Kit and method for detecting Tau protein

Abstract

The present application discloses a kit for detecting Tau protein, which includes: magnetic beads coated with a first antibody targeting Tau protein; and a second antibody targeting Tau protein labeled with a chemiluminescent agent; wherein the magnetic beads are tosyl-activated magnetic beads or carboxyl magnetic beads. Also disclosed is a use of the aforementioned kit in the preparation of a product for detecting Tau protein and / or for diagnosing Alzheimer's disease, and a method for detecting the content of Tau protein in a sample. The kit of the present application has a short detection time, a lower limit value, a wider linear range, and a high detection sensitivity, which is of great significance for the early detection of Alzheimer's disease and the dynamic tracking of the treatment effect.

Description

Technical Field

[0001] The present application belongs to the technical field of immunoassay, and in particular, relates to a kit for detecting Tau protein and a method for detecting Tau protein. Background Art

[0002] Alzheimer's disease (AD) is the most common age-related neurodegenerative disease.

[0003] Alzheimer's disease (AD) is characterized by neuronal death, which is usually associated with the appearance of amyloid plaques and neurofibrillary tangles (NFTs), which are filamentous tangles of the hyperphosphorylated microtubule-associated protein Tau. Under normal physiological conditions, Tau protein promotes microtubule assembly and stability, and also acts as a connecting protein between axonal microtubules and neuronal plasma membrane components. Defective modification of Tau, especially phosphorylation, is believed to cause Tau to lose its microtubule binding function, induce its aggregation to form Tau filamentous tangles, and may ultimately lead to the occurrence of AD.

[0004] The current research results confirm that using total tau in cerebrospinal fluid as an AD biomarker can effectively increase the percentage of correctly diagnosed patients. Compared with imaging methods such as PET-CT, the detection of this biomarker combination will help in the early detection of AD and the dynamic tracking of treatment effects.

[0005] However, the existing kit preparation technology has the following main disadvantages:

[0006] (1) Detection time is too long

[0007] At present, the total Tau detection time based on microfluidic chips is relatively long. For example, the chip kit for early detection of Alzheimer's disease developed by Li Yihong et al. takes about 1.5 hours to detect. A similar situation also exists in the total Tau determination kit based on the chemiluminescence platform. The total Tau determination kit developed by Liu Jianfang et al. also takes no less than 0.5 hours to detect a single sample. Such a long detection time has undoubtedly brought troubles to the high-throughput and high-efficiency total Tau detection in clinical laboratory departments / physical examination departments.

[0008] (2) High detection limit

[0009] The lower the detection limit value, the higher the sensitivity of the kit, and the more reliable it is in quantitatively detecting low-abundance analytes. However, the data of Lins et al. showed that the detection limit of total Tau in their POCT platform kit was 15pg / mL. The detection limit of the total Tau determination kit based on the chemiluminescence platform established by Guo Jianfu et al. was as high as 500pg / mL. We hope that with the help of technical improvements, we can more effectively detect low-abundance total Tau and improve the sensitivity of the kit.

[0010] (3) Narrow linear range

[0011] A wider linear range ensures that the sample can be tested without dilution, which can undoubtedly reduce the number of tests and eliminate the detection bias caused by sample dilution. The upper limit of total Tau detection of the chemiluminescence platform established by Wang Qian et al. is 400pg / mL. Publicly available data show that the upper limit of the linear range of the total Tau detection system established by Yao Tianming et al. is only 1000pg / mL. The upper limit of this linear range is lower than the concentration range of 759±417pg / mL of total Tau in some samples reported by Andreasen et al. Summary of the invention

[0012] Based on the above description, the current total Tau detection system still has many deficiencies. Therefore, the early detection of AD and the dynamic tracking of treatment effects are limited. In order to overcome the deficiencies in the prior art, the present application provides a kit for detecting Tau protein, and the use of the kit in the preparation of products for detecting Tau protein and / or for diagnosing Alzheimer's disease, and also provides a method for detecting the content of Tau protein in a sample.

[0013] The specific technical solutions of this application are as follows:

[0014] 1. A kit for detecting Tau protein, wherein the kit comprises:

[0015] Magnetic beads coated with a primary antibody targeting Tau protein; and

[0016] A secondary antibody targeting Tau protein labeled with a chemiluminescent agent;

[0017] Wherein, the magnetic beads are tosyl-activated magnetic beads or carboxyl magnetic beads.

[0018] 2. The kit according to item 1, wherein the mass ratio of the first antibody targeting Tau protein to the magnetic beads is 1:(20-60), preferably 1:(25-50).

[0019] 3. The kit according to item 1 or 2, wherein the molar ratio of the second antibody targeting Tau protein to the chemiluminescent agent is 1:(6-50), preferably 1:(10-30).

[0020] 4. The kit according to any one of items 1 to 3, wherein the kit further comprises a Tau protein standard, a substrate, a coating buffer, a labeling buffer, a washing solution, a blocking solution and a sample diluent.

[0021] Preferably, the coating buffer and the marker buffer are TBS-T buffer, the TBS-T buffer contains Tween-20 and BSA, and preferably the TBS-T buffer further contains trehalose;

[0022] Preferably, the TBS-T buffer contains 0.7% to 1.2% Tween-20 and 0.5% to 1% BSA;

[0023] Preferably, the TBS-T buffer further comprises 30-50 g / L trehalose.

[0024] 5. The kit according to any one of items 1 to 4, wherein

[0025] The first antibody targeting Tau protein comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0026] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1;

[0027] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 3;

[0028] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 5;

[0029] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 7,

[0030] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 9;

[0031] The amino acid sequence of the CDR-L3 is shown in SEQ ID No: 11;

[0032] The second antibody targeting Tau protein comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0033] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 2;

[0034] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 4;

[0035] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 6;

[0036] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 8;

[0037] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 10;

[0038] The amino acid sequence of the CDR-L3 is shown in SEQ ID No:12.

