Method for the immunoassay of beta amyloid in blood and kit for use in the method

Abstract

Disclosed is an immunoassay method capable of highly sensitively measuring β-amyloid in a blood sample and a kit therefor. The immunoassay method for β-amyloid is a method for immunoassaying β-amyloid in a blood sample, wherein the immunoassay is performed in the presence of an anionic polymer such as a dextran sulfate salt or a polystyrene sulfonate salt. The kit for immunoassaying β-amyloid in a blood sample contains an anti-β-amyloid antibody or antigen-binding fragment thereof and an anionic polymer.

Description

Technical Field

[0001] This invention relates to an immunoassay method for β-amyloid protein in blood and a kit for using the method. Background Technology

[0002] β-amyloid protein is well-known as a biomarker for Alzheimer's disease, and the measurement of β-amyloid protein in samples by immunoassay is also known (Patent Document 1), and kits for this purpose are commercially available. Several commercially available immunoassay kits for β-amyloid protein use cerebrospinal fluid (CSF) as the sample. On the other hand, β-amyloid protein is known to be present in the blood of Alzheimer's patients. Since CSF collection is invasive, it would be advantageous to be able to measure β-amyloid protein in blood samples via immunological methods.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: JP2006-091025A Summary of the Invention

[0006] The technical problem that the invention aims to solve

[0007] Whether measured in CSF or blood, the target protein is the same: β-amyloid. Therefore, it is assumed that immunoassays of β-amyloid in blood can be performed in the same way as with CSF. However, when performing immunoassays of β-amyloid in blood samples, it has been found that the sensitivity is reduced compared to using CSF as the sample.

[0008] Therefore, the object of the present invention is to provide an immunoassay method and a kit for measuring β-amyloid protein in blood samples with high sensitivity.

[0009] Means for solving technical problems

[0010] The inventors of this application conducted in-depth research on the determination of β-amyloid protein in blood and discovered that by reacting β-amyloid protein in blood samples with anti-β-amyloid antibodies or their antigen-binding fragments in the presence of anionic polymers, β-amyloid protein in blood samples can be determined with high sensitivity, thus completing this invention.

[0011] That is, the present invention provides the following solution.

[0012] (1) An immunoassay method for β-amyloid protein, which is a method for immunoassay of β-amyloid protein in a blood sample; wherein the immunoassay is performed in the presence of an anionic polymer.

[0013] (2) According to the method of (1), wherein the above-mentioned anionic polymer is an anionic polymer having a sulfate group or a sulfonic acid group on the side chain.

[0014] (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.

[0015] (4) According to the method described in (3), wherein the above-mentioned anionic polymer is sodium dextran sulfate.

[0016] (5) The method according to any one of (1) to (4), wherein the above immunoassay is a sandwich method, the method comprising: in the presence of the above anionic polymer, contacting the above β-amyloid protein in the above blood sample with an anti-β-amyloid antibody or its antigen-binding fragment solidified on a solid phase, and measuring the β-amyloid protein.

[0017] (6) The method according to any one of (1) to (5), wherein the above-mentioned β-amyloid protein is β-amyloid protein 1-40 or β-amyloid protein 1-42.

[0018] (7) A kit for immunoassay of β-amyloid in a blood sample, comprising an anti-β-amyloid antibody or an antigen-binding fragment thereof and an anionic polymer.

[0019] (8) The kit according to (7), wherein the above immunoassay is a sandwich method, the kit comprising a solid phase and an anionic polymer immobilized with anti-β amyloid antibody or its antigen-binding fragment.

[0020] (9) The kit according to (8), wherein the solid phase is a particle, and the kit comprises a particle liquid containing the particle and the anionic polymer.

[0021] The effects of the invention

[0022] According to the method of the present invention, β-amyloid protein in blood samples can be determined with high sensitivity. Attached Figure Description

[0023] Figure 1 To illustrate the Aβ values ​​measured in Alzheimer's patients and healthy individuals in the following examples 1-42 / Aβ 1-40 A diagram of ratios. Detailed Implementation

[0024] The sample provided in the immunoassay method of the present invention is a blood sample. The blood sample includes any one of whole blood, serum, and plasma. Preferably, it is serum or plasma.

[0025] As specifically described in the following examples, experiments were conducted by adding purified β-amyloid protein to plasma and buffer solutions and comparing the recovery level (count value) of the added β-amyloid protein using an immunoassay. Gel filtration chromatography revealed the presence of a substance (coating material) that inhibits the reaction between β-amyloid protein and anti-β-amyloid antibody, eluted to a fraction equivalent to a molecular weight of 300–400 kDa. Further investigation showed that the binding inhibition caused by the coating material could be mitigated by performing the antigen-antibody reaction in the presence of an anionic polymer. The most significant feature of this invention, based on this novel insight, is the immunoassay performed in the presence of an anionic polymer.

[0026] As anionic polymers, preferred are anionic polymers having sulfate groups in their side chains, such as dextran sulfate, heparin sulfate, chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate C, and their salts; anionic polymers having sulfonic acid groups in their side chains, such as polystyrene sulfonic acid and its salts; and anionic polymers having carboxyl groups, such as poly(meth)acrylic acid and its salts. Among these, anionic polymers having sulfate or sulfonic acid groups in their side chains are preferred, more preferably dextran sulfate, polystyrene sulfonic acid, and heparin sulfate, and their salts, particularly preferred, and especially preferred dextran sulfate and its salts. Examples of salts include alkali metal salts such as sodium and potassium salts. These anionic polymers can be used alone or in combination of two or more.

