Immunoassay method for blood amyloid β, and kit therefor

Abstract

To disclose an immunoassay method capable of measuring amyloid β in a blood specimen with high sensitivity, and a kit therefor.SOLUTION: An immunoassay method for amyloid β is a method for immunoassaying amyloid β in a blood specimen, where the immunoassay is performed in the presence of an anionic polymer such as dextran sulfate or polystyrene sulfonate. A kit for immunoassaying amyloid β in a blood specimen includes an anti-amyloid β antibody or an antigen-binding fragment thereof, and an anionic polymer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for immunoassaying blood amyloid β and a kit therefor. [Background technology]

[0002] Amyloid β is known as a biomarker for Alzheimer's disease, and it is known that amyloid β in a sample can be measured by immunoassay (Patent Document 1), and kits for this purpose are also commercially available. Many commercially available immunoassay kits for amyloid β use cerebrospinal fluid (CSF) as a sample. On the other hand, it is known that amyloid β is also present in the blood of patients with Alzheimer's disease. Since collection of CSF is invasive, it would be advantageous if amyloid β in a blood sample could be immunologically measured. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO 2017 / 179611 Summary of the Invention [Problem to be solved by the invention]

[0004] Because the target of measurement is the same amyloid beta whether it is in CSF or blood, it was thought that it would be possible to immunoassay amyloid beta in blood in the same way as CSF. However, it was found that immunoassay of amyloid beta in blood samples had a problem of lower sensitivity compared to when CSF samples were used.

[0005] Therefore, an object of the present invention is to provide an immunoassay method and a kit therefor that can measure amyloid β in a blood sample with high sensitivity. [Means for solving the problem]

[0006] As a result of intensive research into the measurement of amyloid beta in blood, the inventors of the present application discovered that amyloid beta in a blood sample can be measured with high sensitivity by performing an antigen-antibody reaction between amyloid beta in the blood sample and an anti-amyloid beta antibody or an antigen-binding fragment thereof in the presence of an anionic polymer, and thus arrived at the present invention.

[0007] That is, the present invention provides the following.

[0008] (1) A method for immunoassaying amyloid β in a blood sample, the immunoassay being carried out in the presence of an anionic polymer. (2) The method according to (1), wherein the anionic polymer is an anionic polymer having a sulfate group or a sulfo group in the side chain. (3) The method according to (2), wherein the anionic polymer is at least one selected from the group consisting of dextran sulfate and its salts, polystyrene sulfonic acid and its salts, and heparin sulfate and its salts. (4) The method according to (3), wherein the anionic polymer is dextran sulfate sodium. (5) The method according to any one of (1) to (4), wherein the immunoassay is a sandwich method, and includes measuring the amyloid beta by contacting the amyloid beta in the blood sample with an anti-amyloid beta antibody or an antigen-binding fragment thereof immobilized on a solid phase in the presence of the anionic polymer. (6) The method according to any one of (1) to (5), wherein the amyloid β is amyloid β1-40 or amyloid β1-42. (7) A kit for immunoassaying amyloid beta in a blood sample, comprising an anti-amyloid beta antibody or an antigen-binding fragment thereof, and an anionic polymer. (8) The kit according to (7), wherein the immunoassay is a sandwich method and comprises a solid phase having an anti-amyloid β antibody or an antigen-binding fragment thereof immobilized thereon, and an anionic polymer. (9) The kit according to (8), wherein the solid phase is particles, and the kit comprises a particle liquid containing the particles and the anionic polymer. Effect of the Invention

[0009] According to the method of the present invention, amyloid β in a blood sample can be measured with high sensitivity. [Brief description of the drawings]

[0010] [Figure 1] FIG. 2 shows the Aβ1-42 / Aβ1-40 ratio in Alzheimer's disease patients and healthy subjects, as measured in the Examples below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The sample used in the immunoassay method of the present invention is a blood sample. The blood sample includes whole blood, serum, and plasma. Preferably, the sample is serum or plasma.

[0012] As specifically described in the following examples, an experiment was conducted in which purified amyloid β was added to plasma and a buffer solution, and the recovery levels (count values) of the added amyloid β were compared using an immunoassay. This experiment revealed the presence of a substance (coating substance) that inhibits the reaction between amyloid β and anti-amyloid β antibodies, which are eluted in a fraction corresponding to a molecular weight of 300 to 400 kDa by gel filtration chromatography. As a result of further research, it was found that the binding inhibition by the coating substance can be reduced by performing the antigen-antibody reaction of the immunoassay in the presence of an anionic polymer. The present invention is based on this new finding, and its greatest feature is that the immunoassay is performed in the presence of an anionic polymer.

[0013] As the anionic polymer, those having sulfate groups in the side chain, such as dextran sulfate, heparin sulfate, chondroitin A sulfate, chondroitin B sulfate, and chondroitin C sulfate, and their salts, those having sulfo groups in the side chain, such as polystyrene sulfonic acid and its salts, and those having carboxyl groups, such as poly(meth)acrylic acid and its salts, are preferred. Among these, those having sulfate groups or sulfo groups in the side chain are preferred, and dextran sulfate, polystyrene sulfonic acid, and heparin sulfate, and their salts are more preferred, particularly dextran sulfate, polystyrene sulfonic acid, and their salts, and especially dextran sulfate and its salts are preferred. Examples of salts include alkali metal salts such as sodium salts and potassium salts. These anionic polymers can be used alone or in combination of two or more.