[0039] 6. The kit according to any one of items 1 to 5, wherein

[0040] The first antibody targeting Tau protein comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 15, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 15;

[0041] The second antibody targeting Tau protein comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 14; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 16, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 16.

[0042] 7. The kit according to any one of items 1 to 6, which is used for diagnosing Alzheimer's disease.

[0043] 8. Use of the kit according to any one of items 1 to 7 in the preparation of a product for detecting Tau protein and / or for diagnosing Alzheimer's disease.

[0044] 9. A method for detecting the content of Tau protein in a sample, comprising the following steps:

[0045] The test sample, magnetic beads coated with a first antibody targeting Tau protein, and a second antibody targeting Tau protein labeled with a chemiluminescent agent are mixed, and magnetic separation is performed after incubation;

[0046] After washing, the substrate is added to measure the luminescence value of the sample being tested;

[0047] The luminescence value of the Tau protein standard is used to calculate the content of Tau protein in the tested sample;

[0048] Wherein, the magnetic beads are tosyl-activated magnetic beads or carboxyl magnetic beads.

[0049] 10. The method according to item 9, wherein the mass ratio of the first antibody targeting Tau protein to the magnetic beads is 1:(20-60), preferably 1:(25-50);

[0050] Preferably, the ratio of the amount of the second antibody targeting Tau protein to the amount of the chemiluminescent agent is 1:(6-50), preferably 1:(10-30).

[0051] 11. The method according to item 9 or 10, wherein the sample to be tested is cerebrospinal fluid;

[0052] Preferably, the sample to be tested is at least 50 μL, preferably at least 100 μL;

[0053] More preferably, for each microliter of the sample to be tested, 0.005-0.015 μg of the first antibody targeting the Tau protein and 0.05-0.15 ng of the second antibody targeting the Tau protein are required;

[0054] More preferably, the incubation time is at least 15 min.

[0055] 12. The method according to any one of items 9 to 11, wherein the first antibody targeting Tau protein and the second antibody targeting Tau protein are the first antibody targeting Tau protein and the second antibody targeting Tau protein described in item 5 or 6.

[0056] Effects of the Invention

[0057] The kit for detecting Tau protein of the present application uses a pair of recombinant antibodies as core raw materials, including magnetic beads coated with a first antibody targeting Tau protein, and a second antibody targeting Tau protein labeled with chemiluminescence. The method for detecting Tau protein of the present application is to mix the magnetic beads coated with the first antibody, the labeled second antibody and the sample to be tested, and then magnetically separate them, and detect them on a magnetic particle chemiluminescence platform. The recombinant antibodies contained in the kit of the present application have excellent performance and stability, high affinity, high sensitivity, and significantly shortened production cycle, and are more suitable as core raw materials for application in the field of in vitro diagnostic reagents. The magnetic beads used in the kit and detection method of the present application are specific tosyl-activated magnetic beads or carboxyl magnetic beads, with lower detection limit values, wider linear range, and high detection sensitivity. In addition, the magnetic particle chemiluminescence platform has the advantages of simple operation, high degree of automation, and homogeneous reaction system. The detection time can be further shortened by using a one-step reaction system. The present application further adopts high-affinity and stable recombinant antibodies with stronger antigen epitope recognition ability and higher degree of binding with antigen. Therefore, high-affinity antibodies have stronger ability to capture low-abundance target antigens, providing a lower detection limit for the kit and high detection sensitivity. Without changing the coating process, replacing high-affinity antibodies can enhance the antigen capture ability of magnetic beads or other media per unit mass, thereby significantly improving the linear range of the reagent without producing the Hook effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 For the use of the Tau kit of the present application and the use of Consistency analysis results of Tau content in samples tested by G1200 platform products. DETAILED DESCRIPTION

[0059] The present application is further described in detail below in conjunction with specific implementation methods. The implementation methods given are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to technicians in the field.

[0060] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0061] The main function of Tau protein in the normal human body is to induce and promote tubulin to aggregate into microtubules and maintain its stability. There are 6 subtypes of Tau protein in the adult brain, three of which have three microtubule binding repeat regions (2N3R, 1N3R, 0N3R), and the other three have four microtubule binding repeat regions (2N4R, 1N4R, 0N4R). 2N4R / Tau-441 is the full-length Tau protein, and K18 and K19 are truncated Tau proteins that only contain microtubule binding repeat regions. The microtubule binding ability of Tau protein is controlled by post-translational modification, among which phosphorylation modification is one of the main ways. 20% of the amino acids of Tau protein can be potential phosphorylation sites. Normally, the microtubule binding region of Tau protein is positively charged, so it can attract negatively charged microtubules. When this microtubule binding region is highly phosphorylated, it loses its positive charge, detaches from the tubulin, and no longer has the ability to bind to stabilize microtubules. Next, it causes the failure of tubulin, and the highly phosphorylated Tau aggregates and precipitates, which is toxic to nerve cells. These consequences together cause the cause of neurodegeneration. Since Tau protein has multiple phosphorylation sites and more than one protein kinase and phosphatase are involved in regulation, the pathogenesis of AD is very complicated. Tau precursor fibrils (PFFs) can induce the aggregation of Tau protein in artificially cultured cells and living animals, causing soluble monomers to aggregate to form insoluble fibers. Introducing a small amount of TauPFFs into cells expressing Tau protein can cause a large amount of Tau to aggregate to form filamentous inclusions similar to NFTs. Synthetic TauPFFs can also enter non-neuronal cells to aggregate Tau protein to form NFTs.

[0062] In some embodiments, the Tau protein of the present application is the full-length Tau protein 2N4R / Tau-441.

[0063] As used herein, the term "recombinant antigen" refers to an antigen that is purified by in vitro recombinant expression after the antigen gene is connected to a vector and transferred into prokaryotic or eukaryotic cells.

[0064] In this article, the term "recombinant antibody" refers to an antibody obtained by cloning the immunospecific heavy chain and light chain antibody genes into a high-efficiency expression vector, and then introducing these vectors into an expression host (such as bacteria, yeast or mammals) for antibody expression. The antibody of the present application can be a fully humanized antibody, a humanized antibody, a chimeric antibody, etc.