[0027] The average molecular weight of anionic polymers, measured by mass-average molecular weight, is typically around 1,000–5,000,000, 2,000–4,000,000, 3,000–2,000,000, or 4,000–1,000,000, preferably around 5,000–700,000, and more preferably around 5,000–50,000.

[0028] It has been established that anionic polymers can mitigate the inhibition of antigen-antibody reactions between the coating material and anti-β-amyloid protein antibodies or their antigen-binding fragments (hereinafter, unless explicitly excluded by context, the term "antibody" refers to "antibody or its antigen-binding fragment"), and are therefore effective in any immunoassay method. That is, immunoassays include sandwich assays, competitive assays, agglutination assays, immunochromatography, etc., and this invention includes any of these immunoassays. These immunoassay methods are known in themselves and need not be described in detail here; therefore, they will be briefly described separately.

[0029] Sandwich methods include various techniques such as chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, electrochemiluminescence immunoassay (ECLIA), and fluorescence immunoassay (FIA), and this invention includes any of these methods. The immunoassay of this invention is not particularly limited and can be a sandwich method. Sandwich methods include, for example, one-step sandwich methods and two-step sandwich methods.

[0030] In the two-step sandwich method, for example, firstly, an anti-β-amyloid antibody immobilized on a solid phase, or an anti-β-amyloid antibody to be immobilized on a solid phase (solid-phase antibody), is contacted with β-amyloid in a blood sample to perform an antigen-antibody reaction between the solid-phase antibody and β-amyloid (primary reaction). If the solid-phase antibody is an anti-β-amyloid antibody to be immobilized on a solid phase, after the primary reaction, the solid-phase antibody is immobilized on the solid phase. Then, B / F separation is performed. Next, the β-amyloid bound to the solid-phase antibody is contacted with a labeled anti-β-amyloid antibody (labeled antibody) used for detection to perform an antigen-antibody reaction between β-amyloid and the labeled antibody (secondary reaction). Then, B / F separation is performed, and the signal from the labeled antibody bound to the β-amyloid bound to the solid phase is detected, thereby determining the β-amyloid in the blood sample. After B / F separation, washing can be performed.

[0031] In a one-step sandwich method, a solid-phase antibody is brought into contact with β-amyloid protein and a labeled antibody in the sample. The antigen-antibody reaction between the solid-phase antibody and β-amyloid protein, and the antigen-antibody reaction between β-amyloid protein and the labeled antibody, are performed in a single step. When the solid-phase antibody is an anti-β-amyloid antibody to be immobilized on a solid phase, it is immobilized on the solid phase after or simultaneously with the antigen-antibody reaction. Then, B / F separation is performed, and the signal from the labeling of the labeled antibody bound to the β-amyloid protein on the solid phase is detected, thereby allowing the determination of β-amyloid protein in the blood sample.

[0032] The direct competition method is as follows: An antibody targeting the antigen to be measured (β-amyloid protein in this invention) is immobilized (solid-phase) onto a solid phase. After a blocking treatment to prevent non-specific adsorption (treating the solid phase with a protein solution such as serum albumin), the antibody reacts with a test sample containing the target antigen and a certain amount of labeled antigen. After washing, the label bound to the solid phase is quantified. The antigen in the test sample and the labeled antigen competitively bind to the antibody; therefore, the more antigen in the test sample, the less label binds to the solid phase. Various antigen standard solutions of known concentrations are prepared, and the amount of label immobilized on the solid phase is measured (absorbance, luminescence intensity, fluorescence intensity, etc., are measured according to the properties of the label, the same applies below). A standard curve is constructed with antigen concentration on the horizontal axis and label amount on the vertical axis. For unknown test samples, the label amount is measured, and the measured label amount is substituted into the standard curve, thereby determining the amount of antigen in the unknown test sample. The direct competition method itself is known in this field, for example, as described in US20150166678A.

[0033] In the indirect competitive method, the target antigen (β-amyloid protein in this invention) is immobilized. Next, after blocking the immobilized phase, the 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 immobilized phase is quantified. This can be done by reacting a labeled secondary antibody against the anti-target antigen antibody and measuring the amount of labeling after washing. Various antigen standard solutions of known concentrations are prepared, immobilized on the immobilized phase, and the amount of labeling is measured to create a standard curve. For unknown test samples, the amount of labeling is measured, and the measured amount of labeling is substituted into the standard curve, thereby determining the amount of antigen in the unknown test sample. It should be noted that a labeled primary antibody can also be used instead of a labeled secondary antibody. The indirect competitive method itself is known in this field, for example, as described in US20150166678A mentioned above.

[0034] Among the various immunoassays mentioned above, chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescence immunoassay (FIA) are immunoassays classified based on the types of labels used in the aforementioned direct competition method, indirect competition 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 that generates a chemiluminescent compound (e.g., AMPPD) 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. As the substrate for each enzyme, a compound that can be quantified by absorbance measurement, etc., is used. For example, in the case of peroxidase, 1,2-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB) are used; in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP) is used; in the case of β-galactosidase, MG (4-methylumbelliferyl ketone galactoside) and NG (nitrophenyl galactoside) are used; and in the case of luciferase, luciferin is used. Radioimmunoassay (RIA) is a method that uses radioactive materials as labels. Examples of radioactive materials include... 3 H, 14 C 32 P, 35 S, 125 Radioactive elements such as I. Fluorescent immunoassay (FIA) is a method that uses fluorescent substances or fluorescent proteins as labels. Examples of fluorescent substances or fluorescent proteins include fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, and red fluorescent protein. Immunoassays using these labels are known in the field, for example, as described in US8039223B and US20150309016A1.

