibody complex formed by the binding of the target antigen (in this invention, amyloid beta) to the antibody against the antigen. Various known concentrations of antigen are added to an anti-target antigen-antibody solution, and the turbidity is measured for each to create a calibration curve. The turbidity of a test sample with an unknown antigen amount is similarly measured, and the measured turbidity is applied to the calibration curve to determine the amount of antigen in the unknown test sample. Turbidimetry itself is well known and is described, for example, in US 20140186238 A1. Latex agglutination is similar to turbidimetry, but instead of the antibody solution used in turbidimetry, a suspension of latex particles with anti-target antigen antibodies immobilized on their surfaces is used. Turbidimetry and latex agglutination themselves are well known in the art and are described, for example, in US 7,820,398 B.

[0027] Immunochromatography is a method in which the above-mentioned sandwich method or competitive method is performed on a substrate (also called a matrix or strip) formed of a porous material such as filter paper, cellulose membrane, glass fiber, or nonwoven fabric. For example, in the case of sandwich immunochromatography, a detection zone on which an anti-target antigen antibody is immobilized is provided on the substrate, a test sample containing the target antigen is added to the substrate, and a developer is flowed from the upstream side to migrate the target antigen to the detection zone and immobilize it in the detection zone. The immobilized target antigen is sandwiched with a labeled secondary antibody, and the label immobilized in the detection zone is detected to detect the target antigen in the test sample. By forming a label zone containing a labeled secondary antibody upstream of the detection zone, a conjugate of the target antigen and the labeled secondary antibody is immobilized in the detection zone. If the label is an enzyme, a substrate zone containing a substrate for the enzyme is also provided upstream of the detection zone. In the case of a competitive method, for example, the target antigen is immobilized in the detection zone, and the target antigen in the test sample and the target antigen immobilized in the detection zone can compete with each other. A labeled antibody zone is provided upstream of the detection zone, and the target antigen in the test sample is reacted with the labeled antibody, and the unreacted labeled antibody is immobilized in the detection zone, and the label is detected or quantified, thereby enabling detection or quantification of the target antigen in the test sample. The immunochromatography method itself is well known in this field and is described, for example, in U.S. Pat. No. 6,210,898B.

[0028] The solid phase is not particularly limited, and any solid phase used in known immunoassays can be used. Specific examples of solid phase materials include, but are not limited to, polystyrene, polyethylene, Sepharose, latex, dextran, agarose, gelatin, and polyacrylamide. The solid phase used is preferably one that allows easy immobilization of antibodies to its surface and facilitates separation of immune complexes formed during the assay from unreacted components. Plastic plates, latex particles, and magnetic particles used in conventional immunoassays are particularly preferred. From the perspectives of ease of handling, storage, and separation, magnetic particles made of the aforementioned materials are most preferred. Immobilization of antibodies to these solid phases can be performed by conventional methods well known to those skilled in the art. Immobilization of antibodies to the solid phase can be achieved by physical adsorption or covalent bonding. Alternatively, immobilization of antibodies to the solid phase can be achieved by immobilizing one affinity substance, such as biotin-streptavidin, on the solid phase, binding the other to the antibody, and then mixing the two. This allows the antibody to be immobilized via the affinity substance.

[0029] The labeling substance is not particularly limited, and the same labeling substances as those used in known immunoassays can be used. Specific examples include enzymes, fluorescent substances, chemiluminescent substances, dyes, radioactive substances, etc. Known enzymes such as alkaline phosphatase (ALP), peroxidase, and β-galactosidase can be used, but are not limited thereto.

[0030] Among the various immunoassays mentioned above, from the viewpoints of detection sensitivity and ease of automation, the sandwich method, in particular the chemiluminescent enzyme immunoassay (CLEIA), which is an immunoassay using magnetic particles as a solid phase, an enzyme (e.g., alkaline phosphatase) as a label, and a substrate that generates a chemiluminescent compound (e.g., 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD)), is preferred.