[0014] The average molecular weight of the anionic polymer is usually about 1,000 to 5,000,000, about 2,000 to 4,000,000, about 3,000 to 2,000,000, about 4,000 to 1,000,000, preferably about 5,000 to 700,000, and more preferably about 5,000 to 50,000, as a mass average molecular weight.

[0015] Since the anionic polymer reduces the inhibition of the antigen-antibody reaction between amyloid β and an anti-amyloid β antibody or an antigen-binding fragment thereof by the coating substance (hereinafter, unless otherwise clear from the context, the term "antibody" means "antibody or an antigen-binding fragment thereof"), it is clear that the anionic polymer is effective for any immunoassay method. That is, immunoassays include the sandwich method, competitive method, agglutination method, immunochromatography method, etc., and the present invention includes any of these immunoassays. These immunoassay methods themselves are well known and need not be described in detail here, but each will be briefly described.

[0016] 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 the present invention encompasses all of these techniques. The immunoassay of the present invention is not particularly limited, and may be a sandwich method. The sandwich method includes, for example, a one-step sandwich method and a two-step sandwich method.

[0017] In the two-step sandwich method, for example, first, an anti-amyloid β antibody immobilized on a solid phase or an anti-amyloid β antibody immobilized on a solid phase (solid-phase antibody) is brought into contact with amyloid β in a blood sample, and an antigen-antibody reaction between the solid-phase antibody and amyloid β is carried out (primary reaction). If the solid-phase antibody is an anti-amyloid β 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 carried out. Next, the amyloid β bound to the solid-phase antibody is brought into contact with an anti-amyloid β antibody (labeled antibody) to which a labeling substance for detection is bound, and an antigen-antibody reaction between the amyloid β and the labeled antibody is carried out (secondary reaction). Next, B / F separation is carried out, and amyloid β in the blood sample can be measured by detecting a signal derived from the label of the labeled antibody bound to the amyloid β bound to the solid phase. Washing may be carried out after B / F separation.

[0018] In the one-step sandwich method, the solid-phase antibody is contacted with the amyloid beta in the sample and the labeled antibody, and the antigen-antibody reaction between the solid-phase antibody and the amyloid beta and the antigen-antibody reaction between the amyloid beta and the labeled antibody are carried out in one step. If the solid-phase antibody is an anti-amyloid beta antibody that is immobilized on the solid phase, the solid-phase antibody is immobilized on the solid phase after the antigen-antibody reaction or simultaneously with the antigen-antibody reaction. Then, B / F separation is performed, and the amyloid beta 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.

[0019] The direct competitive method is a method in which an antibody against the target antigen to be measured (amyloid β in this invention) is immobilized on a solid phase (immobilization), and after blocking treatment (treating the solid phase with a protein solution such as serum albumin) to prevent non-specific adsorption, this antibody is reacted with a test sample containing the target antigen and a certain amount of labeled antigen, and after washing, the amount of label bound to the solid phase is quantified. Since the antigen and labeled antigen in the test sample competitively bind to the antibody, the greater the amount of antigen in the test sample, the smaller the amount of label bound to the solid phase. Various antigen standard solutions of 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 below) is measured, and a calibration curve is created with the antigen concentration on the horizontal axis and the amount of label on the vertical axis. The amount of label is measured for an unknown test sample, and the amount of antigen in the unknown test sample can be measured 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.

[0020] In the indirect competitive method, the target antigen (amyloid β in the present invention) is immobilized. Then, after blocking of the solid phase, a test sample containing the target antigen is mixed with a certain amount of anti-target antigen antibody and reacted with the immobilized antigen. After washing, the anti-target antigen antibody bound to the solid phase is quantified. This can be done by reacting a labeled secondary antibody against the anti-target antigen antibody, washing, and measuring the amount of label. Various antigen standard solutions of known concentrations are prepared, and the amount of label immobilized on the solid phase is measured for each, to create a calibration curve. The amount of label for an unknown test sample is measured, and the measured amount of label is applied to the calibration curve, thereby measuring the amount of antigen in the unknown test sample. It is also possible to use a labeled primary antibody without using a labeled secondary antibody. The indirect competitive method itself is well known in the art and is described, for example, in the above-mentioned US20150166678A.

[0021] 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 in 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, etc., 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) is used, in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP) is used, in the case of β-galactosidase, MG: 4-methylumbelliferyl galactoside, NG: nitrophenyl galactoside, in the case of luciferase, luciferin, etc. 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 in, for example, US8039223B and US20150309016A1.