[0035] Immunoturbidimetry (TIA) is an immunoassay that utilizes the following phenomenon: the target antigen (β-amyloid in this invention) binds to an antibody against that antigen, and the resulting antigen-antibody complex increases turbidity. Various known concentrations of antigen are added to the anti-target antigen antibody solution, and the turbidity is measured to create a standard curve. For unknown test samples, the turbidity is measured in the same way, and the measured turbidity is substituted into the standard curve to determine the amount of antigen in the unknown test sample. Immunoturbidimetry itself is known, for example, as described in US 20140186238 A1. Latex agglutination is similar to immunoturbidimetry, but uses a suspension of latex particles with surface-immobilized anti-target antigen antibodies instead of the antibody solution in immunoturbidimetry. Immunoturbidimetry and latex agglutination are known in this field, for example, as described in US 7820398 B.

[0036] Immunochromatography is a method that uses sandwich or competitive methods on a matrix (also called a substrate or test strip) made of porous materials such as filter paper, cellulose membrane, glass fiber, or nonwoven fabric. For example, in the case of sandwich-based immunochromatography, a detection zone immobilized with an antibody against the target antigen is provided on the matrix. A test sample containing the target antigen is added to the matrix, and the developing solvent flows from the upstream side, causing the target antigen to move to and immobilize 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, thereby detecting the target antigen in the test sample. By pre-forming a labeled region containing the labeled secondary antibody on the upstream side of the detection zone, the conjugate of the target antigen and the labeled secondary antibody is immobilized in the detection zone. In the case of an enzyme label, a substrate region containing the enzyme substrate is also provided on the upstream side of the detection zone. In the case of a competitive method, for example, the target antigen can be pre-immobilized in the detection zone, and the target antigen in the test sample competes with the immobilized target antigen in the detection zone. A labeled antibody region is pre-positioned upstream of the detection zone, allowing the target antigen in the test sample to react with the labeled antibody. Unreacted labeled antibody is immobilized in the detection zone, and the label is then detected or quantified. This allows for the detection or quantification of the target antigen in the test sample. Immunochromatography itself is known in this field, for example, as described in US 6210898 B.

[0037] Among the various immunoassays mentioned above, sandwich methods are preferred from the viewpoint of detection sensitivity and ease of automation, especially chemiluminescent enzyme immunoassay (CLEIA), which is an immunoassay that uses magnetic particles as the solid phase, an enzyme (e.g., alkaline phosphatase) as the label, and a substrate that generates a chemiluminescent compound (e.g., 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxane disodium salt (AMPPD)) as the substrate.

[0038] Anionic polymers exist in reaction systems that bring β-amyloid protein in blood samples into contact with anti-β-amyloid antibodies, thus carrying out an antigen-antibody reaction between β-amyloid protein in blood samples and anti-β-amyloid antibodies.

[0039] For example, in a two-step sandwich method, anionic polymers are preferably present in the reaction system in which a solid-phase antibody contacts β-amyloid protein in the sample to carry out an antigen-antibody reaction between the solid-phase antibody and β-amyloid protein. In a one-step sandwich method, anionic polymers are preferably present in the reaction system in which a solid-phase antibody contacts β-amyloid protein in the sample and a labeled antibody to carry out an antigen-antibody reaction.

[0040] The solid phase is not particularly limited, and any solid phase known for use in immunoassays can be used. Specific examples of solid phase materials include polystyrene, polyethylene, agarose, latex, dextran, gelatin, and polyacrylamide, but are not limited to these. The solid phase used is preferably one on which antibodies can be easily immobilized, allowing for easy separation of the immune complex formed during the assay from unreacted components. In particular, plastic plates, latex particles, and magnetic particles commonly used in immunoassays are preferred. From the viewpoint of ease of handling, preservation, and separation, magnetic particles made of the aforementioned materials are most preferred. Antibodies can be immobilized on these solid phases using conventional methods known to those skilled in the art. Immobilization of antibodies on solid phases can be achieved through physical adsorption or covalent bonding. Regarding the immobilization of antibodies on solid phases, one of an affinity substance such as biotin-streptavidin can be immobilized on the solid phase, while the other binds to the antibody; these can be mixed, and the antibody can be immobilized on the solid phase via the affinity substance.

[0041] There are no particular limitations on the labeling substance; any known labeling substance used in immunoassays may be used. Specific examples include enzymes, fluorescent substances, chemiluminescent substances, staining substances, and radioactive substances. For enzymes, known enzymes such as alkaline phosphatase (ALP), peroxidase, and β-galactosidase may be used, but the study is not limited to these.