[0031] The anti-amyloid beta antibody or its antigen-binding fragment may be any antibody or antigen-binding fragment that specifically binds to amyloid beta. The antibody may be a monoclonal or polyclonal antibody. Monoclonal antibodies are generally preferred. Since methods for producing polyclonal and monoclonal antibodies are well known, anti-amyloid beta antibodies may be prepared using an amyloid beta antigen as an immunogen. The "antigen-binding fragment" may be any antibody fragment as long as it maintains the binding ability of the original antibody to its corresponding antigen (antigen-antibody reactivity). Specific examples include, but are not limited to, Fab, Fab', F(ab')2, and scFv. As is well known, Fab, Fab', and F(ab')2 can be obtained by treating antibodies with protease enzymes such as papain and pepsin. Methods for producing scFv (single chain fragment of variable region, single-chain antibody) are also well known and can be produced according to well-known methods.

[0032] The anti-amyloid β antibody or antigen-binding fragment thereof can be appropriately selected depending on the type of amyloid β to be measured. Such antibodies and antibody combinations are known, and commercially available antibodies can be used.

[0033] For example, to measure amyloid β1-42 by the sandwich method, an antibody that specifically binds to the C-terminal region of amyloid βx-42 (anti-Aβ42 antibody) and an antibody that specifically binds to the N-terminal region containing the first amino acid of amyloid β (anti-Aβ N-terminal antibody) can be used. As described above, such antibodies may be commercially available or may be produced by well-known methods. An anti-Aβ42 antibody may be used as the solid-phase antibody and an anti-Aβ N-terminal antibody as the labeled antibody, or conversely, an anti-Aβ N-terminal antibody may be used as the solid-phase antibody and an anti-Aβ42 antibody as the labeled antibody.

[0034] Furthermore, to measure amyloid β1-40 by the sandwich method, an antibody that specifically binds to the C-terminal region of amyloid β1-40 (anti-Aβ40 antibody) and an antibody that specifically binds to the N-terminal region containing the first amino acid of amyloid β (anti-Aβ N-terminal antibody) can be used. As described above, these antibodies may be commercially available or may be produced by well-known methods. An anti-Aβ40 antibody may be used as the solid-phase antibody and an anti-Aβ N-terminal antibody as the labeled antibody, or conversely, an anti-Aβ N-terminal antibody may be used as the solid-phase antibody and an anti-Aβ40 antibody as the labeled antibody.

[0035] Immunoassay methods themselves are well known, and as described above, the present invention is applicable to any immunoassay method. That is, the immunoassay method of the present invention can use well-known immunoassay methods as they are, except for the pretreatment step. Furthermore, since immunoassays using CSF or blood as samples have already been performed, kits used for these immunoassays can also be used as they are.

[0036] The kit of the present invention may be any kit for performing the immunoassay method of the present invention described above, and comprises an anti-amyloid β antibody or an antigen-binding fragment thereof, (i) an acidifying agent, and (ii) at least one selected from the group consisting of cationic surfactants, zwitterionic surfactants, protein denaturants, and chelating agents. When the immunoassay is a sandwich method, the kit also includes a solid phase on which an anti-amyloid β antibody is immobilized or a solid phase capable of immobilizing an anti-amyloid β antibody. When the solid phase is a particle such as a magnetic particle, the kit also includes the particle (usually in the form of a particle liquid). The kit of the present invention can be prepared by adding (i) an acidifying agent and (ii) at least one selected from the group consisting of a cationic surfactant, zwitterionic surfactant, protein denaturant, and chelating agent to a known amyloid β immunoassay kit.

[0037] The present invention will be specifically described below based on examples, although the present invention is not limited to the following examples.