[0065] In some embodiments, the recombinant antibody of the present application is an antibody obtained by immunizing an animal with the recombinant antigen of the present application.

[0066] In some embodiments, the antibody of the present application is a full-length antibody, which typically refers to an antibody composed of two "heavy chains" and two "light chains". A "heavy chain" is generally a polypeptide that consists of a heavy chain variable region (VH), a heavy chain constant region 1 (CH1), a hinge region (HR), an antibody heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3; in some embodiments, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is generally a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.

[0067] In the present application, the light chain constant region and the heavy chain constant region can be any light chain constant region and heavy chain constant region. The light chain constant region (CL) can be κ (kappa) or λ (lambda). The heavy chain constant region can be any one of the heavy chain constant regions of IgG, IgM, IgA, IgE, and IgD.

[0068] It is well known to those skilled in the art that each heavy chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs), and each light chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs). The complementary determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. In some embodiments, from the N-terminus to the C-terminus, the heavy chain variable region and the light chain variable region both include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. There are two common definitions of the CDR sequence of the heavy chain variable region or the light chain variable region, namely the Kabat definition and the Chothia definition, for example, see Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR sequence in the heavy chain variable region or the light chain variable region sequence can be determined according to the Kabat definition or the Chothia definition. In the embodiment of the present application, the Chothia definition is used to determine the CDR sequence.

[0069] As used herein, the term "identity" may be defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps. Methods and computer programs for alignment are well known in the art. As used herein, an amino acid sequence that is referenced to have a percentage identity with an amino acid sequence refers to a sequence that has that percentage identity over the entire length of the referenced amino acid sequence.

[0070] In this article, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0071] In this article, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers in single-stranded or double-stranded form thereof. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides, which have similar binding properties to reference nucleic acids (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include variants modified in a conservative manner thereof, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0072] As used herein, the term "EC50 value" refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that can induce 50% of the maximal effect.

[0073] As used herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be determined by common methods known in the art.

[0074] In this article, the term "monoclonal antibody" means an antibody derived from a population of substantially homologous antibodies, that is, each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody population, and should not be interpreted as requiring the antibody to be produced by any ad hoc method.

[0075] Herein, the term "recombinant antibody targeting Tau protein" refers to a recombinant antibody that can bind to Tau protein with sufficient affinity and can be used as a diagnostic agent and / or therapeutic agent targeting Tau protein.

[0076] The recombinant antibody targeting Tau protein of the present application does not bind to proteins unrelated to the target. Here, "unrelated proteins" refer to proteins other than the Tau protein as the target; here, "no binding" means: when the binding ability of the recombinant antibody targeting Tau protein of the present application to the Tau protein as its target is taken as 100%, the binding ability of the recombinant antibody targeting Tau protein of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0077] Tau protein suitable for producing antibodies can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Other forms of β-amyloid protein may also include Tau protein expressing cells, products or cell extracts or fractions containing Tau protein, and partially purified Tau protein.

[0078] In some embodiments, the present application uses a magnetic particle chemiluminescence immunoassay to determine the level of Tau protein in a sample. The specific principle is: directly labeling an antigen or antibody (chemiluminescent agent marker) with a chemiluminescent agent, reacting with the corresponding antibody or antigen in the sample to be tested, and the antigen or antibody of magnetic particles (magnetic beads), separating the bound state (precipitation part) and the free state of the chemiluminescent agent marker through a magnetic field, and then adding a luminescence promoter (luminescent substrate) for a luminescent reaction, and performing quantitative or qualitative detection by detecting the luminescence intensity.

[0079] In this article, ELISA (enzyme linked immune sorbent assay) refers to a detection method that uses the characteristics of antibody molecules that can specifically bind to antigen molecules to bind free impurities and target proteins bound to solid phase carriers, and uses special markers to conduct qualitative or quantitative analysis. The principle is that antigens or antibodies can be physically adsorbed on the surface of solid phase carriers and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of immune reactions can be determined by the color reaction of adding substrates, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen.

[0080] In a first aspect, the present application provides a kit for detecting Tau protein, the kit comprising: magnetic beads coated with a first antibody targeting Tau protein; and a second antibody targeting Tau protein labeled with a chemiluminescent agent; wherein the magnetic beads are tosyl-activated magnetic beads or carboxyl magnetic beads.

[0081] Tosyl-activated magnetic beads are superparamagnetic pre-activated functional magnetic microspheres. Compared with traditional magnetic beads, they have faster magnetic responsiveness while maintaining good dispersibility, extremely low nonspecific adsorption and pre-activated reaction sites. They can be directly used for high-load binding with a variety of biological ligands (proteins, peptides, oligonucleotides, drug molecules, etc.) to achieve the purpose of covalent bonding on the surface of magnetic beads. The magnetic beads have long hydrophilic spacer arms and relatively mild reaction conditions, making them particularly suitable for the immobilization of bioactive macromolecules.

[0082] Carboxyl magnetic beads have the characteristics of superparamagnetism, rapid magnetic responsiveness, rich carboxyl functional groups, monodispersity and submicron particle size. They can covalently couple biological ligands such as peptides, proteins, antibodies, oligonucleotides to the surface of microspheres under the action of special chemical reagents (such as EDC).

[0083] In some embodiments, the mass ratio of the first antibody targeting Tau protein to the magnetic beads is 1:(20-60), for example, 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:37, 1:40, 1:43, 1:45, 1:48, 1:50, 1:52, 1:55, 1:57, 1:60, etc., preferably 1:(25-50).

[0084] In some embodiments, the ratio of the amount of the second antibody targeting Tau protein to the amount of the chemiluminescent agent is 1:(6-50), for example, 1:6, 1:6.7, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19. .5, 1:20, 1:20.5, 1:21, 1:21.5, 1:22, 1:22.5, 1:23, 1:23.5, 1:24, 1:24.5, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, etc., preferably 1:(10-30).