[0042] β-amyloid protein is formed by the cleavage of its precursor by an enzyme, and there are various types depending on the cleavage enzyme, such as β-amyloid protein composed of amino acids from position 1 to position 42 (β-amyloid 1-42 (Aβ)). 1-42 ), composed of amino acids from position 1 to position 40, β-amyloid protein (β-amyloid 1-40 (Aβ)). 1-40 β-amyloid protein (β-amyloid protein 5-42 (Aβ)) is composed of amino acids from position 5 to position 42. 5-42 β-amyloid protein (β-amyloid protein 5-40 (Aβ)) is composed of amino acids from position 5 to position 40. 5-40 ), composed of amino acids from position 11 to position 42 (β-amyloid protein 11-42 (Aβ) 11-42 ), composed of amino acids from position 11 to 40 (β-amyloid protein 11-40 (Aβ) 11-40 β-amyloid protein (β-amyloid 16-42 (Aβ)) is composed of amino acids from position 16 to position 42. 16-42 ), composed of amino acids from position 16 to 40 (β-amyloid protein 16-40 (Aβ) 16-40), composed of amino acids from position 17 to 42, β-amyloid protein (β-amyloid 17-42 (Aβ)). 17-42 ), composed of amino acids from position 17 to 40, β-amyloid protein (β-amyloid 17-40 (Aβ)). 17-40 β-amyloid protein (β-amyloid protein 20-42 (Aβ)) is composed of amino acids from position 20 to 42. 20-42 β-amyloid protein (β-amyloid 20-40 (Aβ)) is composed of amino acids from position 20 to 40. 20-40 β-amyloid protein (β-amyloid protein 21-42 (Aβ)) is composed of amino acids from position 21 to 42. 21-42 β-amyloid protein (β-amyloid protein 21-40 (Aβ)) is composed of amino acids from position 21 to 40. 21-40 ), composed of amino acids from position 35 to 42, is β-amyloid protein (called β-amyloid 35-42 (Aβ)). 35-42 )) and β-amyloid protein composed of amino acids from position 35 to 40 ("β-amyloid 35-40 (Aβ)"). 35-40 ) ) etc. Aβ 1-42 Aβ 5-42 Aβ 11-42 Aβ 16-42 Aβ 17-42 Aβ 20-42 Aβ 21-42 Aβ 35-42 Collectively referred to as β-amyloid X-42. Additionally, Aβ... 1-40 Aβ 5-40 Aβ 11-40 Aβ 16-40 Aβ 17-40 Aβ 20-40 Aβ 21-40 Aβ 35-40 Collectively referred to as β-amyloid X-40. This invention is useful in the determination of any β-amyloid protein, and the determination of any of these is included within the scope of this invention, with particular preference for the determination of β-amyloid 1-42 and β-amyloid 1-40.

[0043] Anti-β-amyloid antibodies or their antigen-binding fragments can be any antibody or antigen-binding fragment that specifically binds to β-amyloid protein. The antibody can be monoclonal or polyclonal. Monoclonal antibodies are generally preferred. Methods for preparing polyclonal and monoclonal antibodies are known, therefore β-amyloid antigen can be used as an immunogen to prepare anti-β-amyloid antibodies. The "antigen-binding fragment" can be any antibody fragment as long as it maintains the binding of the original antibody to the corresponding antigen (antigen-antibody reactivity). Specific examples include Fab, F(ab')2, and scFv, but these are not limited to. It is well known that Fab and F(ab')2 can be obtained by treating antibodies with proteases such as papain or pepsin. Methods for preparing scFv (single chain fragment of variable region) are also known and can be prepared according to known methods.

[0044] Anti-β-amyloid antibodies or their antigen-binding fragments can be appropriately selected based on the type of β-amyloid protein being measured. Such antibodies and antibody combinations are well-known and commercially available substances can be used.

[0045] For example, when determining β-amyloid 1-42 using a sandwich assay, an antibody that specifically binds to the C-terminal region of β-amyloid x-42 (anti-Aβ42 antibody) and an antibody that specifically binds to the N-terminal region containing the first amino acid of β-amyloid (anti-AβN-terminal antibody) can be used. As mentioned above, such antibodies can be commercially available or manufactured using known methods. Anti-Aβ42 antibody can be used as the solid-phase antibody, and anti-AβN-terminal antibody as the labeling antibody; conversely, anti-AβN-terminal antibody can be used as the solid-phase antibody, and anti-Aβ42 antibody as the labeling antibody.

[0046] Additionally, when determining β-amyloid 1-40 using the sandwich assay, antibodies that specifically bind to the C-terminal region of β-amyloid x-40 (anti-Aβ40 antibody) and antibodies that specifically bind to the N-terminal region containing the first amino acid of β-amyloid (anti-AβN-terminal antibody) can be used. As mentioned above, these antibodies can be commercially available or manufactured using known methods. Anti-Aβ40 antibody can be used as the solid-phase antibody, and anti-AβN-terminal antibody as the labeling antibody; conversely, anti-AβN-terminal antibody can be used as the solid-phase antibody, and anti-Aβ40 antibody as the labeling antibody.

[0047] Anionic polymers can be added separately to the antigen-antibody reaction system, or pre-added to reagents such as solutions containing antibody-immobilized solid phases (e.g., antibody-immobilized particles) or sample diluents for diluting blood samples. Alternatively, they can be pre-added to the blood sample.

[0048] The final concentration of the anionic polymer in the antigen-antibody reaction system is usually around 0.01 g / L to 50 g / L, around 0.02 g / L to 25 g / L, around 0.03 g / L to 10 g / L, around 0.04 g / L to 5 g / L, and preferably around 0.05 g / L to 2.5 g / L.

[0049] Immunoassay methods are known, and as described above, the present invention can be used with any immunoassay method. That is, the immunoassay method of the present invention can directly use known immunoassay methods, except for antigen-antibody reactions in the presence of anionic polymers. Furthermore, as described above, immunoassays using CSF as samples have been implemented, and therefore the kits used in those immunoassays can also be used directly.

[0050] It should be noted that blood, like CSF, also contains Aβ. 1-42 and Aβ 1-40 These two are known to be found in either CSF or blood by obtaining their ratio, i.e., Aβ. 1-42 / Aβ 1-40 This can improve diagnostic specificity, therefore, Aβ is being used. 1-42 and Aβ 1-40 This method quantifies these two factors and calculates their ratio. A ratio lower than that of healthy individuals suggests a high probability of Alzheimer's disease. Typically, the sensitivity of the immunoassay decreases with a longer time elapsed since blood sample collection; however, as specifically described in the examples below, an unexpected effect was observed: by performing the antigen-antibody reaction in the presence of anionic polymers, the sensitivity hardly decreased even after a long period following sample collection (i.e., Aβ...). 1-42 / Aβ 1-40 (It hardly changes over time).