[0038] Reference Example 1 (1) Preparation of Immobilized Particles 1.0 mg / mL of mouse anti-Aβ40 antibody, which specifically binds to the C-terminal region of Aβx-40, was added to 0.02 g / mL of magnetic particles in 50 mM MES buffer (pH 5.0), and the mixture was incubated at 25°C for 1 hour with gentle stirring. After the reaction, the magnetic particles were collected with a magnet and washed to obtain anti-Aβ40 antibody-immobilized particles. The obtained anti-Aβ40 antibody-immobilized particles were diluted with a particle diluent (50 mM MES buffer, 1 mM EDTA 2Na, 0.1% NaN ３ The Aβ40 antibody-immobilized particle solution was prepared by suspending the Aβ40 antibody in a solution containing 0.5% BSA (pH 6.0). Anti-Aβ42 antibody-immobilized particles and an anti-Aβ42 antibody-immobilized particle solution were prepared in the same manner using a mouse anti-Aβ42 antibody that specifically binds to the C-terminal region of Aβx-42.

[0039] (2) Preparation of Enzyme-Labeled Antibody Desalted alkaline phosphatase (ALP) and N-(4-maleimidobutyryloxy)-succinimide (GMBS) were mixed and left to stand at 30°C for 1 hour to perform maleimidation. Next, a Fab'-conjugated mouse anti-Aβ N-terminal antibody that specifically binds to the N-terminal region containing the first amino acid of Aβ was mixed with maleimidated ALP at a molar ratio of 1:1 in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA2Na, pH 6.0) and reacted at 15°C for 1 hour. The desalted coupling reaction solution was purified using column chromatography to obtain alkaline phosphatase-labeled antibody (enzyme-labeled antibody). The enzyme-labeled antibody was diluted with a label diluent (150 mM NaCl, 0.3 mM ZnCl ２ , 1 mM MgCl ２ The antibody was suspended in a buffer containing 1.0% BSA to obtain an enzyme-labeled antibody solution.

[0040] (3) Measurement of Aβ1-40 and Aβ1-42: 80 μL of sample and 50 μL of anti-Aβ40 antibody-immobilized particle solution were dispensed into a cuvette. After stirring, the mixture was incubated at 37°C for 8 minutes. The particles in the cuvette were collected with a magnet, and the cuvette was washed. 50 μL of enzyme-labeled antibody solution was added to the washed cuvette, stirred, and incubated at 37°C for 8 minutes. The particles in the cuvette were collected with a magnet, and the cuvette was washed. 200 μL of Lumipulse® substrate solution (Fujirebio) containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was added, and the mixture was incubated at 37°C for 4 minutes. The luminescence (counts) was then measured using a luminometer. Actual measurements were performed using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (Fujirebio)). A calibration curve was created using the counts of standard solutions measured in the same way, and the Aβ1-40 concentration in the samples was calculated. Aβ1-42 was also measured in the same way, and the Aβ1-42 concentration in the samples was calculated.

[0041] Reference Example 2: Effect of the Presence of Antibodies in Samples on Measurement Values ​​(1) Sample Preparation: Purified Aβ1-40 was added to a plasma sample containing Aβ1-40 and Aβ1-42 at or below the detection limit to prepare a sample containing Aβ1-40. To mimic the Aβ in a sample from a patient administered an antibody drug containing an anti-Aβ antibody, mouse anti-Aβ40 antibody and mouse anti-Aβ N-terminal antibody were added to the Aβ1-40 sample to a total concentration of 130 mg / mL, creating an antibody-binding model sample for Aβ1-40. A plasma sample containing the same amount of Aβ1-40 but without the addition of mouse anti-Aβ40 antibody or mouse anti-Aβ N-terminal antibody was used as a control sample.

[0042] The antibody-binding model specimen for Aβ1-42 was prepared in the same manner as above, except that purified Aβ1-42 was added instead of purified Aβ1-40, and mouse anti-Aβ42 antibody was added instead of mouse anti-Aβ40 antibody.

[0043] Standard solutions were prepared as follows: Purified Aβ1-40 of known concentrations was diluted with Normal Human Plasma (TRINA) to prepare Aβ1-40 standard solutions of 0, 100, 1000, and 5000 pg / mL. Similarly, purified Aβ1-42 of known concentrations was diluted with Normal Human Plasma to prepare Aβ1-42 standard solutions of 0, 30, 100, and 1000 pg / mL.

