Tau protein 289 phosphorylation site-specific binding protein, alzheimer's disease diagnostic kit and preparation method and application thereof

Abstract

This application belongs to the field of immunoassay technology, specifically relating to a Tau protein 289 phosphorylation site-specific binding protein, an Alzheimer's disease diagnostic kit, its preparation method, and its application. This application provides a Tau protein 289 phosphorylation site-specific binding protein with specific CDRs. This specific binding protein specifically recognizes the p-tau-289 site, exhibiting a significant abundance change in the early stages of AD. Compared to p-tau-181 and p-tau-217, it shows higher specificity in differentiating Alzheimer's disease from non-AD neurodegenerative diseases, enabling accurate identification in the preclinical stage and providing a crucial time window for ultra-early intervention.

Description

Technical Field

[0001] This application belongs to the field of immunoassay technology, specifically relating to a Tau protein 289 phosphorylation site specific binding protein, an Alzheimer's disease diagnostic kit, its preparation method and application. Background Technology

[0002] Early diagnosis of Alzheimer's disease (AD) is of great significance for disease intervention and treatment. Currently, phosphorylated tau protein (p-tau) biomarkers are considered as major candidate indicators for clinical applications. Related research mainly focuses on sites such as p-tau-217, p-tau-181, p-tau-205, and p-tau-231, and these biomarkers play a certain role in the diagnosis of AD.

[0003] Existing p-tau biomarkers are mostly associated with late-stage Alzheimer's disease (AD), limiting their ability to diagnose early AD and differentiate it from other dementias. These biomarkers lack sufficient sensitivity and specificity in the preclinical stage, hindering ultra-early identification and accurate diagnosis, thus limiting their value in early intervention. Furthermore, the differential diagnosis between different neurodegenerative diseases remains a challenge.

[0004] Therefore, it is necessary to conduct further research on new phosphorylation sites in Alzheimer's disease to provide new diagnostic methods for Alzheimer's disease, thereby achieving accurate identification in the preclinical stage and providing a critical time window for ultra-early intervention. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a Tau protein 289 phosphorylation site specific binding protein, an Alzheimer's disease diagnostic kit, its preparation method and application.

[0006] This application provides a Tau protein 289 phosphorylation site specific binding protein, wherein the Tau protein 289 phosphorylation site specific binding protein has a Tau protein 289 phosphorylation site binding domain.

[0007] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.15 to SEQ ID NO.17.

[0008] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.4, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.15 to SEQ ID NO.17.

[0009] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.5 to SEQ ID NO.6, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.16, SEQ ID NO.18 to SEQ ID NO.19.

[0010] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.7 to SEQ ID NO.9, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.20 to SEQ ID NO.22.

[0011] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.10 to SEQ ID NO.12, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.23 to SEQ ID NO.25.

[0012] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.10, SEQ ID NO.13 to SEQ ID NO.14, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.16, SEQ ID NO.26 to SEQ ID NO.27.

[0013] In some embodiments, the heavy chain variable region is shown in SEQ ID NO.28, and the light chain variable region is shown in SEQ ID NO.29.

[0014] In some embodiments, the heavy chain variable region is shown in SEQ ID NO.30, and the light chain variable region is shown in SEQ ID NO.31.

[0015] In some embodiments, the heavy chain variable region is shown in SEQ ID NO.32, and the light chain variable region is shown in SEQ ID NO.33.

[0016] In some embodiments, the heavy chain variable region is shown in SEQ ID NO.34, and the light chain variable region is shown in SEQ ID NO.35.

[0017] In some embodiments, the heavy chain variable region is shown in SEQ ID NO.36, and the light chain variable region is shown in SEQ ID NO.37.

[0018] In some embodiments, the heavy chain variable region is shown in SEQ ID NO.38, and the light chain variable region is shown in SEQ ID NO.39.

[0019] In some embodiments, the Tau protein 289 phosphorylation site-specific binding protein further includes a heavy chain constant region and a light chain constant region.

[0020] In some embodiments, the Tau protein 289 phosphorylation site-specific binding protein has a sequence of any one of the constant regions of IgG, IgA, IgM, IgE, and IgD.

[0021] In some embodiments, the heavy chain constant region and the light chain constant region are selected from any one of the following species: human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

[0022] This application, in another respect, provides a nucleic acid molecule encoding a protein that specifically binds to the Tau protein 289 phosphorylation site.