[0085] In some embodiments, the kit further comprises a Tau protein standard, a substrate, a coating buffer, a labeling buffer, a washing solution, a blocking solution, and a sample diluent.

[0086] In some embodiments, the coating buffer and the marker buffer are TBS-T buffer, and the TBS-T buffer contains Tween-20 and BSA. In some embodiments, the coating buffer is TBS-T buffer, and the TBS-T buffer contains Tween-20, BSA and trehalose.

[0087] In some embodiments, the TBS-T buffer contains 0.1% to 2% Tween-20 and 0.2% to 2% BSA, preferably 0.7% to 1.2% Tween-20 and 0.5% to 1% BSA, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1. 3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% etc. of Tween-20 and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% etc. of BSA.

[0088] In some embodiments, the TBS-T buffer further comprises 10-50 g / L trehalose, preferably 30-50 g / L trehalose, for example, 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L, 34 g / L, 36 g / L, 38 g / L, 40 g / L, 42 g / L, 44 g / L, 46 g / L, 48 g / L, 50 g / L and the like of trehalose.

[0089] In some preferred embodiments, the TBS-T buffer contains 0.7% to 1.2% Tween-20, 0.5% to 1% BSA and 30 to 50 g / L trehalose.

[0090] The coating buffer of the present application provides a high pH environment for magnetic bead coating, improves the coating efficiency, limits the contents of Tween-20, BSA and trehalose in the coating buffer to the above range, and can further improve the detection sensitivity and heating stability.

[0091] In some embodiments, the substrate includes luminescent excitation liquid A and luminescent excitation liquid B, wherein luminescent excitation liquid A is an aqueous solution of nitric acid and hydrogen peroxide, luminescent excitation liquid B is an aqueous solution of sodium hydroxide, the coating buffer is TBS-T buffer (phosphate buffer system, containing Tween-20, BSA, trehalose and Proclin300), the marker buffer is TBS-T buffer (phosphate buffer system, containing Tween-20, BSA, trehalose and Proclin300); the washing solution is PBS and Tween-20; the blocking solution is BSA blocking solution, and the sample diluent is PBS buffer. In some embodiments, the blocking solution is 10% BSA blocking solution, the sample diluent is a PBS buffer with a pH of 7.4, and each liter of sample diluent also contains 1% BSA and 0.1% Proclin-300.

[0092] In the above embodiment, the kit further comprises a storage buffer and a quenching buffer, wherein the storage buffer is used to provide a stable pH environment, reduce antibody degradation, and preserve, etc.; the quenching buffer is used to quench the markers that are not bound to the antibody. In some embodiments, the storage buffer is TBS-T buffer, and the quenching buffer is 5% DL-lysine.

[0093] In some embodiments, the first antibody targeting Tau protein comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1 (GFTFTDY); the amino acid sequence of the CDR-H2 is shown in SEQ ID No: 3 (RNKANGYT); the amino acid sequence of the CDR-H3 is shown in SEQ ID No: 5 (EEDYRHGFPY); the amino acid sequence of the CDR-L1 is shown in SEQ ID No: 7 (NSSQSLLNTGNQKNYLA), the amino acid sequence of the CDR-L2 is shown in SEQ ID No: 9 (GALTRES); the amino acid sequence of the CDR-L3 is shown in SEQ ID No:11 (QNDHSYPLT); the second antibody targeting Tau protein comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown in SEQ ID No:2 (GFVFIDY); the amino acid sequence of the CDR-H2 is shown in SEQ ID No:4 (RNKANGYR); the amino acid sequence of the CDR-H3 is shown in SEQ ID No:6 (EGSYPYGFPD); the amino acid sequence of the CDR-L1 is shown in SEQ ID No:8 (KSSQSLLHSGNQKNYLA); the amino acid sequence of the CDR-L2 is shown in SEQ ID No:10 (GASTRES); the amino acid sequence of the CDR-L3 is shown in SEQ ID No:12 (QNDHSYPFT).

[0094] In some embodiments, the first antibody targeting Tau protein comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13 (EVMLQESGG GLVQPGSSLRLTCATSGFTFTDYYMSWVRQPPGKALEWLGVIRNKANGY TTEYSASVKGRFTISRDNTQGILYLHMNTLSAEDSATYYCAREEDYRHGFPYWGQGTLVTVSA), or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 15 (DIIMTQSPSSLSVSVGEKVTMNCNSSQSLLNTGNQKN YLAWYQQKPGQTPKLLIHGALTRESGIPDRFTGRGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGTGTKLELRRAD), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 15;The second antibody targeting Tau protein comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14 (EVKLIESGGGLMQPGSSLRLSCASS GFVFIDYYMSWVRQPPGKALEWLGLVRNKANGYRTEYSSTVKGRFTISR DNSQGILYLHMNTLSAEDSATYYCAREGSYPYGFPDWGQGTLVTVSA), or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 14, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 16 (DIV MTQSPSSLSASVGERVTMNCKSSQSLLHSGNQKNYLAWYQQKPGQQPKL LIHGASTRESGVPDRFTGRGSGTDFTLTISSVQAEDLALYYCQNDHSYPFTFGTGTKLELRRAN), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 16. ;

[0095] In some embodiments, any of the aforementioned kits for detecting Tau protein is used to diagnose Alzheimer's disease.

[0096] In a second aspect, the present application provides a use of any of the aforementioned kits for detecting Tau protein in the preparation of a product for detecting Tau protein and / or for diagnosing Alzheimer's disease.

[0097] In a third aspect, the present application also provides a method for detecting the content of Tau protein in a sample, which comprises the following steps: mixing the test sample, magnetic beads coated with a first antibody targeting Tau protein, and a second antibody targeting Tau protein labeled with a chemiluminescent agent, and performing magnetic separation after incubation; adding a substrate after washing, and determining the luminescence value of the test sample; calculating the content of Tau protein in the test sample using the luminescence value of the Tau protein standard; wherein the magnetic beads are tosyl-activated magnetic beads or carboxyl magnetic beads.