[0022] The immunoturbidimetry (TIA) is an immunoassay that utilizes the phenomenon that turbidity increases due to an antigen-antibody complex generated by binding of a target antigen (amyloid β in the present invention) to be measured and an antibody against the antigen. Various known concentrations of antigens are added to an anti-target antigen-antibody solution, and the turbidity is measured for each, and a calibration curve is created. The turbidity of an unknown test sample is similarly measured, and the measured turbidity is applied to the calibration curve, so that the amount of antigen in the unknown test sample can be measured. The immunoturbidimetry itself is well known, and is described, for example, in US 20140186238 A1. The latex agglutination method is similar to the immunoturbidimetry, but instead of the antibody solution in the immunoturbidimetry, a suspension of latex particles with anti-target antigen-antibody immobilized on the surface is used. The immunoturbidimetry and latex agglutination methods themselves are well known in the art, and are described, for example, in US 7,820,398 B.

[0023] The immunochromatography method 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 the sandwich method of immunochromatography, a detection zone in which an anti-target antigen antibody is immobilized is provided on the substrate, a test sample containing a target antigen is added to the substrate, and a developing solution is flowed from the upstream side to move 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. A label zone containing a labeled secondary antibody is formed upstream of the detection zone, so that a conjugate of the target antigen and the labeled secondary antibody is immobilized in the detection zone. When 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 the competitive method, for example, a 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 be made to 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 to detect or quantify the label, thereby making it possible to detect or quantify the target antigen in the test sample. The immunochromatography method itself is well known in the art and is described, for example, in US 6210898 B.

[0024] 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 that uses magnetic particles as the 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.

[0025] The anionic polymer is present in a reaction system in which amyloid β in a blood sample is contacted with an anti-amyloid β antibody to carry out an antigen-antibody reaction between the amyloid β in the blood sample and the anti-amyloid β antibody.

[0026] For example, in the two-step sandwich method, the anionic polymer is preferably present in a reaction system in which a solid-phase antibody is brought into contact with amyloid β in a specimen to cause an antigen-antibody reaction between the solid-phase antibody and amyloid β. In the one-step sandwich method, the anionic polymer is preferably present in a reaction system in which a solid-phase antibody is brought into contact with amyloid β in a specimen and a labeled antibody to cause an antigen-antibody reaction.

[0027] The solid phase is not particularly limited, and a solid phase used in known immunoassays can be used. Specific examples of the material of the solid phase include, but are not limited to, polystyrene, polyethylene, sepharose, latex, dextran, agarose, gelatin, polyacrylamide, and the like. It is preferable that the solid phase used is one that can easily immobilize an antibody on its surface and can easily separate immune complexes formed during the measurement from unreacted components. In particular, plastic plates, latex particles, and magnetic particles used in ordinary immunoassays are preferable. From the viewpoints of ease of handling, storage, and separation, it is most preferable to use magnetic particles made of the above-mentioned materials. The antibody can be immobilized on these solid phases by a conventional method well known to those skilled in the art. The antibody can be immobilized on the solid phase by physical adsorption or covalent bonding. The antibody can also be immobilized on the solid phase by immobilizing one of affinity substances such as biotin-streptavidin on the solid phase, binding the other to the antibody, and mixing them to immobilize the antibody on the solid phase via the affinity substance.

[0028] The labeling substance is not particularly limited, and the same labeling substances used in known immunoassays can be used. Specific examples include enzymes, fluorescent substances, chemiluminescent substances, dyeing substances, radioactive substances, etc. As the enzyme, known substances such as alkaline phosphatase (ALP), peroxidase, β-galactosidase, etc. can be used, but are not limited thereto.

[0029] Amyloid β is generated by enzymatic cleavage of its precursor. Depending on the cleavage enzyme, there are two types of amyloid β (Aβ1-42), which consists of amino acids 1 to 42. 1-42 )), and those consisting of amino acids 1 to 40 (amyloid β1-40 (Aβ 1-40 )), and those consisting of amino acids 5 to 42 (amyloid β5-42 (Aβ 5-42 )), and those consisting of amino acids 5 to 40 (amyloid β5-40 (Aβ 5-40 )), and those consisting of amino acids 11 to 42 (amyloid β11-42 (Aβ 11-42 )), and those consisting of amino acids 11 to 40 (amyloid β11-40 (Aβ 11-40 )), and those consisting of amino acids 16 to 42 (amyloid β16-42 (Aβ 16-42 )), and those consisting of amino acids 16 to 40 (amyloid β16-40 (Aβ 16-40 )), and those consisting of amino acids 17 to 42 (amyloid β17-42 (Aβ 17-42 )), and those consisting of amino acids 17 to 40 (amyloid β17-40 (Aβ 17-40 )), and those consisting of amino acids 20 to 42 (amyloid β20-42 (Aβ 20-42 )), and those consisting of amino acids 20 to 40 (amyloid β20-40 (Aβ 20-40 )), and those consisting of amino acids 21 to 42 (amyloid β21-42 (Aβ 21-42 )), and those consisting of amino acids 21 to 40 (amyloid β21-40 (Aβ 21-40 )), and those consisting of amino acids 35 to 42 (amyloid β35-42 (Aβ 35-42), and those consisting of amino acids 35 to 40 (called amyloid β35-40 (Aβ 35-40 There are several types of Aβ. 1-42 , Aβ 5-42 , Aβ 11-42 , Aβ 16-42 , Aβ 17-42 , Aβ 20-42 , Aβ 21-42 , Aβ 35-42 These are collectively called amyloid βX-42. 1-40 , Aβ 5-40 , Aβ 11-40 , Aβ 16-40 , Aβ 17-40 , Aβ 20-40 , Aβ 21-40 , Aβ 35-40 These are collectively referred to as amyloid βX-40. The present invention is useful for measuring any of the amyloid β, and the measurement of any of these falls within the scope of the present invention, but it is particularly preferable to use the present invention for measuring amyloid β1-42 and amyloid β1-40.