[0023] This application also provides a carrier comprising the aforementioned nucleic acid molecule.

[0024] This application also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned vector.

[0025] This application also provides a method for preparing the cells, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

[0026] This application also provides a method for preparing the Tau protein 289 phosphorylation site-specific binding protein, the preparation method comprising the following steps:

[0027] The cells were cultured; and a Tau protein 289 phosphorylation site-specific binding protein was isolated from the resulting culture.

[0028] Another aspect of this application provides an Alzheimer's disease diagnostic kit, which includes the Tau protein 289 phosphorylation site-specific binding protein.

[0029] This application also provides a method for detecting the Tau protein 289 phosphorylation site in a sample to be tested. The method uses the Tau protein 289 phosphorylation site-specific binding protein as the detection antibody and determines the presence of the Tau protein 289 phosphorylation site in the sample to be tested through an immune binding reaction.

[0030] This application provides a Tau protein 289 phosphorylation site-specific binding protein with specific CDRs. This specific binding protein specifically recognizes the p-tau-289 site, exhibiting a significant abundance change in the early stages of Alzheimer's disease (AD). Compared with p-tau-181 and p-tau-217, it demonstrates higher specificity in differentiating Alzheimer's disease from non-AD neurodegenerative diseases, enabling precise identification in the preclinical stage and providing a crucial time window for ultra-early intervention. Furthermore, this specific binding protein is prepared using a Single B cell monoclonal antibody development platform, possessing advantages such as high affinity, strong specificity, and short development time. It exhibits excellent diagnostic performance in multiple detection platforms, including chemiluminescence, electrochemiluminescence, flow cytometry, and single-molecule platforms. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The abundance of p-tau-289 at different stages of AD;

[0033] Figure 2 The principle and schematic diagram of Sulfo-SMCC coupling;

[0034] Figure 3 Map of recombinant plasmids into which the target gene is inserted;

[0035] Figure 4 This is the result of the purity test;

[0036] Figure 5 These are the results of Western blot analysis of serum.

[0037] Figure 6Receiver operating characteristic curves of p-tau-289 antibody prepared for testing positive and negative Alzheimer's disease samples in one embodiment of this application;

[0038] Figure 7 The luminescence value of a p-tau-289 antibody prepared for testing positive and negative Alzheimer's disease samples was obtained from an embodiment of this application.

[0039] Figure 8 Receiver operating characteristic curves of p-tau-289 antibody prepared for testing positive and negative Alzheimer's disease samples in one embodiment of this application;

[0040] Figure 9 The luminescence value of a p-tau-289 antibody prepared for testing positive and negative Alzheimer's disease samples was obtained from an embodiment of this application.

[0041] Figure 10 Receiver operating characteristic curves of p-tau-289 antibody prepared for testing positive and negative Alzheimer's disease samples in one embodiment of this application;

[0042] Figure 11 The percentage of positive particles in exosomes of positive and negative Alzheimer's disease samples was measured using a p-tau-289 antibody prepared in one embodiment of this application.

[0043] Figure 12 Receiver operating characteristic curves (electrochemiluminescence) of p-tau-289 antibody prepared for an embodiment of this application for testing positive and negative Alzheimer's disease samples.

[0044] Figure 13 The p-tau-289 antibody prepared for an embodiment of this application was used to test positive and negative Alzheimer's disease samples (electrochemiluminescence method). Detailed Implementation

[0045] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0046] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0047] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0048] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0049] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0050] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0051] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0052] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0053] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0054] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0055] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0056] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0057] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0058] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0059] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site located within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins including antibodies, and any other modified immunoglobulin molecule, provided that the antibody exhibits the desired biological activity. Antibodies may be any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ, respectively, based on the characteristic of their heavy chain constant structure. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies may be naked or conjugated to other molecules (such as toxins, radioisotopes, etc.).

[0060] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that specifically binds to an antigen. An antigen-binding fragment may contain an antigen recognition site of the complete antibody (e.g., a CDR region (complementarity-determining region) sufficient to specifically bind to an antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0061] The terms “anti-Tau protein 289 phosphorylation site antibody” and “antibody binding to Tau protein 289 phosphorylation site” refer to antibodies that specifically bind to the Tau protein 289 phosphorylation site with sufficient affinity, such that the antibody can be used as a diagnostic and / or therapeutic agent targeting the Tau protein 289 phosphorylation site. As used herein, the terms “specifically bind,” “immunely specifically bind,” “immunely specifically recognize,” and “specifically recognize” are similar terms in the context of an antibody or its antigen-binding fragment. These terms indicate that the antibody or its antigen-binding fragment binds to the epitope via its antigen-binding domain, and that binding requires a certain complementarity between the antigen-binding domain and the epitope.