[0051] The kit of the present invention is any kit for performing the immunoassay method of the present invention described above, and the kit contains at least an anti-β-amyloid antibody and an anionic polymer. In the case of a sandwich immunoassay, it also contains a solid phase in which the anti-β-amyloid antibody is immobilized. When the solid phase is composed of particles such as magnetic particles, it also contains these particles (usually in the form of a particle liquid). The kit of the present invention can be prepared by including an anionic polymer in a known β-amyloid immunoassay kit. As described above, the anionic polymer can be included in reagents such as anti-β-amyloid antibody immobilized particle liquid or sample diluent, or it can be included separately in the kit.

[0052] Example

[0053] The present invention will be described in detail below based on embodiments. Of course, the present invention is not limited to the following embodiments.

[0054] <Preparation of solidified particles>

[0055] In 10 mM MES buffer (pH 5.0), 0.2 mg / mL of mouse anti-Aβ42 antibody, which specifically binds to the C-terminal region of β-amyloid protein x-42, was added to magnetic particles at a concentration of 0.01 g / mL. The mixture was incubated at 25 °C with slow stirring for 1 hour. After the reaction, the magnetic particles were collected using a magnet and washed with washing buffer (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-Aβ42 antibody-immobilized particles. The obtained anti-Aβ42 antibody-immobilized particles were then suspended in particle dilution buffer (50 mM Tris buffer, 1 mM EDTA-2Na, 0.1% NaN3, 2.0% BSA, pH 7.2) to obtain an anti-Aβ42 antibody-immobilized particle solution.

[0056] Similarly, using mouse anti-Aβ40 antibodies that specifically bind to the C-terminal region of β-amyloid x-40, solidified anti-Aβ40 antibody particles and solutions were prepared.

[0057] <Preparation of alkaline phosphatase-labeled antibodies>

[0058] Desalted alkaline phosphatase (ALP) and N-(4-maleimidebutyryloxy)-succinimide (GMBS) (final concentration 0.3 mg / mL) were mixed and incubated at 30°C for 1 hour to induce maleimide. Next, in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA-2Na, pH 6.3), Fab'-modified mouse anti-Aβ N-terminal antibody specifically binding to the N-terminal region containing the first amino acid of β-amyloid protein and maleimide-labeled ALP were mixed at a 1:1 molar ratio and reacted at 25°C for 1 hour. Purification was performed using a Superdex 200 10 / 300 (GE) column chromatography to obtain the alkaline phosphatase-labeled antibody (ALP-labeled antibody). The ALP-labeled antibody was suspended in a label dilution buffer (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 the ALP-labeled antibody solution.

[0059] <β-amyloid protein 1-42 (Aβ) 1-42 The determination method of ) >

[0060] 50 μL of sample and 50 μL of anti-Aβ42 antibody solid-phase particle solution were dispensed into the reaction vessel and stirred. The mixture was then incubated at 37°C for 8 minutes, followed by B / F separation using a magnetic field and washing. Next, 50 μL of ALP-labeled antibody solution was dispensed into the reaction vessel, stirred, and incubated at 37°C for 8 minutes, followed by B / F separation using a magnetic field and washing. Then, 200 μL of Lumipulse (registered trademark) substrate solution (manufactured by FUJIREBIO Inc.) containing 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxane disodium salt (AMPPD) as the chemiluminescent substrate was dispensed into the reaction vessel, stirred, and incubated at 37°C for 4 minutes. The luminescence intensity was then measured using a photometer. In actual measurements, a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (manufactured by FUJIREBIO Inc.)) was used.

[0061] <β-amyloid protein 1-40 (Aβ) 1-40 The determination method of ) >

[0062] 30 μL of sample and 50 μL of anti-Aβ40 antibody immobilized particle solution were dispensed into the sample tank and stirred. The mixture was then incubated at 37°C for 8 minutes, followed by B / F separation using a magnetic field and washing. Next, 50 μL of enzyme-labeled antibody solution was dispensed into the reaction tank, stirred, and incubated at 37°C for 8 minutes, followed by separation using a magnetic field and washing. Then, 200 μL of Lumipulse substrate solution containing AMPPD (manufactured by FUJIREBIO Inc.) was dispensed into the reaction tank, stirred, and incubated at 37°C for 4 minutes. The luminescence intensity was measured using a photometer. In actual measurements, a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (manufactured by FUJIREBIO Inc.)) was used.

[0063] Example 1

[0064] The effects of sodium dextran sulfate on Aβ in blood samples were investigated. 1-42 and Aβ 1-40 The effect of addition during the test.

[0065] As a sample, to Aβ 1-42 and Aβ 1-40 For blood samples (serum and EDTA-2K plasma) below the detection limit, Aβ was added to 4.5 mL of each sample at concentrations of approximately 794 pg / mL and approximately 7744 pg / mL, respectively. 1-42 Antigen and Aβ 1-40 500 μL of cerebrospinal fluid (CSF) containing the antigen was used to prepare antigen-containing serum (Serum1, Serum2) and antigen-containing plasma (EDTA2K). Additionally, Aβ at the same concentration as the blood sample was added to Tris-buffered saline (TBS buffer, pH 7.4). 1-42 Antigen and Aβ 1-40 Antigen, thereby preparing antigen-containing TBS buffer. Using the prepared sample, follow the above-described "Aβ" procedure. 1-42 "Determination method of Aβ" and "Aβ" 1-40 The method described in the "Determination Method of Aβ" is used to determine Aβ. 1-42 and Aβ 1-40 As sodium dextran sulfate, sodium dextran sulfate (weight-average molecular weight 5000 (hereinafter referred to as "molecular weight 5000", and the same applies to others)) was added to the antibody immobilization particle solution to a concentration of 0.5 g / L, and its effect was studied. Regarding the final concentration of sodium dextran sulfate in the antigen-antibody reaction system, Aβ... 1-42 0.25 g / L, Aβ 1-40The concentration was 0.31 g / L. Additionally, the same measurements were performed on each sample (serum, EDTA-2K plasma, and TBS buffer) as blanks, and the resulting counts were used as blank values. The blank values ​​were subtracted from the counts of each antigen-containing sample to obtain the final count. Aβ... 1-42 The results are shown in Table 1, with Aβ 1-40 The results are shown in Table 2.