[0098] In some embodiments, the mass ratio of the first antibody targeting Tau protein to the magnetic beads is 1:(20-60), for example, 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:37, 1:40, 1:43, 1:45, 1:48, 1:50, 1:52, 1:55, 1:57, 1:60, etc., preferably 1:(25-50).

[0099] In some embodiments, the amount ratio of the second antibody targeting Tau protein to the amount of the chemiluminescent agent is 1: (6-50), for example, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19.5, 1:11. 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, etc., and preferably 1:(10-30).

[0100] In some embodiments, the sample to be tested is cerebrospinal fluid, and the sample to be tested is at least 50 μL, for example, it can be 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, etc., preferably at least 100 μL, and the incubation time is at least 15 min, for example, it can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, etc.

[0101] In some embodiments, the sample to be tested is cerebrospinal fluid, the sample to be tested is at least 50 μL, preferably at least 100 μL, the incubation time is at least 15 min, and per microliter of the sample to be tested, 0.005-0.015 μg of the first antibody targeting Tau protein and 0.05-0.15 ng of the second antibody targeting Tau protein are required. The first antibody targeting Tau protein required to be used per microliter of the sample to be tested can be, for example, 0.005 μg, 0.006 μg, 0.007 μg, 0.008μg, 0.009μg, 0.01μg, 0.011μg, 0.012μg, 0.013μg, 0.014μg, 0.015μg, etc. The amount of the second antibody targeting Tau protein required per microliter of the tested sample may be, for example, 0.05ng, 0.06ng, 0.07ng, 0.08ng, 0.09ng, 0.1ng, 0.11ng, 0.12ng, 0.13ng, 0.14ng, 0.15ng, etc.

[0102] In some embodiments, the first antibody targeting Tau protein and the second antibody targeting Tau protein are the first antibody targeting Tau protein and the second antibody targeting Tau protein described in the first aspect above.

[0103] Example

[0104] The present application content will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results according to the embodiments.

[0105] Example 1 Preparation of recombinant antigen Tau-ABP

[0106] The method for preparing the recombinant antigen in this embodiment is: confirm the ABP tag amino acid sequence (UniPort, P19909) and the Tau-F amino acid sequence (UniPort, P10636-8) (2N4R / Tau-441 form) with the help of literature and databases, and construct them in series to the pET30a vector after codon optimization. After the vector is sequenced correctly, it is transformed into the competent state of Escherichia coli BL21 (DE3) and the recombinant antigen is prepared and purified. The recombinant antigen is purified by Ni-NTA Agarose (QIAGEN, Cat. No. 30210). Among them, the amino acid sequence of the recombinant antigen Tau-ABP is shown in SEQ ID No: 17, and the nucleotide sequence of the nucleic acid molecule encoding the recombinant antigen Tau-ABP after codon optimization is shown in SEQ ID No: 18.

[0107] Example 2 Preparation of recombinant antibodies rTau-A and rTau-B

[0108] The preparation method of the recombinant antibody in this embodiment is as follows: immunize mice with the recombinant antigen Tau-ABP prepared in the embodiment, obtain the mouse monoclonal antibody cell line combination with the highest sensitivity, and then sequence the antibody variable region sequence. According to the variable region sequence, the chimeric vector is constructed and transfected into the Hek293T cell line for recombinant antibody expression, wherein the light chain vector used is (pFUSE2ss-CLIg-mk, InvivoGen), and the heavy chain vector is (pFUSEss-CHIg-mG1, InvivoGen). The above two vectors are commercial vectors with constant region sequences and no additional processing is required. After the recombinant antibodies are expressed in the Hek293T cell line, the purified recombinant antibodies rTau-A and rTau-B are obtained through ProteinG filler. Among them, the amino acid sequence of the rTau-A heavy chain variable region is shown in SEQ ID No: 13, the amino acid sequence of the rTau-A light chain variable region is shown in SEQ ID No: 15, the amino acid sequence of the rTau-B heavy chain variable region is shown in SEQ ID No: 14, and the amino acid sequence of the rTau-B light chain variable region is shown in SEQ ID No: 16.

[0109] Example 3 Performance Verification of Recombinant Antibodies rTau-A and rTau-B

[0110] (1) Determination of recombinant antibody concentration

[0111] The concentration of multiple batches of different recombinant antibodies was tested by BCA (Bicinchoninic Acid Assay) technology. The concentration statistical results are shown in Table 1 below. Table 1 shows the concentration of the purified recombinant antibodies. The BCA protein concentration determination kit (enhanced) (Biyuntian, item number P0010) was used. The specific operation was carried out according to the instructions of the kit.

[0112] Table 1

[0113] Concentration (mg / mL) 20220801 20220802 rTau-A 12.3 13.2 rTau-B 10.9 12.1

[0114] (2) Determination of recombinant antibody concentration

[0115] Elisa technology was used to test the affinity of multiple batches of recombinant antibodies. Each well of the ELISA plate was coated with 100 ng of Tau antigen, and the recombinant antibody diluted in multiples was added and incubated at 37°C for 1 hour. After the incubation, a goat anti-human secondary antibody labeled with HRP was added, and finally TMB was added for color development. 2M sulfuric acid was added to terminate the reaction, and the wavelength of the ELISA reader was set to 450nM for detection. The test results are shown in Tables 2 to 5 below.

[0116] 20220801 batch rTau-A:

[0117] Table 2

[0118]

[0119]

[0120] 20220802 batch rTau-A:

[0121] Table 3

[0122]

[0123] 20220801 batch rTau-B:

[0124] Table 4

[0125]

[0126] 20220802 batch rTau-B:

[0127] Table 5

[0128]

[0129]

[0130] The EC50 of each batch of recombinant antibodies was calculated using SigmaPlot 13.0 software. The data are summarized in Table 6 below. The results in Table 6 show that the above recombinant antibodies have a higher affinity. D The K value of the recombinant antibody of the present application D The value can reach pM level, which is about one thousandth of the prior art. The affinity of the recombinant antibody of the present application is much higher than that of the antibody in the prior art.