[0062] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which differ in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called CDRs, while more conserved regions within a variable domain are called FR regions (Framework regions). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region is a rodent or mouse variable region.

[0063] The term "heavy chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the heavy chain variable region are defined as the V gene, D gene, and J gene.

[0064] The term "light chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the light chain variable region are defined as the V gene and the J gene.

[0065] The term "heavy chain" generally includes one variable region and three constant regions (CH1 / CH2 / CH3).

[0066] The term "light chain" generally includes one variable region and one constant region (CL).

[0067] The term “Kabat numbering” and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. In some respects, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35 (optionally including one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In one specific embodiment, the CDRs of the antibody described herein have been determined according to the Kabat numbering scheme.

[0068] As used herein, the terms “constant region” and “constant domain” are interchangeable and have their common meanings in the art. A constant region is an antibody portion that does not directly participate in antibody-antigen binding but can exhibit a variety of effector functions, such as an antibody portion interacting with an Fc receptor, or, for example, the carboxyl-terminal portion of the light chain and / or heavy chain. Compared to the variable domains of immunoglobulins, the constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence. In some respects, antibody or antigen-binding fragments include constant regions or portions thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0069] As used herein, based on the amino acid sequence of constant structural domains, the term "heavy chain" in relation to antibody use can refer to any different type, for example, α, δ, ε, γ, and μ that produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of the heavy chain is well known in the art. In a specific embodiment, the heavy chain is a human heavy chain. In a specific embodiment, the heavy chain is a rodent or mouse heavy chain.

[0070] As used herein, based on the amino acid sequence of a constant domain, the term "light chain" can refer to any different type, such as κ or λ, in relation to antibody use. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain. In a specific embodiment, the light chain is a rodent or mouse light chain.

[0071] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies or antigen-binding fragments thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions are homologous to sequences in antibodies or antigen-binding fragments thereof derived from another species (usually human) to avoid triggering an immune response in said species.

[0072] The term "human" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment thereof is prepared using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes complete or full-length antibodies and fragments thereof.

[0073] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y can generally be represented by a dissociation constant (K). Affinity can be measured and / or represented in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant.

[0074] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitopes). In some embodiments, the epitope to which the antibody or its antigen-binding fragment specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / atmosphere exchange in combination with mass spectrometry (e.g., liquid chromatography-electrospray ionization mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody / its antigen-binding fragment: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be improved using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymo1 (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt1): 37-60; Bricogne G (1997) Meth Enzymo276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) JBiol Chem 270:1388-1394 and Cunningham BC and Wells JA (1989) Science 244:1081-108.

[0075] If an antibody binds to a given epitope or overlapping epitope of a reference antibody such that it partially blocks the binding of the reference antibody to the epitope, then it can be said that the antibody "competitively inhibits" the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. It can be said that the antibody competitively inhibits the binding of the reference antibody to the given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0076] In this application, the term "sample" refers to a human sample, but animal samples may also be used in the practice of this application. Non-limiting sources of samples used in this application include, for example, solid tissues, biopsies, ascites, aspirates, fluid leachates, blood (including circulating tumor cells), plasma, serum, cerebrospinal fluid, lymph, external sections of skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, tumors, organs, cell cultures, and / or cell culture components.

[0077] The term "chemiluminescence" refers to the phenomenon where an excited-state intermediate is generated during a chemical reaction, and when it returns to its ground state, it releases photons. In immunoassay, chemiluminescent substances are labeled onto antibodies. After the formation of immune complexes, a luminescent substrate is added, and a chemical reaction generates a light signal. The intensity of the signal is proportional to the concentration of the analyte antigen. In this application, chemiluminescence is a technical platform for achieving high-sensitivity detection. Its function is to amplify minute antigen-antibody binding events and convert them into a precisely measurable light signal, thereby enabling the detection of trace amounts of Tau protein 289 phosphorylation sites in clinical samples. The sub-concept of the chemiluminescent substance used in this application is acridine ester, which is a highly efficient chemiluminescent label with advantages such as high luminescence efficiency, low background signal, and rapid reaction. Combined with high-affinity antibodies, it forms the technical basis for the excellent sensitivity of the kit in this application.