[0066] [Table 1]

[0067]

[0068] [Table 2]

[0069]

[0070] As shown in Table 1, the Aβ values ​​of serum and plasma samples were as follows, without the addition of sodium dextran sulfate. 1-42 The count value is lower than the count value of the buffer sample. If the count value of the buffer sample is set to 100%, the count value of plasma 2, which is a serum sample, drops significantly to 56.9%.

[0071] On the other hand, sodium dextran sulfate was added to the antibody-immobilized particle solution and Aβ was measured. 1-42 At that time, the count values ​​of both serum and plasma samples increased, and the count values ​​recovered to more than 80% of the count values ​​of the buffer sample.

[0072] Additionally, as shown in Table 2, for Aβ 1-40 In contrast, without the addition of sodium dextran sulfate, the Aβ levels in plasma samples were... 1-40 The count values ​​were also lower than those of the buffer sample. Setting the count value of the buffer sample to 100%, the EDTA2K in the plasma sample decreased significantly to 36.4%. On the other hand, Aβ was determined by adding dextran sulfate sodium to the antibody-immobilized particle solution. 1-40 At that time, the count value of the plasma sample increased, and the count value recovered to more than 90% of the count value of the buffer sample.

[0073] Example 2

[0074] The study investigated Aβ in the blood 1-42 The concentration of sodium dextran sulfate added was determined in the assay. Using the method described in Example 1, the concentrations of Aβ in antigen-containing serum (plasma 1, plasma 2), antigen-containing plasma (EDTA2K), and antigen-containing TBS buffer were determined. 1-42Sodium dextran sulfate (molecular weight 5000) was added to the antibody immobilization particle solution to concentrations shown in Table 3 (0.1 g / L to 5.0 g / L), and its effect was investigated. The final concentration of sodium dextran sulfate in the antigen-antibody reaction system was 0.05 g / L to 2.5 g / L. Blank values ​​were obtained as in Example 1, and count values ​​were calculated. The results are shown in Table 3.

[0075] [Table 3]

[0076]

[0077] As shown in Table 3, under all the concentration conditions of sodium dextran sulfate studied, the count values ​​increased compared to the count values ​​under the unadded condition.

[0078] Examples 3-5, Comparative Examples 1-3

[0079] The effects of adding various anionic polymers on the determination of Aβ in blood samples were investigated. Aβ was determined using serum (antigen-containing serum and plasma 2) with significantly decreased counts in the absence of sodium dextran sulfate as described in Examples 1 and 2, and using antigen-containing TBS buffer, according to the method described in Example 1. 1-42 As anionic polymers, sodium dextran sulfate (molecular weight 5000) (Example 3), sodium poly(4-styrene sulfonate) (molecular weight 700000) (Example 4), sodium dodecyl sulfate (SDS) (Comparative Example 1), sodium dodecylbenzene sulfonate (SDBS) (Comparative Example 2), sodium polyacrylate (molecular weight 5100) (Example 5), and sodium N-lauroyl sarcosinate (NLS) (Comparative Example 3) were used. These anionic polymers were added to the antibody-immobilized particle solution at a concentration of 0.5 g / L, and their addition effect was studied. In addition, blank values ​​were obtained as in Example 1, and count values ​​were calculated. The results are shown in Table 4.

[0080] [Table 4]

[0081]

[0082] As shown in Table 4, without the addition of anionic polymers, the count value of serum sample (plasma 2) was 56.9% compared to the count value of buffer sample. In contrast, when sodium dextran sulfate (Example 3), sodium poly(4-styrene sulfonate) (Example 4), and sodium polyacrylate (Example 5) were added as anionic polymers, the count values ​​increased to 82.6%, 77.4%, and 66.9%, respectively. In particular, the recovery of count values ​​was greater when sodium dextran sulfate and sodium poly(4-styrene sulfonate), which are anionic polymers having sulfate or sulfonic acid groups in their side chains, were added, indicating that they are particularly effective. On the other hand, even when SDS (Comparative Example 1), a surfactant having sulfate groups in its side chain, SDBS (Comparative Example 2), a surfactant having sulfonic acid groups in its side chain, or NLS (Comparative Example 3), a surfactant having carboxyl groups in its side chain, were added, the count values ​​did not recover.

[0083] This indicates that adding anionic polymers to the reaction solution improves the decrease in β-amyloid protein counts in blood samples. Furthermore, the improvement effect is particularly pronounced among anionic polymers, especially those with sulfate or sulfonic acid groups in their side chains.