[0131] Table 6

[0132]

[0133] Example 4 Preparation of a kit for detecting Tau protein and detection process

[0134] (1) Antibody coating

[0135] Select tosyl-activated magnetic beads (Thermo Dynabeads TM M-280Tosylactivated, Cat#14204) and carboxyl magnetic beads (Thermo Dynabeads TMM-270 Carboxylic Acid, Cat#14305D) were vortexed and then sonicated for 5 min and fully resuspended. 100 μL of the fully resuspended magnetic bead mother solution (about 10 mg magnetic beads) was taken out with a pipette, and the liquid was removed by magnetic separation for 5 min.

[0136] Tosyl-activated magnetic bead system: Add 165 μL 0.1M borate buffer (pH 9.5), and 100 μg, 200 μg, and 400 μg of rTau-A recombinant antibody, mix well, and incubate at 37°C overnight. After the incubation, remove the liquid by magnetic separation for 5 minutes. Add 10% BSA and block at 37°C for 3 hours. After the blocking is completed, remove the liquid by magnetic separation for 5 minutes. Finally, add TBS-T buffer (0.1M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin300) and store at 4°C for use. In this embodiment, the mass ratios of recombinant antibodies to magnetic beads are 1:25, 1:50, and 1:100, respectively. The mother solution after coating needs to be further diluted 20 times to be configured as the Tau protein antibody coated magnetic bead working solution for use.

[0137] Carboxyl magnetic bead system: add 1mL 0.1M MES buffer (pH 5.0) to resuspend the magnetic beads, add 100μg, 200μg, and 400μg of Tau recombinant antibody and vortex to mix. After rotating and mixing at room temperature for 30 minutes, add 100μL coupling reagent (10mg / mLEDC, dissolved in ice-cold 0.1M MES buffer at pH 5.0, ready for use). Then continue to rotate and mix at room temperature for 3 hours. After the incubation, remove the liquid by magnetic separation for 5min. Finally, add TBS-T buffer (0.1M phosphate buffer system, pH7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin300) and store at 4℃ for use. In this embodiment, the mass ratios of recombinant antibodies to magnetic beads are 1:25, 1:50, and 1:100, respectively. The mother solution after coating needs to be further diluted 20 times to be configured as the Tau protein antibody coated magnetic bead working solution for use.

[0138] (2) Antibody labeling

[0139] Pipette 0.1 mg of rTau-B recombinant antibody and mix thoroughly with 8.25 μL 4mM NSP-SA-NHS (Heliosense, Cat# HS-11015005, dissolved in DMF) solution for 2 hours. Add 200 μL 5% DL-lysine to the solution and continue mixing for 30 minutes. Use Sephadex G-25 for desalting, TBS-T buffer (0.1M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin300). After adding an equal volume of glycerol and mixing thoroughly, store at -20°C for use. In this example, the molar ratio of the recombinant antibody to NSP-SA-NHS is 1:20. The labeled mother solution needs to be further diluted 4000 times to be configured as the β-amyloid protein labeled antibody working solution for use.

[0140] (3) Antigen tracing

[0141] exist G1200 platform is used according to the instruction manual G Total Tau (Fujirebio, Catalog No. 231302) was used to trace the Tau antigen.

[0142] (4) Packaging of calibration and quality control products

[0143] Tau calibrators and quality controls are made from antigen and sample diluent. The concentrations of Tau calibrators are 300pg / mL and 1200pg / mL; the concentrations of Tau quality controls are 300pg / mL and 1500pg / mL. The sample diluent is a PBS buffer at pH 7.4, and each liter of sample diluent also contains 1% BSA and 0.1% Proclin-300. The sample diluent is a PBS buffer at pH 7.4, and each liter of sample diluent also contains 1% BSA and 0.1% Proclin300.

[0144] (5) Kit assembly

[0145] The coated antibodies and labeled antibodies produced in three batches of pilot tests were packaged in the reagent compartment and assembled into a test kit together with the calibration products and quality control products. The specific information is shown in Table 7 below:

[0146] Table 7

[0147]

[0148] (6) On-machine testing

[0149] Testing process: Use the fully automatic chemiluminescence immunoassay analyzer (Smart 500s fully automatic chemiluminescence analyzer, Chongqing Cosma Biotechnology Co., Ltd.) as the testing tool, check and replenish consumables, and complete self-test after powering on. Manually or by scanning the code, enter the parameters and reagent batch number required for the total Tau test, and place the reagent in the corresponding reagent position. Use the Tau calibrator to calibrate the master curve, and then use the Tau quality control product to quality control the detection system. After the quality control is qualified, add the samples to be tested in the sample position in turn for testing. After the test is completed, the data is output and analyzed.

[0150] Reaction system: The machine automatically aspirates 150 μL of the sample and mixes it thoroughly with 100 μL of magnetic bead working solution and 100 μL of labeled antibody working solution, and then incubates at 37°C for 20 minutes before magnetic separation. After discarding the supernatant, add the immunoassay instrument's supporting wash solution (PBS and Tween-20) and repeat the washing three times. The reaction is then sent to a darkroom, and 100 μL of the immunoassay instrument's supporting luminescent excitation solution A (nitric acid and hydrogen peroxide aqueous solution) and luminescent excitation solution B (sodium hydroxide aqueous solution) are added for luminescent reaction, and the luminescent value is recorded. The corresponding concentration value of the sample is calculated in combination with the entered and calibrated calculation curve.

[0151] The above two different magnetic bead systems and multiple different coated antibody concentration combinations were cross-combined and their linearity was tested. Each combination and each antigen concentration were repeated 3 times and the average value was calculated. The test results are shown in Table 8 below:

[0152] Table 8

[0153]

[0154] Example 5 Optimization of the coupling ratio of labeled antibodies

[0155] (1) Antibody coating: The antibody coating of carboxyl magnetic beads was carried out in accordance with Example 4, wherein the amount of rTau-A recombinant antibody used was 200 μg.