[0078] The term “label” as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody in order to produce a “labeled” antibody. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzyme labeling, may catalyze a chemical change in a detectable substrate compound or composition.

[0079] Terms such as “treatment” and “relief” refer to therapeutic measures that can cure, slow down, alleviate, reduce the symptoms of a pathological disease or condition and / or stop its progression. Therefore, those requiring treatment include those already diagnosed with or suspected of having the stated condition. In some embodiments, a subject’s cancer is successfully diagnosed according to the method of this application if the patient exhibits one or more of the following conditions.

[0080] This application utilized single-cell multi-omics sequencing on samples diagnosed using the gold standard pathology method. The results indicated changes in the abundance of p-tau-289 protein at different stages of Alzheimer's disease (AD). HC represents healthy individuals, L represents mild AD, M represents moderate AD, and H represents severe AD. The results are as follows: Figure 1 As shown.

[0081] from Figure 1 Compared with healthy individuals, the proportion of p-tau-289 alterations in patients with different stages of AD is significantly higher in mild AD, suggesting its potential as a diagnostic marker for AD. Therefore, this application develops an antibody targeting p-tau-289.

[0082] The full-length amino acid sequence of the Tau protein (P10636-8) obtained by Uniprot is as follows:

[0083] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKG ADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQ S KCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO. 40).

[0084] This application provides a Tau protein 289 phosphorylation site specific binding protein, wherein the Tau protein 289 phosphorylation site specific binding protein has a Tau protein 289 phosphorylation site binding domain.

[0085] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.15 to SEQ ID NO.17.

[0086] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.4, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.15 to SEQ ID NO.17.

[0087] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.5 to SEQ ID NO.6, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.16, SEQ ID NO.18 to SEQ ID NO.19.

[0088] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.7 to SEQ ID NO.9, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.20 to SEQ ID NO.22.

[0089] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.10 to SEQ ID NO.12, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.23 to SEQ ID NO.25.

[0090] In some embodiments, the Tau protein 289 phosphorylation site binding domain has heavy chain CDR1 to heavy chain CDR3 with amino acid sequences as shown in SEQ ID NO.10, SEQ ID NO.13 to SEQ ID NO.14, and light chain CDR1 to light chain CDR3 with amino acid sequences as shown in SEQ ID NO.16, SEQ ID NO.26 to SEQ ID NO.27.

[0091] The specific sequences are shown in Tables 1 and 2 below:

[0092] Table 1

[0093]

[0094] Table 2

[0095]

[0096] The Tau protein 289 phosphorylation site-specific binding protein of this application may have the aforementioned CDRs or derivative fragments having the aforementioned CDRs. The derivative fragments are formed by replacing amino acids at no more than six sites relative to their corresponding CDRs (“conserved modification” or “conserved substitution”), retaining the biological activity consistent with their corresponding complementarity-determining regions. For example, the derivative fragments may replace one amino acid with another, or one amino acid with multiple (e.g., two) amino acids, at sites 1, 2, 3, 4, 5, or 6 of their corresponding complementarity-determining regions.

[0097] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 3.

[0098] Table 3

[0099]

[0100] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will understand that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.

[0101] In some embodiments, the heavy chain variable region of the Tau protein 289 phosphorylation site-specific binding protein is shown in SEQ ID NO.28, and the light chain variable region is shown in SEQ ID NO.29.

[0102] In some embodiments, the heavy chain variable region of the Tau protein 289 phosphorylation site-specific binding protein is shown in SEQ ID NO.30, and the light chain variable region is shown in SEQ ID NO.31.

[0103] In some embodiments, the heavy chain variable region of the Tau protein 289 phosphorylation site-specific binding protein is shown in SEQ ID NO.32, and the light chain variable region is shown in SEQ ID NO.33.

[0104] In some embodiments, the heavy chain variable region of the Tau protein 289 phosphorylation site-specific binding protein is shown in SEQ ID NO.34, and the light chain variable region is shown in SEQ ID NO.35.

[0105] In some embodiments, the heavy chain variable region of the Tau protein 289 phosphorylation site-specific binding protein is shown in SEQ ID NO.36, and the light chain variable region is shown in SEQ ID NO.37.