[0084] Example 6

[0085] The effect of the concentration of anionic polymer added on the determination of Aβ in blood samples was investigated. 1-42 Add synthetic Aβ at a concentration of 50,000 pg / mL to 5 mL of blood samples (serum and EDTA-2K plasma) below the detection limit. 1-42 50 μL of antigen was used to prepare antigen-containing serum (plasma 1, plasma 2) and antigen-containing plasma (EDTA2K). Additionally, blood samples and the same concentration of synthetic Aβ were added to Tris-buffered saline (TBS buffer, pH 7.4). 1-42 Antigen was used to prepare an antigen-containing TBS buffer. Aβ was then determined using the prepared samples according to the method described in Example 1. 1-42 Sodium heparin sulfate, an anionic polymer having sulfate groups in its side chain, was added instead of sodium dextran sulfate to the antibody-immobilized particle solution at concentrations (0.5 g / L and 5.0 g / L) as shown in Table 5, and its effect was investigated. The final concentrations of sodium heparin sulfate in the antigen-antibody reaction system were 0.25 g / L and 2.5 g / L. Blank values ​​were obtained and count values ​​were calculated, as in Example 1. The results are shown in Table 5.

[0086] [Table 5]

[0087]

[0088] As shown in Table 5, under all the concentrations of sodium heparin sulfate added in the study, the count values ​​increased compared to the values ​​under the unadded conditions.

[0089] Example 7

[0090] The effects of adding sodium dextran sulfate with different molecular weights on the determination of Aβ in blood samples were investigated. In Examples 1 and 2, 5 mL of serum (plasma 2) with significantly decreased count values ​​under conditions without the addition of sodium dextran sulfate were treated with synthetic Aβ at a concentration of 50,000 pg / mL. 1-42 50 μL of antigen was used to prepare antigen-containing serum, and Aβ was determined using the method described in Example 1. 1-42 Additionally, synthetic Aβ at the same concentration as the blood sample was added to the TBS buffer. 1-42 Antigen, and thus prepare antigen-containing TBS buffer, and similarly measure Aβ. 1-42 Two types of dextran sulfate with average molecular weights of 5000 and 50000 were used, and the concentration added to the antibody-immobilized particle solution was set to 7 g / L. The final concentration of sodium dextran sulfate in the antigen-antibody reaction system was 3.5 g / L. Blank values ​​were then obtained and count values ​​were calculated in the same manner as in Example 1. The results are shown in Table 6.

[0091] [Table 6]

[0092]

[0093] As shown in Table 6, without the addition of sodium dextran sulfate, the count value of serum sample (plasma 2) was 66.7% compared with the count value of buffer sample. In contrast, when sodium dextran sulfate (molecular weight 5000) and sodium dextran sulfate (molecular weight 50000) were added, the count values ​​increased to 80.9% and 93.8%, respectively.

[0094] This indicates that adding anionic polymers, such as sodium dextran sulfate with an average molecular weight of over 5000, to the reaction solution can improve the decrease in the count value of β-amyloid protein in blood samples.

[0095] Example 8

[0096] To investigate the reason for the decrease in count values ​​in blood samples compared to buffered samples, EDTA-2K plasma and cerebrospinal fluid (CSF) were fractionated using a gel filtration column. The conditions were as follows: AKTA FPLC (manufactured by GE HealthCARE), Superose 6 10×300 column (manufactured by GE HealthCARE), flow rate 0.3 mL / min, D-PBS (-) buffer, 1 mL sample volume, and 1 mL volume for each fraction. For each fraction, Aβ was added at 1000 pg / mL. 1-42 Antigen, thereby preparing a sample. Using the prepared sample, Aβ was determined according to the method described in Example 1. 1-42 The effect of adding sodium dextran sulfate (molecular weight 5000) to the antibody-immobilized particle solution at a concentration of 2.5 g / L was investigated. The final concentration of sodium dextran sulfate in the antigen-antibody reaction system was 1.3 g / L. As a control sample, a solution containing 1000 pg / mL of Aβ was used. 1-42 The antigen was also measured in the same way using TBS buffer. The results are shown in Table 7 as the percentage (%) of the count values ​​for each sample relative to the control sample. For molecular weight, an approximate formula was prepared by operating on the molecular weight marker (Bio-Rad) under the above conditions to determine the molecular weight in each fraction.

[0097] [Table 7]

[0098]

[0099] As shown in Table 7, in the case of EDTA2K plasma, the fraction around 300-400 kDa showed a decrease in count value to 61% without the addition of sodium dextran sulfate, while the count value recovered to the same level as the control sample (106%) upon the addition of sodium dextran sulfate. On the other hand, in the case of CSF, the count value in any fraction did not decrease compared to the control sample without the addition of sodium dextran sulfate, and no fractions were observed to show a significant difference in the count value ratio due to the presence or absence of sodium dextran sulfate.

[0100] Therefore, it is believed that the component that causes the count to decrease exists only in blood samples, and the effect of this component can be avoided by adding anionic polymers.

[0101] Example 9

[0102] The presence of Aβ in anionic polymers in serum samples and EDTA-Na plasma samples from healthy individuals was investigated. 1-42 and Aβ 1-40的Effects during the assay. Blood samples were prepared as follows: Blood was collected using vacuum blood collection tubes (10 mL, Terumo-made) and EDTA-2Na (7 mL, Terumo-made), respectively. The samples were allowed to stand at room temperature for 30 minutes, then centrifuged at 1200×g for 10 minutes. The supernatant was collected to obtain the blood samples.

[0103] To evaluate the impact of sample preservation, samples prepared and stored at 4°C for 0, 1, 2, and 3 days were used, following the aforementioned "Aβ" method. 1-42 "Determination method of Aβ" and "Aβ" 1-40 The determination method for Aβ 1-42 and Aβ 1-40 Dextran sulfate sodium (molecular weight 5000), an anionic polymer, was added at 0.5 g / L to the antibody-immobilized particle solution to investigate its effect. The measured values ​​and the ratios of measured values ​​under each condition to the storage period of 0 days are shown in Table 8. Regarding the calculation method for the measured values, the synthesized Aβ... 1-42 Peptides and the synthesis of Aβ 1-40 The peptides were adjusted to concentrations of 0 pg / mL, 10 pg / mL, 100 pg / mL, and 1000 pg / mL, respectively, to serve as calibrators. An approximate formula was constructed based on the count values ​​obtained from these calibrators, and the count values ​​from each sample were embedded into the approximate curve. The measured values ​​were then calculated.