[0156] (2) Antibody labeling: 0.1 mg, 0.25 mg, 0.5 mg and 0.75 mg of rTau-B recombinant antibody were taken and mixed thoroughly with 8.25 μL 4 mM NSP-SA-NHS (Heliosense, Cat# HS-11015005, dissolved in DMF) solution for 2 h. 200 μL 5% DL-lysine was added to the solution and the mixture was mixed for 30 min. Sephadex G-25 was used for desalting, and TBS-T buffer (0.1 M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA, 0.5% Tween-20 and 0.1% Proclin 300) was used. After adding an equal volume of glycerol and mixing thoroughly, the mixture was stored at -20°C for later use. In this example, the molar ratios of the recombinant antibody rTau-B to NSP-SA-NHS were 1:50, 1:20, 1:10 and 1:6.7, respectively. The labeled mother solution needs to be diluted 4000 times with TBS-T buffer to prepare the working solution.

[0157] (3) Antigen tracing: refer to Example 4.

[0158] (4) Machine testing: refer to Example 4.

[0159] The above labeled antibody coupling ratio and dilution ratio combinations were cross-combined and their linearity was tested. Each combination and each antigen concentration were repeated 3 times and the average value was calculated. The test results are shown in Table 9 below:

[0160] Table 9

[0161]

[0162] Example 6 Optimization of coating buffer and marker buffer

[0163] (1) Antibody coating: refer to Example 5.

[0164] (2) Antibody labeling: The method was carried out in accordance with Example 5, wherein the amount of rTau-B recombinant antibody used was 0.25 mg.

[0165] (3) Antigen tracing: refer to Example 5.

[0166] (4) Machine testing: refer to Example 5.

[0167] In this example, the compositions of TBS-T buffer used as the coating buffer and the labeling buffer are the same.

[0168] Five TBS-T buffers were selected for the kit sensitivity test, wherein the five TBS-T buffers were all 0.1M phosphate buffer system, pH 7.4, supplemented with 0.1% BSA and 0.1% Proclin300, and supplemented with 0%, 0.1%, 0.5%, 1%, and 2% Tween-20, respectively. The test results are shown in Table 10 below.

[0169] Table 10

[0170] Tween-20 content 0 0.1％ 0.5％ 1％ 2％ Mean 490.8 483.5 356.6 216.6 327.5 SD 26.376 24.160 19.162 11.639 35.107 Mean+2SD 543.552 531.770 394.924 239.878 397.714 Corresponding antigen concentration pg / mL 5.541 5.421 4.018 2.448 4.050

[0171] 12 kinds of TBS-T buffers were selected for accelerated stability analysis of the kit, and the kit was placed at 37°C for 7 days before testing, and compared with the control group that was not heated. Among them, the 12 kinds of TBS-T buffers were all 0.1M phosphate buffer system, pH 7.4, supplemented with 1% Tween 20, 0.1% Proclin 300, and supplemented with 0%, 0.1%, 0.5%, 1% BSA, and 0g / L, 20g / L, 40g / L trehalose. The test results are shown in Table 11 below.

[0172] Table 11

[0173]

[0174] Example 7 Optimization of on-machine testing program

[0175] (1) Antibody coating: refer to Example 6, wherein the TBS-T buffer is: 0.1 M phosphate buffer system, pH 7.4, supplemented with 0.5% BSA, 1% Tween-20, 40 g / L trehalose and 0.1% Proclin300.

[0176] (2) Antibody labeling: refer to Example 6, wherein the TBS-T buffer is: 0.1 M phosphate buffer system, pH 7.4, supplemented with 0.5% BSA, 1% Tween-20, 40 g / L trehalose and 0.1% Proclin300.

[0177] (3) Antigen tracing: refer to Example 6.

[0178] (4) On-machine detection: The machine automatically aspirates 50 μL, 100 μL, and 150 μL of the sample and mixes it thoroughly with 100 μL of magnetic bead working solution and 100 μL of labeled antibody working solution, and then incubates at 37°C for 10 min, 15 min, 20 min, and 40 min before magnetic separation. After discarding the supernatant, add the immunoassay instrument's supporting wash solution (PBS and Tween-20) and repeat the washing three times. The reaction is then sent to a darkroom, and 100 μL of the immunoassay instrument's supporting luminescence excitation solution A (nitric acid and hydrogen peroxide aqueous solution) and luminescence excitation solution B (sodium hydroxide aqueous solution) are added to perform a luminescence reaction, and the luminescence value is recorded. The corresponding sample concentration value (pg / mL) is calculated in combination with the entered and calibrated calculation curve.

[0179] The above different computer procedures were performed on 10 cases The G1200 platform traces the cerebrospinal fluid sample test and analyzes the deviation between the test results and the assigned values. The test results are shown in Tables 12 to 14 below:

[0180] Table 12

[0181]

[0182] Table 13

[0183]

[0184] Table 14

[0185]

[0186]

[0187] Example 8 Kit Performance Evaluation

[0188] (1) Accuracy

[0189] The traced Tau antigen was prepared into a 800 pg / mL solution, and the test was repeated 3 times and the data was analyzed. The results of the Tau kit detection accuracy are shown in Table 15 below (concentration unit is pg / mL).

[0190] Table 15

[0191] Kit batch Test result 1 Test result 2 Test result 3 Mean Indicated value deviation 20221001 805.657 819.301 858.511 827.823 800 3.48％ 20221002 828.331 820.399 839.22 829.317 800 3.66％ 20221003 801.535 806.473 779.436 795.815 800 -0.52％

[0192] In summary, the relative deviation of the measurement results is within the range of ±10%, and all three results meet the requirements. The accuracy of the above three batches of test kits is qualified.