[0044] (2) Measurement of Samples and Results The concentrations of Aβ1-40 and Aβ1-42 in each sample were measured as described in Reference Example 1 (3).

[0045] The results of Aβ1-40 and Aβ1-42 measurements are shown in Table 1. The Aβ1-40 concentration in the Aβ1-40 control sample was calculated to be 855.0 pg / mL, while the measured Aβ1-40 concentration in the antibody-bound model sample was calculated to be 17.8 pg / mL, indicating a decrease in the measured values ​​due to the presence of antibodies in the sample. Furthermore, the ratio of the antibody-bound model sample concentration to the control sample concentration ((antibody-bound model sample concentration - base value) / (control sample concentration - base value) × 100%) was calculated as the discrepancy rate, which was very low at approximately 2.1%. Note that the base value was measured in a similar manner using a plasma sample without added purified Aβ1-40. For Aβ1-42, the control sample had an Aβ1-42 concentration of 621.9 pg / mL, while the measured Aβ1-42 concentration in the antibody-bound model sample was 4.6 pg / mL. For Aβ1-42, the discrepancy rate was approximately 0.7%, indicating a significant decrease in the measured value due to the presence of antibodies in the sample. This is thought to be due to the antibodies present in the sample binding to Aβ, inhibiting the measurement.

[0046]

[0047] Reference Example 3: Investigation of Treatment Conditions for Antibody-Binding Samples (1) Sample Pretreatment: 80 μL of the antibody-binding model sample containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and the control sample were each mixed with 90 μL of a pretreatment solution containing 160 mM hydrochloric acid and heated at 37°C for 6.5 minutes. Next, 80 μL of a neutralization solution (containing 700 mM HEPES, 20 mM EDTA-3Na, and NaOH (pH 10.0 or less)) was added to prepare the pretreated sample. Standard solutions were prepared in the same manner as in Reference Example 2, and each standard solution was treated in the same manner as described above.

[0048] (2) Measurement of Samples and Results The concentrations of Aβ1-40 and Aβ1-42 in each sample were measured as described in Reference Example 1 (3).

[0049] The measurement results for Aβ1-40 and Aβ1-42 are shown in Table 2. The concentration of hydrochloric acid during treatment was 84.7 mM (0.0847 N), and the pH during treatment was 2.59.

[0050] The deviation rate calculated from the Aβ1-40 measurement value of the acid-treated control sample and the Aβ1-40 measurement value of the acid-treated antibody-bound model sample was 11.0%, which was found to be an improvement over the deviation rate before acid treatment (Reference Example 2). Note that the closer the deviation rate is to 100%, the more the influence of antibody present in the sample in inhibiting measurement is reduced and improved.

[0051] For Aβ1-42, the control sample and the antibody-bound model sample were similarly treated with acid, and the deviation rate was determined to be 10.5%, which was found to be an improvement over the deviation rate before acid treatment (Reference Example 2).

[0052]

[0053] Example 1: Examination of Treatment Conditions for Antibody-Binding Samples 2 As shown in Reference Example 3, acid treatment of samples increased the deviation rate, but this was only about 11%, leaving room for further improvement. Therefore, the effect of adding other compounds in addition to the acid used during pretreatment was examined.

[0054] (1) Sample Pretreatment Eighty microliters of the antibody-bound model sample containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and the control sample were each mixed with 90 μL of a pretreatment solution containing 160 mM hydrochloric acid (treatment concentration: 84.7 mM (0.0847 N)) and an additional compound, and the mixture was heated at 37°C for 6.5 minutes. Next, 80 μL of a neutralization solution (containing 700 mM HEPES, 20 mM EDTA3Na, and NaOH (pH 10.0 or less)) was added to prepare the pretreated sample. The compounds added to the pretreatment solution were urea, citric acid monohydrate, hexadecyltrimethylammonium chloride (C16TAC), N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C14APS), or N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS), and were added to the treatment solution so that the concentrations at the time of treatment were as shown in Tables 3 and 4. For Aβ1-40, a pretreatment solution containing 160 mM hydrochloric acid and 2.0% Tween® 80 was used as a comparative example. Standard solutions were prepared as in Reference Example 2, and the standard solutions were also pretreated in the same manner for each treatment condition. Calibration curves for each treatment condition were created, and the Aβ1-40 and Aβ1-42 concentrations in the samples were calculated.