[0030] The anti-amyloid β antibody or its antigen-binding fragment may be an antibody or its antigen-binding fragment that specifically binds to amyloid β. The antibody may be a monoclonal antibody or a polyclonal antibody. Generally, a monoclonal antibody is preferably used. Since the method for producing a polyclonal antibody and a monoclonal antibody is well known, an anti-amyloid β antibody may be prepared using an amyloid β antigen as an immunogen. The "antigen-binding 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, but are not limited to, Fab, F(ab')2, scFv, etc. As is well known, Fab and F(ab')2 can be obtained by treating an antibody with a protease such as papain or pepsin. The method for producing a scFv (single chain fragment of variable region, single chain antibody) is also well known, and can be produced according to a well-known method.

[0031] 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.

[0032] 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-AβN-terminal antibody) can be used. As described above, such antibodies may be commercially available or may be produced by a well-known method. An anti-Aβ42 antibody may be used as the solid-phase antibody and an anti-AβN-terminal antibody as the labeled antibody, or conversely, an anti-AβN-terminal antibody may be used as the solid-phase antibody and an anti-Aβ42 antibody as the labeled antibody.

[0033] In addition, to measure amyloid β1-40 by the sandwich method, an antibody that specifically binds to the C-terminal region of amyloid βx-40 (anti-Aβ40 antibody) and an antibody that specifically binds to the N-terminal region containing the first amino acid of amyloid β (anti-AβN-terminal antibody) can be used. As described above, these antibodies may be commercially available or may be produced by a well-known method. An anti-Aβ40 antibody may be used as the solid-phase antibody and an anti-AβN-terminal antibody as the labeled antibody, or conversely, an anti-AβN-terminal antibody may be used as the solid-phase antibody and an anti-Aβ40 antibody as the labeled antibody.

[0034] The anionic polymer can be added separately to an antigen-antibody reaction system, or can be added in advance to a solution containing an antibody-immobilized solid phase such as antibody-immobilized particles, or to a reagent such as a sample dilution solution for diluting a blood sample, or can be added to a blood sample.

[0035] The final concentration of the anionic polymer in the antigen-antibody reaction system is usually about 0.01 g / L to 50 g / L, about 0.02 g / L to 25 g / L, about 0.03 g / L to 10 g / L, about 0.04 g / L to 5 g / L, and preferably about 0.05 g / L to 2.5 g / L.

[0036] The immunoassay method itself is 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 a well-known immunoassay method as it is, except that the antigen-antibody reaction is carried out in the presence of an anionic polymer. Furthermore, as described above, immunoassays using CSF as a sample have already been carried out, and therefore kits used for such immunoassays can be used as they are.

[0037] In addition, Aβ is also present in the blood as in the CSF. 1-42 and Aβ 1-40 It is known that both Aβ and Aβ are present in the CSF and blood. 1-42 / Aβ 1-40 It is known that diagnostic specificity increases by taking Aβ 1-42 and Aβ 1-40 and Aβ are quantified and their ratio is calculated. If this ratio is smaller than that of a healthy individual, it is determined that there is a high possibility of Alzheimer's disease. Generally, the sensitivity of immunoassay decreases as the time elapsed from the time of blood sample collection to immunoassay increases. However, as specifically described in the Examples below, by carrying out the antigen-antibody reaction in the presence of an anionic polymer, the sensitivity hardly decreases even if the time elapsed from the time of sample collection is long (i.e., Aβ 1-42 / Aβ 1-40 We also found an unexpected effect: the effect of the stimulant stimuli on the stimuli was almost unchanged over time.

[0038] The kit of the present invention may be any kit for carrying out the immunoassay method of the present invention described above, and includes at least an anti-amyloid β antibody and an anionic polymer. In the case where the immunoassay is a sandwich method, the kit also includes a solid phase on which the anti-amyloid β antibody is immobilized. In the case where the solid phase is a particle such as a magnetic particle, the solid phase also includes the particle (usually in the form of a particle liquid). The kit of the present invention can be obtained by including an anionic polymer in a well-known amyloid β immunoassay kit. As described above, the anionic polymer may be included in reagents such as the anti-amyloid β antibody immobilized particle liquid or the sample dilution liquid, or may be included separately and independently in the kit. EXAMPLES

[0039] The present invention will be described in detail below with reference to examples, although the present invention is not limited to the following examples.

[0040] <Preparation of solid-phase particles> In 10 mM MES buffer (pH 5.0), 0.01 g / mL of magnetic particles was added with 0.2 mg / mL of mouse anti-Aβ42 antibody, which specifically binds to the C-terminal region of amyloid βx-42, and incubated at 25 ° C. for 1 hour with gentle stirring. After the reaction, the magnetic particles were collected with a magnet and washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-Aβ42 antibody solid-phased particles. The obtained anti-Aβ42 antibody solid-phased particles were suspended in a particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN3, 2.0% BSA, pH 7.2) to obtain an anti-Aβ42 antibody solid-phased particle solution.