[0193] (2) Precision

[0194] According to the EP5-A2 protocol of the National Committee for Clinical Laboratory Standards (NCCLS / CLSI), the intra-batch precision and inter-laboratory precision were tested using a multi-factor integrated nested design. Different operators, different equipment, and different locations were tested once in the morning and afternoon each day. Each sample was tested twice for 20 consecutive days. A total of 160 data results were collected for each batch of test kits to calculate their precision. At the same time, the traced Tau-ABP antigen was configured into 200pg / mL and 1500pg / mL solutions, and the test was repeated 3 times and the data was analyzed. The test results are shown in Table 16 below:

[0195] Table 16

[0196]

[0197] In summary, the results of repeated testing of low and high concentration samples by the above-mentioned kit showed that the intra-batch precision and inter-batch precision CV were both less than 5%, indicating that the above-mentioned kit met the requirements for precision performance evaluation.

[0198] (3) Linear range

[0199] One sample of cerebrospinal fluid supplemented with Tau-ABP antigen was selected, and 13 concentration levels were diluted with diluent. Each diluted sample was tested 3 times, and the mean was calculated. According to the results, the concentration points were gradually reduced to calculate the corresponding linear relationship, and the widest linear range of the kit was determined. The Tau kit test results are shown in Table 17 below (concentration units are pg / mL).

[0200] Table 17

[0201]

[0202] Table 17 above shows that the correlation coefficient r of the Tau kit is not less than 0.9900 in the range of [10-4000] pg / mL and there is no outlier. Therefore, the linear range of the Tau kit is [10-4000] pg / mL.

[0203] (4) Sensitivity

[0204] The blank sample was tested with the above kit and repeated 20 times to determine the sensitivity. The results of the Tau kit test are shown in Table 18 below (concentration units are pg / mL):

[0205] Table 18

[0206] project parameter Mean 224.9 SD 12.964 Mean+2SD 250.828 Corresponding concentration value 2.549

[0207] The average luminescence value of the blank sample measured by the Tau kit was 224.9. The Mean+2SD value was substituted into the reaction curve, and the sensitivity of the total Tau kit was 2.549 pg / mL.

[0208] (5) Sample test results

[0209] Test 60 samples were assigned values ​​on the G1200 platform to determine the consistency of the kit with the platform products. For the part beyond the linear range of the benchmark reagent, the dilution method was used for testing. The analysis results are as follows Figure 1 As shown (concentration unit is pg / mL), regression analysis data showed that y = 0.9788x + 79.612, and R 2 =0.9106, which proves that this product has good consistency with the commercial product.

[0210] It can be seen from the above embodiments that the Tau kit of the present application has good stability, high accuracy, high precision, and high sensitivity. The linear range of the kit is [10-4000] pg / mL, which is much wider than the linear range of the prior art. The upper limit of detection of the kit is more than 8 times that of the prior art. At the same time, the kit of the present application has good consistency with commercial products.

[0211] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A kit for detecting Tau protein, in, The kit comprises: Magnetic beads coated with a primary antibody targeting Tau protein; and A secondary antibody targeting Tau protein labeled with a chemiluminescent agent; Wherein, the magnetic beads are tosyl-activated magnetic beads or carboxyl magnetic beads; The first antibody targeting Tau protein comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1; The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 3; The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 5; The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 7, The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 9; The amino acid sequence of the CDR-L3 is shown in SEQ ID No: 11; The second antibody targeting Tau protein comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 2; The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 4; The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 6; The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 8; The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 10; The amino acid sequence of the CDR-L3 is shown in SEQ ID No:

12.

2. The kit according to claim 1, in, The mass ratio of the first antibody targeting Tau protein to the magnetic beads is 1:(20-60).

3. The kit according to claim 1, in, The mass ratio of the first antibody targeting Tau protein to the magnetic beads is 1:(25~50).

4. The kit according to claim 1, wherein the molar ratio of the second antibody targeting Tau protein to the chemiluminescent agent is 1:(6-50).

5. The kit according to claim 1, in, The molar ratio of the second antibody targeting Tau protein to the chemiluminescent agent is 1:(10-30).

6. The kit according to claim 1, in, The kit also includes Tau protein standards, substrates, coating buffer, marker buffer, washing solution, blocking solution and sample diluent.

7. The kit according to claim 6, in, The coating buffer and the marker buffer are TBS-T buffer, and the TBS-T buffer contains Tween-20 and BSA.

8. The kit according to claim 7, in, The TBS-T buffer also contains trehalose.

9. The kit according to claim 7, in, The TBS-T buffer contains 0.7% to 1.2% Tween-20 and 0.5% to 1% BSA.

10. The kit according to claim 7, in, The TBS-T buffer further comprises 30-50 g / L trehalose.

11. The kit according to any one of claims 1 to 10, in, The first antibody targeting Tau protein comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 15, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 15; The second antibody targeting Tau protein comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No: 14; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 16, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No:

16.

12. The kit according to any one of claims 1 to 10, in, It is used to diagnose Alzheimer's disease.

13. The kit according to any one of claims 1 to 10, in, When the kit is used to detect Tau protein in a sample, The test sample, magnetic beads coated with a first antibody targeting Tau protein, and a second antibody targeting Tau protein labeled with a chemiluminescent agent are mixed, and magnetic separation is performed after incubation; After washing, the substrate is added to measure the luminescence value of the sample being tested; The luminescence value of the Tau protein standard was used to calculate the content of Tau protein in the tested sample.

14. The kit according to claim 13, in, The sample to be tested is cerebrospinal fluid.

15. The kit according to claim 13, in, The sample to be tested is at least 50 μL.

16. The kit according to claim 13, in, The sample to be tested is at least 100 μL.

17. The kit according to claim 13, in, For every microliter of sample tested, 0.005~0.015μg of the first antibody targeting Tau protein and 0.05~0.15ng of the second antibody targeting Tau protein are required.

18. The kit according to claim 13, in, The incubation time was at least 15 min.

19. Use of the kit according to any one of claims 1 to 18 in preparing a product for detecting Tau protein and / or for diagnosing Alzheimer's disease.

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