[0055] (2) Measurement and Results of Aβ1-40 and Aβ1-42 in Samples The concentrations of Aβ1-40 and Aβ1-42 in each sample were measured as described in Reference Example 1 (3).

[0056] Table 3 shows the measured values ​​and dissociation rates for Aβ1-40 under each treatment condition. When urea was added to hydrochloric acid, further improvement in dissociation rate was observed at urea concentrations of 0.26M or higher. Significant improvement was observed especially at 1.06M or higher. When citric acid was added, further improvement was observed at treatment concentrations of 10.59mM or higher, with significant improvement in dissociation rate especially at 26.47mM or higher. Furthermore, further improvement in dissociation rate was observed at all concentrations tested with the cationic surfactant C16TAC and the amphoteric surfactants C14APS and C16APS. On the other hand, no further improvement was observed when the nonionic surfactant Tween 80 was added at the same concentration (treatment concentration 1.06mM).

[0057] Table 4 shows the dissociation rate for Aβ1-42 under each treatment condition. When urea was added to hydrochloric acid, further improvement in the dissociation rate was observed at treatment concentrations of 0.26M or higher. When citric acid was added, further improvement in the dissociation rate was observed at all concentrations tested, with a particularly significant improvement observed at treatment concentrations of 10.59mM or higher. Furthermore, further improvement in the dissociation rate was observed at all concentrations tested with the cationic surfactant C16TAC and the zwitterionic surfactants C14APS and C16APS.

[0058]

[0059]

[0060] Example 2: Investigation of Treatment Conditions for Antibody-Binding Samples 3 As shown in Example 1, it was shown that the dissociation rate could be further improved by combining other compounds in addition to the acid used in the pretreatment. Therefore, effective combinations of compounds were investigated.

[0061] (1) Sample Pretreatment: Pretreatment and neutralization were performed using a fully automated Lumipulse L2400 (Fujirebio). Eighty microliters of antibody-bound model samples containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and control samples were each mixed with 90 μL of pretreatment solution (2.0 M urea, 160 mM hydrochloric acid, 16 mM citric acid dihydrate, 0.32% C16TAC, 2.0% C14APS, 3.0% C16APS, pH 1.51 (pH 3.17 at treatment)) and heated at 37°C for 6.5 minutes. Next, 80 μL of neutralization solution (containing 700 mM HEPES, 20 mM EDTA3Na, and NaOH (pH ∼10.0)) was added to prepare the combined treatment sample (Condition 2). In addition, 80 μL of the same sample was mixed with 90 μL of acidification pretreatment solution (160 mM hydrochloric acid, pH 1.10 (pH 2.59 at time of treatment)) and heated at 37°C for 6.5 minutes. Next, 80 μL of neutralization solution was added to prepare an acid-treated sample (Condition 1). Standard solutions were prepared in the same manner as in Reference Example 2, and each standard solution was treated with the pretreatment solution or acidification pretreatment solution in the same manner as above.

[0062] (2) Measurement and Results of Aβ1-40 and Aβ1-42 in Samples Measurement of Aβ1-40 and Aβ1-42 concentrations in each sample was performed as described in Reference Example 1 (3). Table 5 shows the measured values ​​and deviation rates for the antibody-bound model samples of Aβ1-40 and Aβ1-42. For Aβ1-40, the combined pretreatment (condition 2) showed nearly identical values ​​for the control sample and the antibody-bound model sample compared to acid treatment with hydrochloric acid alone (condition 1). Since the deviation rate was 97.0%, close to 100%, it is believed that pretreatment with these compounds in addition to acid can significantly reduce and improve the influence of antibody-mediated measurement inhibition in samples. Similarly to Aβ1-40, the combined pretreatment (condition 2) showed nearly identical values ​​for the control sample and the antibody-bound model sample. The deviation rate was 101.5%, a value close to 100%, suggesting that pretreatment with a combination of these compounds in addition to acid can significantly reduce and improve the effects of measurement inhibition by antibodies present in the sample.