[0106] In some embodiments, the heavy chain variable region of the Tau protein 289 phosphorylation site-specific binding protein is shown in SEQ ID NO.38, and the light chain variable region is shown in SEQ ID NO.39.

[0107] The specific amino acid sequences are shown in Table 4 below:

[0108] Table 4

[0109]

[0110]

[0111] In some embodiments, the Tau protein 289 phosphorylation site-specific binding protein further includes a heavy chain constant region and a light chain constant region.

[0112] In some embodiments, the Tau protein 289 phosphorylation site specific binding protein has a sequence of any one of the constant regions of IgG, IgA, IgM, IgE, and IgD.

[0113] The Tau protein 289 phosphorylation site-specific binding protein provided in this application can be an antibody, an antigen-binding fragment of an antibody, or a small modular immunodrug. Further, the binding protein can be a monoclonal antibody, an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, a ScFv fragment, a biclonal antibody, a multispecific antibody, a microantibody, a chelated recombinant antibody, an internal antibody, a nanobody, a binding domain immunoglobulin fusion protein, or a small modular immunodrug.

[0114] In some embodiments, the heavy chain constant region and light chain constant region of the Tau protein 289 phosphorylation site-specific binding protein are selected from any one of the following species: human, mouse, rabbit, sheep, cattle, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck, and goose.

[0115] This application, in another respect, provides a nucleic acid molecule encoding a protein that specifically binds to the Tau protein 289 phosphorylation site.

[0116] This application also provides a vector comprising the aforementioned nucleic acid molecule. This application does not specifically limit the type of recombinant expression vector, which may include, but is not limited to, antibody expression vectors. This application does not specifically limit the antibody expression vector, which may include, but is not limited to, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or combinations thereof.

[0117] This application also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned vector. This application does not specifically limit the type of host cell, including but not limited to CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0118] This application also provides a method for preparing the cells, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

[0119] In one example, the method of introduction is transfection. The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chuetal, 1981, Gene 13:197.

[0120] This application also provides a method for preparing the Tau protein 289 phosphorylation site-specific binding protein, the method comprising the following steps: culturing the cells; and isolating the Tau protein 289 phosphorylation site-specific binding protein from the resulting culture.

[0121] This application also provides an Alzheimer's disease diagnostic kit, the kit comprising the Tau protein 289 phosphorylation site-specific binding protein.

[0122] This application does not specifically limit the type of reagent kit, such as diagnostic reagent, test strip, test plate, or kit. In the Alzheimer's disease diagnostic kit, a protein that specifically binds to the Tau protein 289 phosphorylation site competes with the Tau protein 289 phosphorylation site in the test sample.

[0123] This application also provides a method for detecting the Tau protein 289 phosphorylation site in a sample. The method uses a protein that specifically binds to the Tau protein 289 phosphorylation site as a detection antibody, and determines the presence of the Tau protein 289 phosphorylation site in the sample through an immunobinding reaction. It is understood that the detection method of this application can be for diagnostic purposes or for non-diagnostic purposes. In non-disease diagnosis and treatment scenarios, it can be used for the study of gene polymorphism.

[0124] The term "immunobinding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen (for which the antibody is specific). The strength or affinity of the immunobinding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The properties of the immunobinding between two molecules can be quantified using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex.

[0125] This application relates to methods for immunodetection or determination of target antigens (e.g., Tau protein 289 phosphorylation site), reagents for immunodetection or determination of target antigens (e.g., Tau protein 289 phosphorylation site), methods for immunodetection or determination of cells expressing target antigens (e.g., Tau protein 289 phosphorylation site), and diagnostic agents for diagnosing diseases associated with cells positive for target antigens (e.g., Tau protein 289 phosphorylation site), comprising, as active ingredients, antibodies or antibody fragments that specifically recognize the target antigen (e.g., Tau protein 289 phosphorylation site) and bind to the amino acid sequence or its three-dimensional structure in the extracellular region.

[0126] In this application, the method for detecting or determining the amount of a target antigen (e.g., the phosphorylation site of Tau protein 289) can be any known method. For example, it includes immunoassay or assay methods. Immunoassay or assay methods are methods that use labeled antigens or antibodies to detect or determine the amount of antibody or antigen. Examples of immunoassay or assay methods include radiolabeled antibody immunoassays (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0127] To detect cells expressing peptides, known immunoassay methods can be used, such as immunoprecipitation, fluorescent cell staining, and immunohistochemical staining. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0128] The application does not impose any particular restrictions on the test sample used to detect or determine the target antigen (e.g., the phosphorylation site of Tau protein 289), as long as it has the potential to include cells expressing the target antigen (e.g., the phosphorylation site of Tau protein 289), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0129] Depending on the required detection method, the detection product may also contain reagents for performing antigen-antibody reactions or for detecting the reaction. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragments, or conjugates, and substrates corresponding to the labeled antibodies.