[0041] Similarly, anti-Aβ40 antibody-immobilized particles and an anti-Aβ40 antibody-immobilized particle solution were prepared using a mouse anti-Aβ40 antibody that specifically binds to the C-terminal region of amyloid βx-40.

[0042] <Preparation of alkaline phosphatase-labeled antibody> Desalted alkaline phosphatase (ALP) and N-(4-maleimidobutyryloxy)-succinimide (GMBS) (final concentration 0.3 mg / mL) were mixed and left to stand at 30°C for 1 hour to perform maleimide conversion. Next, in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA2Na, pH 6.3), a Fab'-conjugated mouse anti-Aβ N-terminal antibody that specifically binds to the N-terminal region containing the first amino acid of amyloid β was mixed with maleimide-conjugated ALP in a molar ratio of 1:1 and reacted at 25°C for 1 hour. The mixture was purified using column chromatography on Superdex200 10 / 300 (GE) to obtain an alkaline phosphatase-labeled antibody (ALP-labeled antibody). The ALP-labeled antibody was suspended in a label diluent (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl2, 1 mM MgCl2, 0.1% NaN3, 2.0% BSA, pH 6.8) to obtain an ALP-labeled antibody solution.

[0043] <Amyloid β1-42 (Aβ 1-42 ) Measurement method> 50μL of sample and 50μL of anti-Aβ42 antibody solid-phase particle solution were dispensed into the reaction vessel and stirred. After that, it was incubated at 37℃ for 8 minutes, and B / F separation and washing were performed using a magnetic field. 50μL of ALP-labeled antibody solution was further dispensed into this reaction vessel, and after stirring, it was incubated at 37℃ for 8 minutes, and B / F separation and washing were performed using a magnetic field. Then, 200μL of Lumipulse (registered trademark) substrate solution (manufactured by Fujirebio Co., Ltd.) containing 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane·disodium salt (AMPPD), which is a chemiluminescent substrate, was dispensed into the reaction vessel, and after stirring, it was incubated at 37℃ for 4 minutes, and the amount of luminescence was measured with a luminometer. The actual measurement was performed using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (manufactured by Fujirebio Co., Ltd.)).

[0044] <Amyloid β1-40 (Aβ 1-40 ) Measurement method> 30 μL of sample and 50 μL of anti-Aβ40 antibody solid-phase particle solution were dispensed into the reaction vessel and stirred. After that, it was incubated at 37 ° C for 8 minutes, and B / F separation and washing were performed using a magnetic field. 50 μL of enzyme-labeled antibody solution was further dispensed into this reaction vessel, and after stirring, it was incubated at 37 ° C for 8 minutes, and B / F separation and washing were performed using a magnetic field. Then, 200 μL of Lumipulse substrate solution (manufactured by Fujirebio) containing AMPPD was dispensed into the reaction vessel, and after stirring, it was incubated at 37 ° C for 4 minutes, and the amount of luminescence was measured with a luminometer. The actual measurement was performed using a fully automated chemiluminescence enzyme immunoassay system (Lumipulse L2400 (manufactured by Fujirebio)).

[0045] Example 1 Aβ using blood samples 1-42 and Aβ 1-40 The effect of adding dextran sulfate sodium on the measurement of .

[0046] As a sample, Aβ 1-42 and Aβ 1-40 Aβ was added to 4.5 mL of blood samples (serum and EDTA 2K plasma) with a concentration of below the detection limit. 1-42 Antigens and Aβ 1-40 500 μL of cerebrospinal fluid (CSF) containing antigen at a concentration of approximately 794 pg / mL and approximately 7744 pg / mL, respectively, was added to prepare antigen-containing serum (Serum 1, Serum 2) and antigen-containing plasma (EDTA2K). In addition, Tris-buffered saline (TBS buffer, pH 7.4) was added with Aβ at the same concentration as the blood sample. 1-42 Antigens and Aβ 1-40 The antigen was added to prepare an antigen-containing TBS buffer solution. The prepared sample was used to carry out the above-mentioned "Aβ 1-42 "Method of measuring Aβ 1-40 According to the method described in "Method of measuring Aβ 1-42 and Aβ 1-40As for dextran sulfate sodium, dextran sulfate sodium (mass average molecular weight 5000 (hereinafter referred to as "molecular weight 5000" and the same applies to other substances)) was added to the antibody-immobilized particle solution to a concentration of 0.5 g / L, and the effect of adding it was examined. The final concentration of dextran sulfate sodium in the antigen-antibody reaction system was 1-42 0.25g / L, Aβ 1-40 The count was 0.31 g / L. As blanks, each sample (serum, EDTA 2K plasma, and TBS buffer) to which no antigen had been added was measured in the same manner, and the count obtained was used as the blank value. The count value was calculated by subtracting the blank value from the count of each antigen-containing sample. Aβ 1-42 The results are shown in Table 1. 1-40 The results are shown in Table 2.