[0063]

[0064] Example 3: Examination of Treatment Conditions for Antibody-Binding Samples 4 Regarding the acid used in pretreatment, it was examined whether similar effects would be obtained if other acids or other concentrations were used.

[0065] (1) Sample Pretreatment: Pretreatment and neutralization were performed using a fully automated Lumipulse L2400 (Fujirebio). 80 μL of antibody-bound model samples containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and control samples were pretreated and neutralized in the same manner as in Example 2, except for changing the hydrochloric acid concentration to sulfuric acid or acetic acid in the pretreatment solution of Example 2 (2.0 M urea, 160 mM hydrochloric acid, 16 mM citric acid dihydrate, 0.32% C16TAC, 2.0% C14APS, 3.0% C16APS). The concentrations and pH values ​​at each treatment step are shown in Table 6. Standard solutions were prepared in the same manner as in Reference Example 2, and each standard solution was also treated with the same pretreatment solution.

[0066] (2) Measurement and Results of Aβ1-40 and Aβ1-42 in Samples Measurement of the concentrations of Aβ1-40 and Aβ1-42 in each sample was performed as described in Reference Example 1 (3). Table 6 shows the effects of each acid and hydrochloric acid concentration on the deviation rate.

[0067] For Aβ1-40 and Aβ1-42, similar improvement effects were observed when hydrochloric acid was replaced with acetic acid (84.7 mM (0.0847 N)) or sulfuric acid (84.7 mM (0.169 N)). Furthermore, as shown in Reference Examples 2 and 3, the deviation rate in the untreated case was approximately 1-2%, and even when treated with hydrochloric acid alone, the deviation rate was approximately 11%. At all concentrations examined, the deviation rate was 50% or higher, demonstrating an improvement effect. In particular, a significant improvement effect in the deviation rate was observed for both Aβ1-40 and Aβ1-42 at treatment concentrations of 84.7 mM or higher. The treatment pH is as shown in Table 6. A significant improvement effect in the deviation rate was observed at treatment pH 4.72 or lower, and a significant improvement effect in the deviation rate was observed at treatment pH 4.03 or lower.

[0068]

[0069] Example 4: Investigation of Treatment Conditions for Antibody-Bound Samples 5 Regarding the surfactant to be added to the acid during pretreatment, it was investigated whether the same effect could be obtained by using other surfactants.

[0070] (1) Sample Pretreatment: Pretreatment and neutralization were performed using a fully automated Lumipulse L2400 (Fujirebio). 80 μL of the antibody-bound model sample containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and the control sample were pretreated and neutralized in the same manner as in Example 2, except that the surfactant used in the pretreatment solution (2.0 M urea, 160 mM hydrochloric acid, 16 mM citric acid dihydrate, 0.32% C16TAC, 2.0% C14APS, 3.0% C16APS) was changed. Specifically, C16TAC was replaced with other alkyltrimethylammonium salts, such as octyltrimethylammonium chloride (C8TAC), decyltrimethylammonium chloride (C10TAC), dodecyltrimethylammonium chloride (C12TAC), tetradecyltrimethylammonium chloride (C14TAC), and octadecyltrimethylammonium chloride (C18TAC), and C16APS was replaced with other quaternary ammonium sulfonate salts, such as N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C10APS) and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C12APS). Standard solutions were prepared as in Reference Example 2, and each standard solution was also treated with the pretreatment solution. The concentrations of each compound in the treatment solution are shown in Table 7.

[0071] (2) Measurement and Results of Aβ1-40 and Aβ1-42 in Samples Measurement of Aβ1-40 and Aβ1-42 concentrations in each sample was performed as described in Reference Example 1 (3). The effect of each component on the deviation rate is shown in Table 7. For Aβ1-40 and Aβ1-42, even when C16TAC was replaced with C8TAC, C10TAC, C12TAC, C14TAC, or C18TAC, the deviation rates were similar, and an improvement in the deviation rate was observed. C16APS and C14APS were also fully functionally substitutable for C10APS and C12APS.

[0072]

[0073] Reference Example 4: Investigation of Treatment Conditions for Antibody-Binding Samples 6 As shown in Reference Example 3, the dissociation rate increases when the sample is treated with hydrochloric acid. Therefore, we investigated whether a similar effect could be achieved by using acids other than hydrochloric acid.

[0074] (1) Sample Pretreatment: Pretreatment and neutralization were performed using a fully automated Lumipulse L2400 (Fujirebio). Eighty microliters of antibody-bound model samples containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and control samples were mixed with 90 μL of acid-containing pretreatment solution and heated at 37°C for 6.5 minutes. The acids used for sample treatment were acetic acid and sulfuric acid, with the respective concentrations shown in Table 8. Next, 80 μL of neutralization solution (containing 700 mM HEPES, 20 mM EDTA-3Na, and NaOH (pH ∼10.0)) was added to prepare the pretreated samples. Standard solutions were prepared as in Reference Example 2, and the same treatment method was used for each standard solution.

[0075] (2) Measurement and Results of Aβ1-40 and Aβ1-42 in Samples The concentrations of Aβ1-40 and Aβ1-42 in each sample were measured as described in Reference Example 1 (3). The effect of each component on the dissociation rate is shown in Table 8. For Aβ1-40 and Aβ1-42, the dissociation rate was improved even when hydrochloric acid was replaced with acetic acid or sulfuric acid.

[0076]

[0077] Example 5: Investigation of Processing Conditions for Antibody-Binding Samples 7 As shown in Example 1, the dissociation rate was further improved by combining a cationic surfactant such as C16TAC with an acid during pretreatment. Therefore, the effects of other cationic surfactants were also investigated.

[0078] (1) Sample Pretreatment: 80 μL of antibody-bound model samples containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and control samples were mixed with 90 μL of a pretreatment solution containing 160 mM hydrochloric acid (84.7 mM concentration during treatment) and an additional compound, and the mixture was heated at 37°C for 6.5 minutes. Next, 80 μL of a neutralization solution (containing 700 mM HEPES, 20 mM EDTA3Na, and NaOH (pH ∼10.0)) was added to prepare the pretreated sample. The compounds added to the pretreatment solution were C8TAC, C10TAC, C12TAC, C14TAC, or C18TAC, and were added to the treatment solution so that the concentrations at treatment were as shown in Tables 9 and 10. A pretreatment solution containing 160 mM hydrochloric acid and 2.0% Tween® 80 was used as a comparative example. In addition, standard solutions were prepared in the same manner as in Reference Example 2, and the standard solutions were also pretreated in the same manner for each treatment condition. Calibration curves were created for each treatment condition, and the Aβ1-40 and Aβ1-42 concentrations in the samples were calculated.

[0079] (2) Measurement and Results of Aβ1-40 and Aβ1-42 in Samples The concentrations of Aβ1-40 and Aβ1-42 in each sample were measured as described in Reference Example 1 (3).

[0080] The results for Aβ1-40 and Aβ1-42 under each treatment condition are shown in Table 9 and Table 10, respectively. Addition of the nonionic surfactant Tween 80 at the same concentration did not result in any improvement. However, under conditions containing the cationic surfactants C8TAC, C10TAC, C12TAC, C14TAC, and C18TAC, further improvements in the dissociation rate were observed at all concentrations tested.

[0081]

[0082]

[0083] Example 6: Investigation of Treatment Conditions for Antibody-Binding Samples 8 As shown in Example 1, the dissociation rate was further improved by combining an amphoteric surfactant such as C14APS or C16APS with an acid during pretreatment. Therefore, the effects of other amphoteric surfactants were also investigated.