[0130] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0131] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0132] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0133] Example 1

[0134] 1. Peptide design and synthesis:

[0135] The peptide design principles included: ① phosphorylation modification of serine (Ser) at position 289 of the protein; ② peptide length of approximately 10-20 amino acids; ③ the peptide should contain a cysteine ​​residue (Cys) to utilize the specific reaction between thiol groups for coupling with the carrier protein. Observation revealed that the cysteine ​​residue at position 289 was followed by cysteine ​​at position 291, requiring no additional addition; therefore, peptide b:LSNVQ(pS)KCGSKD was designed. However, the cysteine ​​residue at position 291 was too close to the serine residue at position 289, which might affect the production of specific antibodies due to steric hindrance after coupling with the carrier. Therefore, peptide a:C-DLSNVQ(pS)KCGSK was designed simultaneously with peptide b. Both peptides were synthesized at 20 mg each by Shanghai Bioengineering Co., Ltd.

[0136] 2. Preparation of immunogens and screening proteins:

[0137] 2.1 Immunogen Design

[0138] Because peptides are haptens and lack immunogenicity, they must be conjugated to a suitable vector to elicit a specific immune response in the body. Therefore, we chose KLH as the vector to conjugate two peptides as immunogens: KLH-peptide 1 and KLH-peptide 2. To ensure the smooth progress of subsequent screening, BSA was also used as the vector to conjugate two peptides: BSA-peptide 1 and BSA-peptide 2.

[0139] 2.2 The SMCC method is selected for coupling. The coupling principle is as follows: Figure 2 :

[0140] ① Coupling of carrier protein and Sulfo-SMCC: Place an appropriate amount of carrier protein in a conical flask and dissolve it in 10 mM PBS buffer (pH 7.4). Weigh the required amount of Sulfo-SMCC according to a molar ratio of carrier protein to Sulfo-SMCC of 1:30, and dissolve it in pure water to a final concentration of 5 mg / mL. Slowly add the completely dissolved Sulfo-SMCC dropwise to the carrier protein solution, mix gently, and incubate at room temperature for 1-2 hours. Then, dialyze overnight to remove excess Sulfo-SMCC. After dialyzing, transfer to a centrifuge tube, bring to a final volume, and calculate the concentration.

[0141] ② Weigh an appropriate amount of peptide according to a carrier protein to peptide mass ratio of 1:1 or 5:4, and dissolve it in pure water to a concentration of 5 mg / ml. After complete dissolution, add the peptide dropwise to the above solution, mix gently at room temperature, and react for 4 hours. After the reaction is complete, the concentration of the conjugate is calculated based on the carrier protein concentration.

[0142] 2.3. Prokaryotic expression of Tau protein:

[0143] The Tau protein gene sequence was obtained from the NCBI database and, after codon optimization, was inserted into the PET-28a vector. Simultaneously, elements such as an antibiotic resistance gene, promoter, terminator, restriction enzyme sites, and a 6XHis purification tag were inserted at appropriate sites. Figure 2 The plasmid was synthesized by a third party (Nanjing Genscript Biotech Co., Ltd.). The plasmid was transformed into BL21(DE3) *E. coli*, and single clones were selected through plate selection with the corresponding antibiotics for expansion culture. After IPTG induction for 6 hours, bacterial cells were collected. Tau protein was purified using a His-tagged protein purification kit (Beyotime Biotechnology, p2226) with a purity ≥90%. The recombinant plasmid map with the inserted target gene is shown below. Figure 3 As shown, purity testing is as follows: Figure 4 As shown.