[0047] [Table 1]

[0048] [Table 2]

[0049] As shown in Table 1, in the absence of dextran sulfate sodium, the Aβ levels in serum and plasma samples were 1-42 The count value of the serum sample Serum2 was significantly lower than that of the buffer solution sample, at 56.9%, assuming the buffer solution sample count value to be 100%.

[0050] On the other hand, dextran sulfate sodium was added to the antibody-immobilized particle solution to obtain Aβ 1-42 When the counts were measured, the count values ​​increased in both the serum and plasma samples, and the count values ​​recovered to 80% or more of the count values ​​in the buffer sample.

[0051] In addition, as shown in Table 2, Aβ 1-40Regarding Aβ, when dextran sulfate sodium was not added, 1-40 The count value of the plasma sample EDTA2K was lower than that of the buffer sample. If the count value of the buffer sample is taken as 100%, the count value of the plasma sample EDTA2K was significantly lower at 36.4%. On the other hand, when dextran sulfate sodium was added to the antibody-immobilized particle solution, the Aβ 1-40 When the counts were measured, the counts in the plasma samples increased and recovered to 90% or more of the counts in the buffer sample.

[0052] Example 2 Aβ in the blood 1-42 The concentration of dextran sulfate sodium added in the measurement of Aβ was examined. 1-42 was measured. As for dextran sodium sulfate, dextran sodium sulfate (molecular weight 5000) was added to the antibody-immobilized particle solution to the concentration (0.1 g / L to 5.0 g / L) shown in Table 3, and the effect of adding it was examined. The final concentration of dextran sodium sulfate in the antigen-antibody reaction system was 0.05 g / L to 2.5 g / L. Furthermore, blank values ​​were obtained in the same manner as in Example 1, and count values ​​were calculated. The results are shown in Table 3.

[0053] [Table 3]

[0054] As shown in Table 3, under all conditions of dextran sulfate sodium concentration examined, the count value increased compared to the count value under the condition where no dextran sulfate sodium was added.

[0055] Examples 3 to 5, Comparative Examples 1 to 3 The effect of adding various anionic polymers on the measurement of Aβ in blood samples was examined. In Examples 1 and 2, serum (antigen-containing serum, Serum 2) in which the count value was particularly significantly decreased without the addition of dextran sulfate sodium, and antigen-containing TBS buffer (Buffer) were used to measure Aβ by the method described in Example 1. 1-42 was measured. As the anionic polymer, sodium dextran sulfate (molecular weight 5,000) (Example 3), poly(sodium 4-styrenesulfonate) (molecular weight 700,000) (Example 4), sodium dodecyl sulfate (SDS) (Comparative Example 1), sodium dodecylbenzenesulfonate (SDBS) (Comparative Example 2), polyacrylic acid sodium salt (molecular weight 5,100) (Example 5), and sodium N-lauroylsarcosine (NLS) (Comparative Example 3) were used. These anionic polymers were added to the antibody solid-phase particle solution to a concentration of 0.5 g / L, and the effect of adding them was examined. In addition, a blank value was obtained in the same manner as in Example 1, and the count value was calculated. The results are shown in Table 4.

[0056] [Table 4]

[0057] As shown in Table 4, in the absence of an anionic polymer, the count value of the serum sample (Serum2) was 56.9% of the count value of the buffer sample, whereas the addition of the anionic polymers dextran sulfate sodium (Example 3), poly(sodium 4-styrenesulfonate) (Example 4), and polyacrylic acid sodium salt (Example 5) increased the count value to 82.6%, 77.4%, and 66.9%, respectively. In particular, the anionic polymers dextran sulfate sodium and poly(sodium 4-styrenesulfonate) having sulfate or sulfo groups in their side chains showed a large recovery of the count value, and were particularly effective. On the other hand, the addition of SDS (Comparative Example 1), a surfactant having a sulfate group in its side chain, SDBS (Comparative Example 2), a surfactant having a sulfo group in its side chain, or NLS (Comparative Example 3), which has a carboxyl group in its side chain, did not restore the count value.

[0058] Therefore, it was shown that the addition of an anionic polymer to the reaction solution has the effect of improving the decrease in the count value when measuring amyloid β in a blood sample. Furthermore, it was shown that, among the anionic polymers, the improvement effect is particularly large when the anionic polymer has a sulfate group or a sulfo group in the side chain.

[0059] Example 6 The effect of the concentration of anionic polymer added on the measurement of Aβ in blood samples was examined. 1-42 A 50,000pg / mL concentration of synthetic Aβ was added to 5mL of blood samples (serum and EDTA·2K plasma) with a concentration below the detection limit. 1-42 50 μL of antigen was added to prepare antigen-containing serum (Serum 1, Serum 2) and antigen-containing plasma (EDTA2K). In addition, synthetic Aβ was added to Tris-buffered saline (TBS buffer, pH 7.4) at the same concentration as the blood sample. 1-42 The antigen was added to prepare an antigen-containing TBS buffer solution. Each prepared sample was used to detect Aβ using the method described in Example 1. 1-42was measured. As the anionic polymer, instead of dextran sodium sulfate, heparin sodium sulfate, an anionic polymer having sulfate groups in the side chains, was added to the antibody-immobilized particle solution to the concentrations shown in Table 5 (0.5 g / L, 5.0 g / L), and the effect of adding it was examined. The final concentrations of heparin sodium sulfate in the antigen-antibody reaction system were 0.25 g / L and 2.5 g / L. Furthermore, blank values ​​were obtained and count values ​​were calculated in the same manner as in Example 1. The results are shown in Table 5.