[0104] [Table 8-1]

[0105]

[0106] [Table 8-2]

[0107]

[0108] As shown in Table 8, the count and measured values ​​of serum and plasma samples increased when sodium dextran sulfate was added. Over time, a tendency for the measured values ​​to decrease was observed.

[0109] In the determination of β-amyloid protein, Aβ was obtained. 1-42 With Aβ 1-40 The ratios were compared. Therefore, in this embodiment, Aβ was calculated under each measured condition. 1-42 Relative to Aβ 1-40 The ratio is shown in Table 9.

[0110] [Table 9]

[0111]

[0112] As shown in Table 9, when serum was measured using reagents without added dextran sulfate, Aβ was confirmed to be... 1-42 / Aβ 1-40 The ratio changes over time. However, when serum is measured using a reagent containing sodium dextran sulfate, Aβ is confirmed. 1-42 / Aβ 1-40 The value of the ratio does not change over time, and the resulting Aβ 1-42 / Aβ 1-40 It is not affected by the shelf life.

[0113] This confirms that the addition of anionic polymers such as sodium dextran sulfate not only affects Aβ... 1-42 and Aβ 1-40 The count value increased, and the resulting Aβ 1-42 / Aβ 1-40 It is not affected by the shelf life.

[0114] Example 10

[0115] Aβ levels in serum samples from Alzheimer's disease (AD) patients and non-AD patients were determined using reagents supplemented with sodium dextran sulfate. 1-42 and Aβ 1-40 .

[0116] Aβ levels in serum samples from AD patients (26 cases) and non-AD patients (22 cases) were measured using the following method. 1-42 and Aβ 1-40 First, 50 μL of sample and 250 μL of antibody-stabilized particle solution with 0.5 g / L sodium dextran sulfate (molecular weight 5000) were aliquoted into the reaction vessel and stirred. The final concentration of sodium dextran sulfate in the antigen-antibody reaction system was 0.42 g / L. Then, the mixture was incubated at 37°C for 8 minutes, separated using a magnetic field, and washed. Next, 250 μL of enzyme-labeled antibody solution was aliquoted into the reaction vessel, stirred, and incubated at 37°C for 8 minutes, separated using a magnetic field, and washed. Then, 200 μL of Lumipulse (registered trademark) substrate solution (manufactured by FUJIREBIO Inc.) containing AMPPD as the chemiluminescent substrate was aliquoted into the reaction vessel, stirred, and incubated at 37°C for 4 minutes. The luminescence intensity was then measured using a photometer. In the actual assay, a fully automated chemiluminescent enzyme immunoassay system (Lumipulse G1200 (manufactured by FUJIREBIO Inc.)) was used. The results are shown below. Figure 1 An approximation formula was prepared 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.

[0117] Figure 1 Aβ is calculated from the obtained measurement values. 1-42 / Aβ 1-40Box plot of the ratio. For results obtained using reagents containing sodium dextran sulfate, Aβ in AD patients... 1-42 / Aβ 1-40 Comparison of Aβ between the non-AD patient group and the non-AD patient group 1-42 / Aβ 1-40 The significantly lower p-value (p=0.0022 (Wilcoxon-Mann-Whitney test) confirms the usefulness of adding anionic polymers to the reaction solution in Aβ assays using patient samples.

[0118] This indicates that reagents containing anionic polymers such as sodium dextran sulfate are useful for the diagnosis of Alzheimer's disease.

Claims

1. A kit for immunoassay of β-amyloid protein in a blood sample, the kit comprising a solid phase immobilized with an anti-β-amyloid antibody or its antigen-binding fragment, an anionic polymer, and an ALP-labeled antibody specifically binding to the N-terminal region of β-amyloid protein. The anionic polymer is an anionic polymer having sulfate or sulfonic acid groups on its side chains.

2. The kit according to claim 1, wherein, The solid phase is particles, and the kit comprises a particle liquid containing the particles and the anionic polymer.

3. The kit according to claim 1 or 2, wherein, The immunoassay method for β-amyloid protein is a sandwich method, which includes: In the presence of the anionic polymer, the β-amyloid protein in the blood sample is contacted with an anti-β-amyloid antibody, i.e., a solid-phase antibody or its antigen-binding fragment, which is immobilized on a solid phase. The solid-phase antibody or its antigen-binding fragment reacts with the antigen-antibody of the β-amyloid protein, followed by B / F separation; and An antigen-antibody reaction is performed between the β-amyloid protein and the ALP-labeled antibody that specifically binds to the N-terminal region of the β-amyloid protein. Then, B / F separation is performed and the mixture is washed. The signal of the labeling source of the ALP-labeled antibody that specifically binds to the N-terminal region of the β-amyloid protein bound to the β-amyloid protein on the solid phase is detected, thereby determining the β-amyloid protein.

4. The kit according to claim 3, wherein, The sandwich method is a two-step sandwich method.

5. The kit according to claim 1 or 2, wherein, The anionic polymer is selected from at least one of dextran sulfate and its salts, polystyrene sulfonic acid and its salts, and heparin sulfate and its salts.

6. The kit according to claim 5, wherein, The anionic polymer is sodium dextran sulfate.

7. The kit according to claim 1 or 2, wherein, The β-amyloid protein is either β-amyloid protein 1-40 or β-amyloid protein 1-42.

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