[0144] The Tau protein gene sequence is as follows:

[0145]

[0146] 3. Animal immunization:

[0147] Healthy adult rabbits were selected as antibody production hosts. For the first immunization, the immunogen was emulsified by mixing the immunogen with Freund's complete adjuvant (Sigma, F5881) at a 1:1 volume ratio. The emulsified antigen (until it did not disperse when dropped into water) was injected subcutaneously into the nape of the neck of the rabbits in small, multiple doses, at a dose of 150 μg per rabbit. Approximately three weeks later, a second immunization was performed, using Freund's incomplete adjuvant (Sigma, F5506) instead of the first immunization. Subsequent immunizations were performed every two weeks, using the same adjuvant, method, and dosage as the second immunization, until an immune plateau was reached (the serum titer no longer increased with each immunization).

[0148] 4. Detection potency:

[0149] Starting from the third immunization, approximately 10 μL of serum was collected per rabbit on the seventh day after the immunization period, and serum titer was detected using ELISA. Based on the titer data, two rabbits (492# and 494#) were selected for Western blotting to verify the binding of antibodies in their serum to native proteins (AD human plasma, AD human brain tissue solution, AD mouse brain tissue solution, and wild-type mouse brain tissue solution, with a total protein content of 50 μg on SDS-PAGE). Figure 5 As shown, rabbit #494 was ultimately selected for B cell enrichment.

[0150] 5. B cell enrichment, screening, and antibody expression:

[0151] Peripheral blood was collected from selected rabbits for B cell enrichment. The enriched B cells were then sorted for antigen specificity to identify those capable of secreting specific antibodies. Individual B cells were lysed to release their nucleic acids, which were then subjected to RT-PCR to reverse the mRNA. Primers were designed based on the genes expressing the antibody's variable heavy chain (VH) and variable light chain (VL) regions. The variable region sequences were amplified by PCR and inserted into a vector plasmid containing the constant region genes to obtain the monoclonal antibody expression plasmid. A suitable expression system was then selected for expression.

[0152] 6. Screening of positive clone supernatant (ELISA):

[0153] Seventy-nine cell supernatants were obtained after transfection and expression. Forward selection using BSA-peptide 1 and BSA-peptide 2, and reverse selection using Tau protein, were performed. Screening method: Each of the three screening antigens was coated with 1 μg / mL of the cell supernatant. 50 μL of the cell supernatant was added to each well, and the cells were incubated at 37°C for 30 min. After washing three times, 50 μL of goat anti-rabbit-HRP (Solepro Biosciences, SE134) diluted 1:5000 was added to each well, and the cells were incubated at 37°C for 30 min. After washing three times, 100 μL of single-component TMB chromogenic buffer was added to each well, and the cells were incubated at 30°C for 10 min. The OD450 was then measured using a microplate reader.

[0154] The results are shown in Table 5. Six cell clones were selected that simultaneously met the following criteria: ① BSA-peptide 1 and BSA-peptide 2 ELISA OD / negative well OD≥3, ② Tau protein ELISA OD value as close as possible to the negative value. These clones are 1M1C1, 1M1B2, 1M1B7, 1M1C8, 1M1B11, and 1M1C11.

[0155] Table 5: Cell supernatant ELISA screening data

[0156]

[0157]

[0158] Example 2

[0159] This embodiment provides the application of the monoclonal antibody provided in this application in chemiluminescence platform detection.

[0160] Chemiluminescence platform:

[0161] The concentration of phosphorylated tau-289 protein (p-tau-289) in the sample was detected using magnetic microparticle chemiluminescence method and double antibody sandwich method, combined with acridinium ester.

[0162] Step 1: The test sample, magnetically microparticle-labeled monoclonal antibody, and acridine ester-labeled antibody are mixed thoroughly to form a solid-phase antibody-antigen-antibody-acidine ester sandwich complex. Unbound acridine ester-labeled antibody and other substances are removed through washing.

[0163] Step 2: An excitation solution and a pre-excitation solution are added to the reaction mixture. The acridine esters on the immune complexes of the magnetic microparticles are excited and emit photons. The luminescence intensity is proportional to the concentration of p-tau-289 in the analyte.

[0164] 2.1. p-tau-289 antibody obtained by coating with magnetic microparticles

[0165] 1) Take 32μL of M300 / Carboxyl 10% magnetic beads (Hangzhou Boyue Biotechnology Co., Ltd., product number M2800C), and discard the liquid after magnetic separation.

[0166] 2) Add 160 μL of 50 mM morpholine ethanesulfonic acid (Suzhou Yake Technology Co., Ltd., catalog number M0006), mix thoroughly, discard the liquid after magnetic separation, repeat 3 times, and finally add 160 μL of MES.

[0167] 3) