Antibody conjugate

Abstract

To provide a technique to increase sensitivity in immunological measurement of tau protein.SOLUTION: In immunological measurement, sensitivity in detecting tau protein is increased by using an antibody conjugate obtained by coupling two or more types of anti-tau protein antibodies to a carrier, compared with a case where an individual anti-tau protein antibody is used.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibody conjugates and the like. [Background technology]

[0002] Alzheimer's disease (AD) is one of the most common dementias and is a neurodegenerative disease characterized histologically by the accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques throughout the cerebral cortex and limbic system. The ultrastructural morphology of neurofibrillary tangles is composed of paired helical filaments (PHFs) that are mainly composed of abnormally phosphorylated tau protein (pTau). The usefulness of tau protein (including phosphorylated tau protein) as a diagnostic marker for neurodegenerative diseases such as Alzheimer's disease, and an immunological method for measuring tau protein in body fluids have been reported (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 8-502898 [Patent Document 2] Special Publication No. 9-506771 Summary of the Invention [Problem to be solved by the invention]

[0004] In the immunological measurement of tau protein in samples such as body fluids, there is a demand for further improvement in sensitivity compared to conventional methods. [Means for solving the problem]

[0005] As a result of intensive research, the inventors have found that in immunological measurements, the use of an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier improves the detection sensitivity of tau protein compared to the use of individual anti-tau protein antibodies, and have thus completed the present invention.

[0006] That is, the present invention is as follows. [1] An antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier. [2] The antibody conjugate of [1], wherein the two or more types of anti-tau protein antibodies are two or more types of anti-human tau protein antibodies. [3] The antibody conjugate of [2], wherein the two or more types of anti-tau protein antibodies include a first antibody that recognizes an epitope located between positions 153 and 169 in the amino acid sequence of SEQ ID NO: 1, and a second antibody that recognizes an epitope located between positions 188 and 207 in the amino acid sequence of SEQ ID NO: 1. [4] The antibody conjugate of [3], wherein the epitope recognized by the first antibody is PPGQK (sequence number 2) and the epitope recognized by the second antibody is DRSGYS (sequence number 3). [5] The antibody conjugate according to any one of [1] to [4], wherein the carrier is a labeled carrier. [6] A kit comprising: (i) an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier; and (ii) An anti-tau protein antibody that recognizes an epitope different from that of the anti-tau protein antibody contained in the antibody conjugate. [7] The kit according to [6], wherein the another epitope is an epitope present between positions 170 and 187 or between positions 209 and 233 in the amino acid sequence of SEQ ID NO:1. [8] The kit according to [7], wherein the other epitope is PPAPKTP (sequence number 4) or PPTREPK (sequence number 5). [9] A method for detecting tau protein, comprising detecting tau protein in a sample using an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier.

[10] The method according to [9], wherein the sample is a body fluid sample taken from a human.

[11] The method of [9] or

[10] , further comprising contacting the sample with an anti-tau protein antibody that recognizes an epitope different from that of the anti-tau protein antibody contained in the antibody conjugate. Effect of the Invention

[0007] Use of the antibody conjugate of the present invention improves the detection sensitivity of tau protein in immunological measurements such as sandwich immunoassays, as compared with the use of individual anti-tau protein antibodies. Detection of tau protein using the antibody conjugate of the present invention is useful for highly accurate diagnosis of neurodegenerative diseases such as Alzheimer's disease. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows a graph plotting the concentration of each sample (LoQ1 to LoQ5) calculated based on a standard curve created from the average count value and concentration of each standard solution in the measurement of total tau protein using a hybrid conjugate, the coefficient of variation (CV) of the count value when each sample (LoQ1 to LoQ5) was measured 10 times (n=10), and the limit of quantification (LoQ (CV20%)) calculated based on the precision profile. [Diagram 2] Figure 2 shows a graph plotting the concentration of each sample (LoQ1 to LoQ5) calculated based on a standard curve created from the average count value and concentration of each standard solution in the measurement of total tau protein using an antibody mixture, the coefficient of variation (CV) of the count value when each sample (LoQ1 to LoQ5) was measured 10 times (n = 10), and the limit of quantification (LoQ (CV 20%)) calculated based on the precision profile. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention provides antibody conjugates (also referred to as "hybrid conjugates") in which two or more types of anti-tau protein antibodies are bound to a carrier.

[0010] The tau protein is preferably human tau protein. Human tau protein includes multiple isoforms, and it is known that 4 to 6 isoforms are detected in the adult brain, and only one isoform is detected in the fetal brain. This diversity of isoforms is generated by selective mRNA splicing from one gene present in human chromosome 17. These isoforms differ from each other in the presence or absence of insertion of three or four repeat domains in the C-terminal portion and a region consisting of 29 or 58 amino acid residues in the N-terminal portion. In this specification, the amino acid numbers in tau protein are represented as the amino acid numbers corresponding to the longest isoform (441 amino acids) shown in SEQ ID NO: 1. Tau proteins include modified tau proteins. Examples of modified tau proteins include phosphorylated tau proteins. Phosphorylated tau proteins refer to tau proteins in which hydroxyl-containing amino acid residues (serine residues, threonine residues, tyrosine residues) are phosphorylated.

[0011] The anti-tau protein antibody is an antibody that recognizes at least a part of the amino acid sequence of tau protein as an epitope. The two or more types of anti-tau protein antibodies may be, for example, 2 to 5 types, preferably 2 to 4 types, more preferably 2 to 3 types, and particularly preferably 2 types of anti-tau protein antibodies. It is preferable that the two or more types of anti-tau protein antibodies each recognize a non-overlapping epitope in tau protein.

[0012] The two or more anti-tau protein antibodies include a first antibody and a second antibody. The epitope recognized by the first antibody may be an epitope present preferably between positions 153 and 169, more preferably between positions 155 and 167, even more preferably between positions 157 and 165 in the amino acid sequence of SEQ ID NO: 1, and particularly preferably PPGQK (between positions 159 and 163, SEQ ID NO: 2). The epitope recognized by the second antibody may be an epitope present preferably between positions 188 and 207, more preferably between positions 190 and 204, even more preferably between positions 192 and 201 in the amino acid sequence of SEQ ID NO: 1, and particularly preferably DRSGYS (between positions 193 and 198, SEQ ID NO: 3).

[0013] The number of amino acid residues between the epitope recognized by the first antibody and the epitope recognized by the second antibody in the amino acid sequence of SEQ ID NO: 1 may be, for example, 18 amino acids or more, preferably 20 amino acids or more, more preferably 22 amino acids or more, even more preferably 24 amino acids or more, and particularly preferably 26 amino acids or more. The number of amino acid residues between the epitopes may be, for example, 44 amino acids or less, preferably 41 amino acids or less, more preferably 38 amino acids or less, even more preferably 35 amino acids or less, and particularly preferably 33 amino acids or less. More specifically, the number of amino acid residues between the epitopes may be, for example, 18 to 44 amino acids, preferably 20 to 41 amino acids, more preferably 22 to 38 amino acids, even more preferably 24 to 35 amino acids, and particularly preferably 26 to 33 amino acids.

[0014] The epitopes recognized by two or more anti-tau protein antibodies may be either unmodified peptides or modified peptides. Modified peptides include, for example, phosphorylated peptides. Phosphorylated tau protein refers to peptides in which hydroxyl-containing amino acid residues (serine residues, threonine residues, tyrosine residues) are phosphorylated.

[0015] The anti-tau protein antibody may be either a polyclonal antibody or a monoclonal antibody. The anti-tau protein antibody may be any isotype of immunoglobulin (e.g., IgG, IgM, IgA, IgD, IgE, IgY). The anti-tau protein antibody may also be a full-length antibody. A full-length antibody refers to an antibody comprising a heavy chain and a light chain, each of which comprises a variable region and a constant region (e.g., an antibody comprising two Fab portions and an Fc portion). The anti-tau protein antibody may also be an antibody fragment derived from such a full-length antibody. An antibody fragment is a portion of a full-length antibody, for example, a constant region-deleted antibody (e.g., F(ab') 2 The anti-tau protein antibody may also be a modified antibody such as a single chain antibody.

[0016] The anti-tau protein antibody can be prepared by using a conventional method. For example, the anti-tau protein antibody can be prepared by using the above-mentioned epitopes as antigens. In addition, since many anti-tau protein antibodies that recognize the above-mentioned epitopes are commercially available, such commercially available products can also be used.

[0017] The carrier may be a linker compound or a solid phase. A linker compound refers to a compound capable of binding to multiple antibodies (e.g., full-length antibodies, antibody fragments). The linker compound may be a linker compound capable of binding to a label, a linker compound bound to a label, or a linker compound that is itself a label. Examples of linker compounds include maleimide, haloacetyl, isothiocyanate, sulfonyl chloride, N-hydroxysuccinimide, azide, polysaccharides (e.g., dextran), peptides, polypeptides, proteins (e.g., bovine serum albumin (BSA)), nucleic acids (DNA, RNA), and polyethylene glycol. Examples of solid phases include solid phases that can be suspended or dispersed in a liquid phase (e.g., solid phase carriers such as particles and beads), and solid phases that can accommodate or carry a liquid phase (e.g., supports such as plates, membranes, and test tubes, and containers such as well plates, microchannels, glass capillaries, nanopillars, and monolith columns). The solid phase for the carrier is preferably a solid phase that can be suspended or dispersed in a liquid phase. Examples of the solid phase material include glass, silica, polymeric compounds (e.g., polystyrene, plastic), metals, and carbon. The solid phase material can also be a non-magnetic material or a magnetic material.

[0018] The antibody can be bound to the linker compound by a method known in the art. Such methods include, for example, physical adsorption, covalent bonding, affinity substances (e.g., biotin, streptavidin), and ionic bonding. When using covalent bonding, the antibody can be covalently bound to the linker compound by, for example, the periodic acid method, glutaraldehyde method, maleimide method, or N-hydroxysuccinimide method. The antibody can be bound to the solid phase by the same method as the antibody is bound to the linker compound.

[0019] The antibody conjugate of the present invention may contain a label. When the antibody conjugate of the present invention contains a label, the carrier may be a labeled carrier, or the antibody may be bound to a label, or both of these may be the case. A labeled carrier refers to a carrier bound to a label, or a carrier that is itself a label. Examples of labels include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of the nucleic acids of the sense strand and the antisense strand that are complementary to each other), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioactive substances (e.g., 3 H, 14 C. 32 P, 35 S, 125 I), and gold colloid. The carrier bound to the label may be, for example, a particle or bead containing a fluorescent substance inside (e.g., fluorescent beads). The material of the particle and bead can be the same as that of the solid phase for the carrier. The carrier itself that is a label can be, for example, an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of the nucleic acids of the sense strand and the antisense strand that are complementary to each other), a fluorescent substance (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), and gold colloid. The antibody conjugate containing the label may be prepared by binding the label to the antibody conjugate, or may be prepared by binding the antibody to the labeled carrier. As the binding method, a method previously known in the art can be used.

[0020] The antibody conjugate of the present invention can be used for detecting tau protein by immunoassay. Examples of such immunoassays include direct competitive assays, indirect competitive assays, sandwich assays, Western blot assays, and immunohistochemical staining assays. Such immunoassays may preferably be sandwich assays. Examples of such immunoassays include chemiluminescent immunoassays (CLIA) (e.g., chemiluminescent enzyme immunoassays (CLEIA)), turbidimetric immunoassays (TIA), enzyme immunoassays (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassays (RIA), latex agglutination assays, fluorescent immunoassays (FIA), and immunochromatography. In the sandwich assays, the antibody conjugate of the present invention may be used as a detection antibody or a capture antibody, and may preferably be used as a detection antibody. When the antibody conjugate of the present invention is used as a detection antibody, a label is bound to the antibody conjugate, and the label bound to the antibody conjugate is detected to detect tau protein. Alternatively, when an antibody conjugate in which the carrier is a labeled carrier is used as a detection antibody, tau protein can be detected by detecting the label in the antibody conjugate bound to tau protein.

[0021] The detection of the label can be carried out based on a method appropriately selected from known methods according to the type of label. When the label is an enzyme, the label can be detected by detecting the enzyme activity using a signal generating substrate (e.g., a fluorescent substrate, a luminescent substrate, a chromogenic substrate). When the label is an affinity substance, the label can be detected by detecting the enzyme or signal generating substance bound to the affinity substance using an enzyme or signal generating substance capable of binding to the affinity substance. Such an enzyme or signal generating substance capable of binding to an affinity substance may be an enzyme or signal generating substance bound to a substance capable of binding to an affinity substance. When the label is a fluorescent substance, a luminescent substance, or a radioactive substance, the label can be detected by detecting the signal generated from these labels.

[0022] The antibody conjugate of the present invention may be provided in the form of a composition (e.g., a solution). Alternatively, the antibody conjugate of the present invention may be provided in the form of a device (e.g., the antibody conjugate is contained in a device).

[0023] The present invention also provides a kit comprising: (i) an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier; and (ii) An anti-tau protein antibody that recognizes an epitope different from that of the anti-tau protein antibody contained in the antibody conjugate.

[0024] The "antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier" is as described above.

[0025] The epitope recognized by the "anti-tau protein antibody that recognizes an epitope different from that of the anti-tau protein antibody contained in the antibody conjugate" (hereinafter referred to as "another anti-tau protein antibody") may be, for example, an epitope that does not overlap with the epitope recognized by the anti-tau protein antibody contained in the antibody conjugate, and may be an epitope present preferably between positions 170 and 187 or between positions 209 and 233, more preferably between positions 172 and 185 or between positions 212 and 230, more preferably between positions 174 and 183 or between positions 215 and 227 in the amino acid sequence of SEQ ID NO: 1, and may be particularly preferably PPAPKTP (between positions 176 and 182, preferably T is a phosphorylated threonine residue, SEQ ID NO: 4) or PPTREPK (between positions 218 and 224, SEQ ID NO: 5). The other epitope may be either an unmodified peptide or a modified peptide. The modified peptide may be, for example, any of those described above. The modified amino acid residue in the other epitope may be, for example, phosphorylated threonine at position 181 in the amino acid sequence of SEQ ID NO: 1. When the other epitope is a modified peptide, the kit of the present invention may be used to detect modified tau protein.

[0026] Another anti-tau protein antibody may be immobilized on a solid phase. The method for immobilizing (binding) the antibody on the solid phase can be the same as the method for binding the antibody to the linker compound described above.

[0027] The kit of the present invention can be used in an immunoassay for tau protein. Such an immunoassay can be, for example, the above-mentioned method, and preferably a sandwich method. In the sandwich method, preferably, an antibody conjugate and another anti-tau protein antibody can be used as a detection antibody and a capture antibody, respectively.

[0028] The kit of the present invention preferably includes (i) an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier (antibody conjugate); and (ii) an anti-tau protein antibody (another anti-tau protein antibody) that recognizes an epitope different from that of the anti-tau protein antibody contained in the antibody conjugate, in a form isolated from each other. Specifically, the antibody conjugate and the other anti-tau protein antibody may be contained in different containers (e.g., tubes, plates). Alternatively, the antibody conjugate and the other anti-tau protein antibody may be provided in the form of a composition (e.g., solution). Alternatively, the kit of the present invention may be provided in the form of a device. Specifically, all of the components, including the antibody conjugate and the other anti-tau protein antibody, may be contained in the device. Alternatively, some of the components may be contained in the device, and the remaining ones may not necessarily be contained in the device (e.g., in a form contained in a different container). In this case, the components not contained in the device may be injected into the device during detection of tau protein.

[0029] In a preferred embodiment, the kit of the present invention may have a configuration according to the type of immunoassay to be adopted. For example, when the sandwich method is adopted, the kit of the present invention may contain, as optional components, a label, a diluent (buffer), a substrate that reacts with the label, and a tau protein preparation. Preferably, the antibody conjugate or another anti-tau protein antibody may be immobilized on magnetic particles. Specific examples of the configuration of the kit of the present invention include an antibody conjugate containing a label carrier, another anti-tau protein antibody immobilized on a solid phase (e.g., magnetic particles, a support, a container), a substrate that reacts with the label, and a tau protein preparation.

[0030] The present invention also provides a method for detecting tau protein. The method of the present invention comprises detecting tau protein in a sample using an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier. The "antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier" is as described above.

[0031] The detection of tau protein in a sample may be performed by immunoassay. Such immunoassays may include, for example, those described above, and may preferably be a sandwich method. In the method of the present invention, the antibody conjugate may be used as a detection antibody or a capture antibody, and may preferably be used as a detection antibody.

[0032] The detection of tau protein may be carried out, for example, by treating a sample with an antibody conjugate to bind the antibody conjugate to tau protein in the sample (e.g., contacting the sample with the antibody conjugate) and detecting the antibody conjugate bound to tau protein. The detection of tau protein may further include a step of removing the antibody conjugate that is not bound to tau protein (e.g., a washing step). The detection of the antibody conjugate bound to tau protein may be carried out by binding a label to the antibody conjugate and detecting the label bound to the antibody conjugate. If the antibody conjugate contains a label, the detection of the antibody conjugate bound to tau protein may be carried out by detecting the label in the antibody conjugate.

[0033] The method of the present invention may further include contacting the sample with an anti-tau protein antibody ("another anti-tau protein antibody") that recognizes an epitope different from that of the anti-tau protein antibody contained in the antibody conjugate. The "another anti-tau protein antibody" is as described above. In this case, detection of tau protein may be performed, for example, by treating the sample with the other anti-tau protein antibody to bind (capture) tau protein in the sample to the other anti-tau protein antibody (e.g., contacting the sample with the other anti-tau protein antibody), treating the tau protein bound to the other anti-tau protein antibody with the antibody conjugate to bind the antibody conjugate to the tau protein bound to the other anti-tau protein antibody (e.g., contacting the tau protein bound to the other anti-tau protein antibody with the antibody conjugate), and detecting the antibody conjugate bound to the tau protein. The detection of tau protein may further include a step of removing free tau protein or another anti-tau protein antibody (B / F separation or washing step), or a step of removing the antibody conjugate not bound to tau protein (e.g., a washing step), or both. Detection of the antibody conjugate bound to tau protein is as described above.

[0034] The sample may be, for example, a liquid sample (e.g., body fluid, preparation), a tissue sample, or a blotting sample. In particular, the sample may be a body fluid sample taken from a human. The body fluid sample may be, for example, a blood sample (e.g., whole blood, plasma, serum), cerebrospinal fluid, urine, and saliva, and may be preferably cerebrospinal fluid, plasma, or serum.

[0035] Examples of detection of tau protein include evaluation of the presence or absence or amount of tau protein, evaluation of the presence or absence or amount of modified tau protein (e.g., phosphorylated tau protein), and evaluation of the degree of modification (e.g., phosphorylation) of tau protein. Based on these evaluations of tau protein, neurodegenerative diseases such as Alzheimer's disease can be diagnosed. Diagnosis of neurodegenerative diseases such as Alzheimer's disease based on the evaluation of tau protein can be performed based on known methods (e.g., JP-A-8-502898, JP-A-9-506771). EXAMPLES

[0036] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0037] Example 1: Preparation of alkaline phosphatase-labeled hybrid conjugates Monoclonal antibody BT2 (epitope: DRSGYS (SEQ ID NO: 3), Fujirebio Europe) that specifically recognizes tau protein was mixed with pepsin in a citrate buffer (pH 3.5) and incubated at 37°C for 1 hour to perform pepsin digestion. After the reaction was stopped, gel filtration purification was performed using a Superdex200 column (GE Healthcare Biosciences) to obtain F(ab') 2 The purified F(ab') fragment was then 2 The concentration of the fragments was determined and adjusted, if necessary, to a concentration of 2.5 mg / mL. The monoclonal antibody HT7 (epitope: PPGQK (SEQ ID NO: 2), Fujirebio Europe) that specifically recognizes tau protein was mixed with bromelain and incubated at 37°C for 1 hour to perform bromelain digestion. After the reaction was stopped, gel filtration purification was performed to obtain F(ab') 2 The purified F(ab') fragment was then 2 The concentration of the fragments was determined and adjusted, if necessary, to a concentration of 2.5 mg / mL. The obtained F(ab') of BT2 2 Fragment and F(ab') of HT7 2 The fragments were mixed at a weight ratio of 1:1, and 2-mercaptoethylamine (2-MEA) hydrochloride was added and incubated at 37°C for 90 minutes to reduce (thiolate) both fragments. After further desalting, a mixture of BT2 Fab' fragment and HT7 Fab' fragment was obtained.

[0038] Meanwhile, desalted alkaline phosphatase (ALP) and N-(4-maleimidobutyryloxy)-succinimide (GMBS) were mixed in a molar ratio of 1:10 and incubated at 30°C for 1 hour to perform maleimidation of ALP. After desalting, a mixture of BT2 Fab' fragment and HT7 Fab' fragment was mixed with maleimidated ALP in a molar ratio of 2:1 and incubated at 25°C for 1 hour to perform coupling. The coupling reaction was stopped by adding 2-MEA hydrochloride and further incubated at 25°C for 30 minutes. The reaction was then quenched by adding 0.5M iodoacetamide and incubating at 25°C for 30 minutes to obtain a conjugate reaction mixture.

[0039] The resulting conjugate reaction mixture was desalted and purified, then loaded onto a HiLoad Superdex200 column (GE Healthcare Biosciences) for gel filtration purification of the antibody. Among the multiple peaks at 280 nm absorbance of the resulting fractions, fractions in which two or more Fab's were bound to one ALP were pooled to obtain the hybrid conjugate. After purification of the hybrid conjugate, it was diluted with 0.1 M MES buffer (1 mM MgCl 2, 0.1 mM ZnCl 2 , 0.1% NaN 3 The antibody was adjusted to a concentration of 0.1 mg / mL in 0.1% BSA, pH 6.8.

[0040] Comparative Example 1: Preparation of alkaline phosphatase-labeled single monoclonal antibody Monoclonal antibody BT2 and pepsin were mixed in a citrate buffer (pH 3.5) and incubated at 37° C. for 1 hour for pepsin digestion. After the reaction was stopped, gel filtration purification was performed using a Superdex 200 column (GE Healthcare Biosciences) to obtain F(ab') 2 The purified F(ab') fragment was then 2 The concentration of the fragments was determined and adjusted, if necessary, to a concentration of 2.5 mg / mL. The obtained F(ab') of BT2 2 The fragment was reduced by incubation with 2-MEA hydrochloride for 90 minutes at 37° C. to obtain the Fab′ fragment of BT2.

[0041] On the other hand, desalted alkaline phosphatase (ALP) and N-(4-maleimidobutyryloxy)-succinimide (GMBS) were mixed in a molar ratio of 1:10 and incubated at 30°C for 1 hour to maleimidate ALP. After desalting, BT2 Fab' fragment and maleimidate ALP were mixed in a molar ratio of 1:1 and incubated at 25°C for 1 hour to perform coupling. The coupling reaction was stopped by adding 2-MEA hydrochloride and incubating at 25°C for 30 minutes to obtain ALP-labeled BT2 Fab' fragment.

[0042] The resulting ALP-labeled BT2 Fab' fragment was purified by gel filtration using a Superdex200 column (GE Healthcare Biosciences). Of the multiple peaks at 280 nm absorbance of the resulting fractions, those in which two or more Fab's were bound to one ALP were divided into two groups, low molecular weight and high molecular weight, and pooled. The low molecular weight fraction was designated the first ALP-labeled BT2 Fab' fragment (1:X), and the high molecular weight fraction was designated the second ALP-labeled BT2 Fab' fragment (1:XX). After purification of each ALP-labeled BT2 Fab' fragment, it was diluted with 0.1 M MES buffer (1 mM MgCl 2 , 0.1 mM ZnCl 2 , 0.1% NaN 3 The concentration was adjusted to 0.05 mg / mL in 0.1% BSA, pH 6.8. The first and second ALP-labeled HT7 Fab' fragments were prepared in the same manner as the first and second ALP-labeled BT2 Fab' fragments. The first ALP-labeled BT2 Fab' fragment solution and the first ALP-labeled HT7 Fab' fragment solution prepared above were mixed in a volume ratio of 1:1 to obtain a first ALP-labeled Fab' fragment mixture. The second ALP-labeled BT2 Fab' fragment solution and the second ALP-labeled HT7 Fab' fragment solution prepared above were mixed in a volume ratio of 1:1 to obtain a second ALP-labeled Fab' fragment mixture.

[0043] Example 2: Preparation of antibody-bound magnetic particles As solid-phase-bound antibodies, mouse monoclonal antibody AT270 (epitope: PPAPKT(p)P (sequence number 4), where T(p) represents the phosphorylated threonine residue), which specifically recognizes tau protein, and mouse monoclonal antibody AT120 (epitope: PPTREPK (sequence number 5)), which specifically recognizes tau protein, were each bound to magnetic ferrite particles. Specifically, ethyl(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to carboxylated magnetic ferrite particles (manufactured by Fujirebio Inc.), and the mixture was incubated at 25°C for 30 minutes with gentle stirring. After washing, mouse monoclonal antibody AT270 was added, and the mixture was incubated at 25°C for 1 hour with gentle stirring. After the reaction, trishydroxymethylaminomethane was added, and the mixture was incubated at 25°C for 30 minutes with gentle stirring to stop the reaction. After the reaction was stopped, the magnetic ferrite particles were collected with a magnet and separated from the reaction solution, and the particles were washed with 50 mM Tris buffer (containing 0.15 M NaCl and 3% BSA, pH 7.2) to obtain AT270-bound magnetic particles. The obtained AT270-bound magnetic particles were suspended in 50 mM MOPS buffer to a concentration of 0.25 mg / mL to obtain an AT270-bound magnetic particle solution. The AT120-bound magnetic particles were also prepared in a manner similar to that for the AT270-bound magnetic particles.

[0044] Example 3: Measurement of phosphorylated tau peptides A phosphorylated tau peptide antigen, in which the 181st threonine of tau protein is phosphorylated, was diluted with 100 mM Tris buffer to prepare phosphorylated tau standard solutions at concentrations of 0, 4.5, 9, 18, 36, 72, and 150 pM. 150 μL of the AT270-bound magnetic particle solution prepared in Example 2 and 100 μL of the diluted phosphorylated tau standard solution were dispensed into the reaction vessel, stirred, and then incubated at 37° C. for 10 minutes. Then, B / F separation and washing were performed. For washing, a dedicated washing solution for "Lumipulse G (registered trademark)" (Lumipulse G Wash Solution, Fujirebio Inc.) was used. After washing, 250 μL of a solution in which the hybrid conjugate prepared in Example 1 was diluted to 0.2 μg / mL with 50 mM MOPS buffer (containing 1% BSA, pH 6.8) was dispensed into the reaction vessel, and incubated at 37° C. for 10 minutes. Then, B / F separation and washing were performed. After washing, 200μL of substrate solution (Lumipulse G Substrate Solution, Fujirebio) containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was dispensed into the reaction vessel, stirred, and reacted at 37℃ for 5 minutes, and the amount of luminescence was measured with a luminometer to obtain a count value. The actual measurement was performed using a fully automated chemiluminescent enzyme immunoassay system "Lumipulse G" (Fujirebio).

[0045] As a control, the standard solution was similarly measured using the ALP-labeled single antibodies (first ALP-labeled Fab' fragment mixture and second ALP-labeled Fab' fragment mixture) prepared in Comparative Example 1 instead of the hybrid conjugate solution, and the count value was calculated.

[0046] The results are shown in Table 1. The count value of each phosphorylated tau standard solution is shown as the average of the count values ​​measured in duplicate. In addition, the ratio of the count value at 4.5 pM to the count value at 0 pM (S / N ratio) was calculated for each. As a result, the first ALP-labeled Fab' fragment mixture produced low counts. The second ALP-labeled Fab' fragment mixture produced count values ​​similar to those of the hybrid conjugate only in the high-concentration standard solution. On the other hand, only the hybrid conjugate showed a very high count value even for the low-concentration standard solution. As a result, it was revealed that the ratio of the count value of the lowest standard solution (4.5 pM) to the count value of the blank (0 pM) (S / N ratio) was about 6 times higher when the hybrid conjugate was used than when the ALP-labeled Fab' fragment mixture (ALP-labeled single antibody) was used.

[0047] A high S / N ratio is an important factor for obtaining a stable assay system and high sensitivity, and it has been shown that the use of hybrid conjugates makes it possible to construct a highly sensitive and stable assay system.

[0048] [Table 1]

[0049] Example 4: Measurement of total tau protein 150 μL of the AT120-bound magnetic particle solution prepared in Example 2, 75 μL of a calibrator reagent for measuring total tau protein ("LUMIPULSE G Total Tau Calibrators set", Fujirebio Inc.) or a tau protein standard solution prepared by diluting the calibrator reagent with 50 mM Tris buffer, and 50 μL of a solution obtained by diluting the hybrid conjugate prepared in Example 1 to 0.2 μg / mL with 50 mM Tris buffer were dispensed into the reaction vessel, stirred, and incubated at 37° C. for 20 minutes. Then, B / F separation and washing were performed. After washing, 200 μL of a substrate solution containing AMPPD (Lumipulse G Substrate Solution, Fujirebio Inc.) was dispensed into the reaction vessel, stirred, and reacted at 37° C. for 5 minutes, and the amount of luminescence was measured with a luminometer to obtain a count value. The actual measurement was performed using a fully automated chemiluminescence enzyme immunoassay system "Lumipulse G" (Fujirebio Inc.).

[0050] As controls, standard solutions were measured as follows using biotinylated monoclonal antibody BT2 (Fujirebio Europe), biotinylated monoclonal antibody HT7 (Fujirebio Europe), and streptavidin-labeled ALP instead of the hybrid conjugate solution. 150 μL of the AT120-bound magnetic particle solution prepared in Example 2, 75 μL of the above tau protein standard solution, and 50 μL of a solution (antibody mixture) containing biotinylated monoclonal antibody BT2 and biotinylated monoclonal antibody HT7 (total antibody 8 μg / mL) were dispensed into the reaction vessel, stirred, and then incubated at 37° C. for 10 minutes. Then, B / F separation and washing were performed. After washing, 250 μL of a 0.08 μg / mL streptavidin-labeled ALP (SA-ALP) solution was dispensed into the reaction vessel, and incubated at 37° C. for 10 minutes. Then, B / F separation and washing were performed. After washing, 200 μL of a substrate solution (Lumipulse G Substrate Solution, Fujirebio) containing AMPPD was dispensed into the reaction vessel, stirred, and reacted at 37° C. for 5 minutes, and the amount of luminescence was measured with a luminometer to obtain a count value.

[0051] Moreover, samples (LoB, LoQ1 to LoQ5) prepared by diluting cerebrospinal fluid specimens with a buffer solution were also measured in the same manner to obtain count values.

[0052] The results are shown in Table 2. Table 2 shows the measured count values, S / N ratios, and LoQs (limit of quantification) determined for both assay systems. The count values ​​for each standard solution (CAL) are shown as the average of the count values ​​obtained by measuring the standard solutions in duplicate. The count values ​​for the samples are shown as the average of the count values ​​obtained by measuring the samples with n=10. The S / N ratio was calculated as the ratio of the count value of 600 pg / mL to the count value of the blank (0 pg / mL). The count value of 600 pg / mL was calculated from the count value of the standard solution.

[0053] Figure 1 (hybrid conjugate) and Figure 2 (antibody mixture) plot the concentration of each sample (LoQ1-LoQ5) calculated based on a standard curve created from the average count value and concentration of each standard solution, and the coefficient of variation (CV) of the count value when each sample (LoQ1-LoQ5) was measured with n = 10. In Figures 1 and 2, the LoQ (CV20%) was calculated for both assay systems based on the precision profile.

[0054] As a result, for total tau protein, the use of the hybrid conjugate produced much higher count values ​​than the mixture of each labeled antibody, and the S / N ratio was about 6-fold higher for the hybrid conjugate than for the mixture of each labeled antibody. Furthermore, the LoQ when using the hybrid conjugate was about 1 / 10 of the LoQ when using the mixture of each labeled antibody. Therefore, it was found that the use of the hybrid conjugate allows the quantification of low concentrations of total tau protein.

[0055] The above results demonstrate that a highly sensitive and stable assay system can be constructed by using the hybrid conjugate, even in a total tau protein assay system.

[0056] [Table 2]

[0057] Example 5: Preparation of bovine serum albumin (BSA) linker hybrid conjugate 1 Bovine serum albumin (BSA) and GMBS were mixed in a molar ratio of 1:10 and incubated at 30°C for 1 hour to perform maleimidation of BSA. After desalting, the mixture of BT2 Fab' fragment and HT7 Fab' fragment obtained in Example 1 was mixed with maleimidized BSA in a molar ratio of 2:1 and incubated at 25°C for 1 hour to perform coupling. The coupling reaction was stopped by adding 2-MEA hydrochloride and further incubated at 25°C for 30 minutes. The reaction was then quenched by adding 0.5M iodoacetamide and incubated at 25°C for 30 minutes to obtain a conjugate reaction mixture.

[0058] The resulting conjugate reaction mixture was loaded onto a HiLoad Superdex200 column (GE Healthcare Biosciences) equilibrated with phosphate buffer (pH 6.8) and the antibody was purified by gel filtration. Among the multiple peaks at 280 nm absorbance of the resulting fractions, fractions in which two or more Fab's were bound to one BSA were pooled to obtain the BSA linker hybrid conjugate.

[0059] The obtained BSA linker hybrid conjugate was mixed with NHS-PEG4-Bition (Thermo Fisher) at a molar ratio of 1:30 and incubated at 25°C for 45 minutes to biotinylate the BSA linker hybrid conjugate. Further desalting was performed to obtain biotinylated BSA linker hybrid conjugate 1. Furthermore, it was diluted to 0.4 μg / mL in 50 mM MOPS buffer (150 mM sodium chloride, 1.0% BSA, pH 6.8), and alkaline phosphatase-labeled streptavidin (ALP-SA) (Streptavidin, Alkaline Phosphatase Conjugate, Invitrogen) was added at a concentration of 0.4 μg / mL to obtain a reaction solution containing BSA linker hybrid conjugate 1 labeled with ALP (hybrid conjugate reaction solution 1).

[0060] Example 6: Preparation of BSA linker hybrid conjugate 2 2-MEA hydrochloride was added to BSA, and the mixture was incubated at 37°C for 60 minutes to reduce (thiolate). The mixture was further desalted to obtain thiolated BSA. The thiolated BSA was mixed with NHS-PEG4-Bition (Thermo Fisher Scientific) at a molar ratio of 1:25, and the thiolated BSA was mixed with 1,2-bis(maleimide)ethane (Tokyo Chemical Industry Co., Ltd.) at a molar ratio of 1:350, and incubated at 30°C for 1 hour to simultaneously biotinylate and maleimidate BSA. After desalting, the mixture of the Fab' fragment of BT2 and the Fab' fragment of HT7 obtained in Example 1 was mixed with biotinylated maleimidate BSA at a molar ratio of 2:1, and incubated at 25°C for 1 hour to perform coupling. The coupling reaction was stopped by adding 2-MEA hydrochloride, and further incubated at 25°C for 30 minutes. The reaction was then quenched by adding 0.5 M iodoacetamide and incubating at 25° C. for 30 minutes to obtain biotinylated BSA linker hybrid conjugate 2.

[0061] The obtained biotinylated BSA linker hybrid conjugate 2 was diluted to 0.2 μg / mL in 50 mM MOPS buffer (150 mM sodium chloride, 1.0% BSA, pH 6.8), and ALP-SA (Streptavidin, Alkaline Phosphatase Conjugate, Invitrogen) was added to a concentration of 0.4 μg / mL to obtain a reaction solution containing BSA linker hybrid conjugate 2 labeled with ALP (hybrid conjugate reaction solution 2).

[0062] Comparative Example 2: Preparation of monoclonal antibody with a single BSA linker Monoclonal antibody BT2 and pepsin were mixed in a citrate buffer (pH 3.5) and incubated at 37° C. for 1 hour for pepsin digestion. After the reaction was stopped, gel filtration purification was performed using a Superdex 200 column (GE Healthcare Biosciences) to obtain F(ab') 2 The purified F(ab') fragment was then 2 The concentration of the fragments was determined and adjusted, if necessary, to a concentration of 2.5 mg / mL. The obtained F(ab') of BT2 2 The fragment was reduced by incubation with 2-MEA hydrochloride for 90 minutes at 37° C. to obtain the Fab′ fragment of BT2.

[0063] Meanwhile, desalted BSA and GMBS were mixed in a molar ratio of 1:10 and incubated at 30°C for 1 hour to maleimidate BSA. After desalting, BT2 Fab' fragment and maleimidate BSA were mixed in a molar ratio of 2:1 and incubated at 25°C for 1 hour to perform coupling. The coupling reaction was stopped by adding 2-MEA hydrochloride and further incubated at 25°C for 30 minutes. Next, the reaction was quenched by adding 0.5M iodoacetamide and incubating at 25°C for 30 minutes to obtain BSA linker BT2 Fab' fragment.

[0064] The obtained BSA linker BT2 Fab' fragment was purified by gel filtration using a Superdex200 column (GE Healthcare Biosciences) equilibrated with phosphate buffer (pH 6.8). Among the multiple peaks at absorbance 280 nm of the obtained fractions, fractions in which two or more Fab's were bound to one ALP were pooled. The BSA linker HT7 Fab' fragment was prepared similarly to the BSA linker BT2 Fab' fragment.

[0065] The obtained BSA linker BT2 Fab' fragment and BSA linker HT7 Fab' fragment were each diluted to 0.2 μg / mL in 50 mM MOPS buffer (150 mM sodium chloride, 1.0% BSA, pH 6.8), and ALP-SA (Streptavidin, Alkaline Phosphatase Conjugate, Invitrogen) was added to a concentration of 0.4 μg / mL to obtain a mixture of the ALP-labeled BSA linker BT2 Fab' fragment and the ALP-labeled BSA linker BT2 Fab' fragment (mixed conjugate reaction solution).

[0066] Example 7: Measurement of phosphorylated tau peptides A phosphorylated tau peptide antigen, in which the 181st threonine of tau protein is phosphorylated, was diluted with 100 mM Tris buffer to prepare phosphorylated tau standard solutions at concentrations of 0, 40, 100, 200, and 400 pg / mL. 150 μL of the AT270-bound magnetic particle solution prepared in Example 2 and 30 μL of the diluted phosphorylated tau standard solution were dispensed into the reaction vessel, stirred, and then incubated at 37° C. for 10 minutes. Then, B / F separation and washing were performed. For washing, a dedicated washing solution for "Lumipulse G (registered trademark)" (Lumipulse G Wash Solution, Fujirebio Inc.) was used. After washing, 250 μL of the hybrid conjugate reaction solution 1 prepared in Example 5 or the hybrid conjugate reaction solution 2 prepared in Example 6 was dispensed into the reaction vessel, and incubated at 37° C. for 10 minutes. Then, B / F separation and washing were performed. After washing, 200 μL of a substrate solution containing AMPPD (Lumipulse G Substrate Solution, Fujirebio Inc.) was dispensed into the reaction vessel, stirred, and reacted at 37° C. for 5 minutes, and the amount of luminescence was measured with a luminometer to obtain a count value. The actual measurements were performed using a fully automated chemiluminescent enzyme immunoassay system "Lumipulse G" (manufactured by Fujirebio Inc.).

[0067] As a control, the standard solution was similarly measured using the mixed conjugate reaction solution prepared in Comparative Example 2 instead of the hybrid conjugate reaction solution, and the count value was calculated.

[0068] The results are shown in Table 3. The count value of each phosphorylated tau standard solution is shown as the average of the count values ​​measured in duplicate. In addition, the ratio of the count value at 40 pg / mL to the count value at 0 pg / mL (S / N ratio) was calculated for each. As a result, when the mixed conjugate reaction solution was used, low counts were generated. On the other hand, when the hybrid conjugate reaction solution 1 and the hybrid conjugate reaction solution 2 were used, very high count values ​​were shown even for low-concentration standard solutions. As a result, it was revealed that the ratio of the count value of the lowest standard solution (40 pg / mL) to the count value of the blank (0 pg / mL) (S / N ratio) was about 9 times higher when the hybrid conjugate reaction solution 1 was used than when the mixed conjugate reaction solution (mixture of ALP-labeled single antibodies) was used, and about 13 times higher when the hybrid conjugate reaction solution 2 was used than when the mixed conjugate reaction solution was used.

[0069] A high S / N ratio is an important factor for obtaining a stable assay system and high sensitivity, and it has been shown that the use of hybrid conjugates makes it possible to construct a highly sensitive and stable assay system.

[0070] [Table 3]

Claims

1. An antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier.

2. The antibody conjugate of claim 1 , wherein the two or more anti-tau protein antibodies are two or more anti-human tau protein antibodies.

3. The antibody conjugate according to claim 2, wherein the two or more types of anti-tau protein antibodies comprise a first antibody recognizing an epitope located between positions 153 and 169 in the amino acid sequence of SEQ ID NO:1, and a second antibody recognizing an epitope located between positions 188 and 207 in the amino acid sequence of SEQ ID NO:

1.

4. The antibody conjugate according to claim 3 , wherein the epitope recognized by the first antibody is PPGQK (SEQ ID NO: 2) and the epitope recognized by the second antibody is DRSGYS (SEQ ID NO: 3).

5. The antibody conjugate according to any one of claims 1 to 4, wherein the carrier is a labeled carrier.

6. The antibody conjugate described in claim 5, wherein the labeling carrier is an enzyme, an affinity substance, a fluorescent substance, a luminescent substance, or a radioactive substance.

7. The antibody conjugate described in claim 5, wherein the labeled carrier is an enzyme.

8. An antibody conjugate described in any one of claims 1 to 4, wherein the carrier is a solid phase.

9. The kit includes: (i) an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier; and (ii) An anti-tau protein antibody that recognizes an epitope different from that of the anti-tau protein antibody contained in the antibody conjugate.

10. The kit according to claim 9, wherein the additional epitope is an epitope present between positions 170 and 187 or between positions 209 and 233 in the amino acid sequence of SEQ ID NO:

1.

11. The kit of claim 10, wherein the additional epitope is PPAPKTP (SEQ ID NO: 4) or PPTREPK (SEQ ID NO: 5).

12. A method for detecting tau protein, comprising detecting tau protein in a sample using an antibody conjugate in which two or more types of anti-tau protein antibodies are bound to a carrier.

13. The method of claim 12, wherein the sample is a body fluid sample taken from a human.

14. The method of claim 12 or 13, further comprising contacting the sample with an anti-tau protein antibody that recognizes an epitope distinct from that of the anti-tau protein antibody contained in the antibody conjugate.

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