[0060] [Table 5]

[0061] As shown in Table 5, under all of the conditions in which sodium heparin sulfate was added at different concentrations, the count value increased compared to the count value under the condition in which no heparin sulfate was added.

[0062] Example 7 The effect of adding dextran sulfate sodium with different molecular weights on the measurement of Aβ in blood samples was examined. In Examples 1 and 2, synthetic Aβ at a concentration of 50,000 pg / mL was added to 5 mL of serum (Serum2), which showed a particularly significant decrease in count value when dextran sulfate sodium was not added. 1-42 50 μL of antigen was added to prepare antigen-containing serum, and Aβ 1-42 In addition, synthetic Aβ was added to the TBS buffer at the same concentration as that of the blood sample. 1-42 The antigen was added to prepare an antigen-containing TBS buffer solution, and Aβ 1-42 were measured. Two types of dextran sulfate with average molecular weights of 5,000 and 50,000 were used, and the concentration added to the antibody-immobilized particle solution was 7 g / L. The final concentration of dextran sulfate sodium in the antigen-antibody reaction system was 3.5 g / L. Furthermore, a blank value was obtained in the same manner as in Example 1, and the count value was calculated. The results are shown in Table 6.

[0063] [Table 6]

[0064] As shown in Table 6, in the absence of added dextran sulfate sodium, the count value of the serum sample (Serum 2) was 66.7% of the count value of the buffer sample, whereas when dextran sulfate sodium (molecular weight 5000) and dextran sulfate sodium (molecular weight 50,000) were added, the count value increased to 80.9% and 93.8%, respectively.

[0065] Therefore, it was shown that adding an anionic polymer such as dextran sulfate sodium with an average molecular weight of 5,000 or more to the reaction solution has the effect of improving the decrease in count values ​​when measuring amyloid beta in blood samples.

[0066] Example 8 To investigate the reason why the count value in blood samples was lower than that in buffer samples, EDTA·2K plasma and cerebrospinal fluid (CSF) were fractionated using a gel filtration column. The conditions were as follows: the apparatus was an AKTA FPLC (GE Healthcare), the column was a Superose6 10x300 (GE Healthcare), the flow rate was 0.3 mL / min, the buffer was D-PBS(-), the application volume was 1 mL, and the volume of each fraction was 1 mL. Aβ was measured for each fraction obtained. 1-42 The antigen was added to a concentration of 1000 pg / mL to prepare a sample. The prepared sample was used to measure Aβ 1-42 The effect of adding dextran sulfate sodium (molecular weight 5000) to the antibody-immobilized particle solution was examined by adding it to the antibody-immobilized particle solution at a concentration of 2.5 g / L. The final concentration of dextran sulfate sodium in the antigen-antibody reaction system was 1.3 g / L. As a control sample, Aβ 1-42The same measurement was performed using TBS buffer to which the antigen was added at 1000 pg / mL. As a result, the ratio (%) of the count value of each sample to the count value of the control sample is shown in Table 7. The molecular weight of each fraction was calculated by using a molecular weight marker (manufactured by Bio-Rad) under the above conditions and creating an approximation equation.

[0067] [Table 7]

[0068] As shown in Table 7, in EDTA2K plasma, the ratio of count values ​​in the fractions around 300-400 kDa decreased to 61% without the addition of dextran sulfate sodium, but it was confirmed that the count value recovered to the same level as the control sample (106%) by adding dextran sulfate sodium. On the other hand, in CSF, the count value did not decrease compared to the control sample in the absence of dextran sulfate sodium in any fraction, and no fractions were observed in which the ratio of count values ​​significantly differed depending on whether dextran sulfate sodium was added or not.

[0069] Therefore, it is believed that the component that reduces the count value is present only in the blood sample, and the influence of this component can be avoided by adding an anionic polymer.

[0070] Example 9 Aβ in serum samples and EDTA2Na plasma samples from healthy subjects 1-42 and Aβ 1-40 The effect of anionic polymers on the measurement was examined. Blood samples were collected using plain (10 mL blood collection tube, Terumo) and EDTA2Na (7 mL blood collection tube, Terumo), left to stand at room temperature for 30 minutes, centrifuged at 1200 × g for 10 minutes, and the supernatant was collected.

[0071] To evaluate the effect of sample storage, the above-mentioned “Aβ 1-42 "Method of measuring Aβ 1-40 According to the "Method of Measuring Aβ 1-42 and Aβ 1-40 As an anionic polymer, dextran sulfate sodium (molecular weight 5000) was added to the antibody-immobilized particle solution at a concentration of 0.5 g / L, and the effect of adding it was examined. The measured values ​​and the ratio of the measured values ​​to the value on day 0 of storage under each condition are shown in Table 8. The measured values ​​were calculated using the synthetic Aβ 1-42 Peptides and synthetic Aβ 1-40 The peptides were prepared as calibrators at 0 pg / mL, 10 pg / mL, 100 pg / mL, and 1000 pg / mL, respectively, and the measurements were performed. An approximation formula was created based on the count values ​​obtained by measuring the calibrators, and the count values ​​obtained from each sample were fitted to the approximation curve to calculate the measured values.

[0072] [Table 8-1]

[0073] [Table 8-2]

[0074] As shown in Table 8, when dextran sulfate sodium was added to the reagent, the count value and the measured value increased in both serum and plasma samples. When measured over time, the measured values ​​tended to decrease.

[0075] In the measurement of amyloid beta, Aβ 1-42 and Aβ 1-40 The ratio of Aβ measured under each condition in this example is taken and compared. 1-40 Aβ against 1-42 The ratio was calculated. The results are shown in Table 9.

[0076] [Table 9]

[0077] As shown in Table 9, when serum was measured using a reagent without dextran sulfate sodium, Aβ 1-42 / Aβ 1-40 However, when serum was measured using a reagent containing dextran sulfate sodium, the Aβ ratio changed over time. 1-42 / Aβ 1-40 The ratio value did not change and was not affected by the storage period. 1-42 / Aβ 1-40 It was confirmed that the ratio could be obtained.

[0078] Therefore, the addition of an anionic polymer such as sodium dextran sulfate reduces Aβ 1-42 and Aβ 1-40 In addition to increasing the count value of Aβ, the Aβ 1-42 / Aβ 1-40 It was confirmed that the ratio could be obtained.

[0079] Example 10 Using a reagent containing sodium dextran sulfate, Aβ levels in serum samples from Alzheimer's disease (AD) and non-AD patients were measured. 1-42 and Aβ 1-40 was measured.

[0080] Aβ in serum samples from AD patients (26 cases) and non-AD patients (22 cases) 1-42 and Aβ 1-40was measured by the following method. First, 50 μL of sample and 250 μL of antibody solid-phase particle solution to which dextran sulfate sodium (molecular weight 5000) was added so as to be 0.5 g / L were dispensed into a reaction vessel and stirred. The final concentration of dextran sulfate sodium in the antigen-antibody reaction system was 0.42 g / L. Then, the mixture was incubated at 37° C. for 8 minutes, and B / F separation and washing were performed using a magnetic field. 250 μL of enzyme-labeled antibody solution was further dispensed into this reaction vessel, and after stirring, the mixture was incubated at 37° C. for 8 minutes, and B / F separation and washing were performed using a magnetic field. Then, 200 μL of Lumipulse (registered trademark) substrate solution (manufactured by Fujirebio Co., Ltd.) containing AMPPD, which is a chemiluminescent substrate, was dispensed into the reaction vessel, and after stirring, the mixture was incubated at 37° C. for 4 minutes, and the amount of luminescence was measured with a luminometer. The actual measurement was performed using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse G1200 (manufactured by Fujirebio Co., Ltd.)). The results are shown in FIG. 1. An approximate equation was created using the same calibrator as in Example 6, and Aβ was calculated from the sample count. 1-42 and Aβ 1-40 The measured value was calculated.

[0081] Figure 1 shows the Aβ 1-42 / Aβ 1-40 The ratio of Aβ in AD patients was measured using a reagent containing dextran sulfate sodium. 1-42 / Aβ 1-40 The ratio of Aβ to non-AD patients 1-42 / Aβ 1-40 This was significantly lower than the ratio (p value 0.0022 (Wilcoxon-Mann-Whitney test)), confirming that the addition of an anionic polymer to the reaction solution is also useful in measuring Aβ using patient samples.

[0082] Therefore, it was demonstrated that reagents containing anionic polymers such as dextran sulfate sodium are useful for diagnosing Alzheimer's disease.

Claims

1. A method for immunoassaying amyloid β in a blood sample, the immunoassay being carried out in the presence of an anionic polymer.

2. The method according to claim 1 , wherein the anionic polymer is an anionic polymer having sulfate or sulfo groups in the side chains.

3. 3. The method according to claim 2, wherein the anionic polymer is at least one selected from the group consisting of dextran sulfate and its salts, polystyrene sulfonic acid and its salts, and heparin sulfate and its salts.

4. The method of claim 3, wherein the anionic polymer is sodium dextran sulfate.

5. The method according to any one of claims 1 to 4, wherein the immunoassay is a sandwich method, and comprises measuring the amyloid beta by contacting the amyloid beta in the blood sample with an anti-amyloid beta antibody or an antigen-binding fragment thereof immobilized on a solid phase in the presence of the anionic polymer.

6. The method according to any one of claims 1 to 5, wherein the amyloid β is amyloid β1-40 or amyloid β1-42.

7. A kit for immunoassaying amyloid β in a blood sample, comprising an anti-amyloid β antibody or an antigen-binding fragment thereof, and an anionic polymer.

8. The kit according to claim 7 , wherein the immunoassay is a sandwich method and comprises a solid phase having an anti-amyloid β antibody or an antigen-binding fragment thereof immobilized thereon, and an anionic polymer.

9. The kit according to claim 8 , wherein the solid phase is a particle, and the kit comprises a particle liquid containing the particle and the anionic polymer.

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