[0084] (1) Sample Pretreatment: 80 μL of the antibody-bound model sample containing Aβ1-40 or Aβ1-42 prepared in Reference Example 2 and the control sample were each mixed with 90 μL of a pretreatment solution containing 160 mM hydrochloric acid (84.7 mM concentration during treatment) and an additional compound, and the mixture was heated at 37°C for 6.5 minutes. Next, 80 μL of a neutralization solution (containing 700 mM HEPES, 20 mM EDTA3Na, and NaOH (pH ∼10.0)) was added to prepare a pretreated sample. The compounds added to the pretreatment solution were C10APS, C12APS, and C8APS, and they were added to the treatment solution so that the concentrations at treatment were as shown in Tables 11 and 12. As a comparative example, a pretreatment solution containing 160 mM hydrochloric acid and 2.0% Tween® 80 was used. In addition, standard solutions were prepared in the same manner as in Reference Example 2, and the standard solutions were also pretreated in the same manner for each treatment condition. Calibration curves were created for each treatment condition, and the Aβ1-40 and Aβ1-42 concentrations in the samples were calculated.

[0085] (2) Measurement and Results of Aβ1-40 and Aβ1-42 in Samples The concentrations of Aβ1-40 and Aβ1-42 in each sample were measured as described in Reference Example 1 (3).

[0086] The results for Aβ1-40 and Aβ1-42 under each treatment condition are shown in Table 11 and Table 12, respectively. Addition of the nonionic surfactant Tween 80 at the same concentration did not result in any improvement. However, under conditions containing the zwitterionic surfactants C8APS, C10APS, and C12APS, further improvements in the dissociation rate were observed at all concentrations tested.

[0087]

[0088]

[0089] According to the present invention, a method and kit for relatively accurately measuring the Aβ concentration in a sample, even if the sample is derived from a patient receiving an anti-Aβ antibody drug, are provided.

Claims

1. A method for immunoassaying amyloid beta in a sample isolated from a living body, comprising a pretreatment step of mixing the sample isolated from a living body with (1) an acidifying agent and (2) at least one selected from the group consisting of cationic surfactants, zwitterionic surfactants, protein denaturants, and chelating agents.

2. The immunoassay method according to claim 1, wherein the sample is derived from a patient who has been administered an anti-amyloid beta antibody.

3. The immunoassay method according to claim 1 or 2, wherein the acidifying agent is an acid, and the final concentration of the acid in the pretreatment step is 0.01N to 1.0N.

4. The immunoassay method according to claim 3, wherein the acid is at least one selected from the group consisting of hydrochloric acid, sulfuric acid and acetic acid.

5. The immunoassay method according to claim 1, wherein the cationic surfactant comprises a quaternary ammonium salt having an alkyl group bonded thereto.

6. The immunoassay method according to claim 5, wherein the cationic surfactant is at least one selected from the group consisting of hexadecyltrimethylammonium chloride, octyltrimethylammonium chloride, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, octyltrimethylammonium bromide, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide.

7. The immunoassay method according to claim 1, wherein the zwitterionic surfactant comprises a quaternary ammonium salt having an alkyl group bonded thereto.

8. The immunoassay method according to claim 7, wherein the zwitterionic surfactant is at least one selected from the group consisting of N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and N-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.

9. The immunoassay method according to claim 1, wherein the protein denaturant is at least one selected from the group consisting of urea and guanidine hydrochloride.

10. The immunoassay method according to claim 1, wherein the chelating agent is at least one selected from the group consisting of citric acid, ethylenediaminetetraacetic acid, glycoletherdiaminetetraacetic acid, and salts thereof.

11. A kit for immunoassay of amyloid beta in a sample isolated from a living body, comprising (1) an acidifying agent and (2) at least one selected from the group consisting of cationic surfactants, zwitterionic surfactants, protein denaturants, and chelating agents.

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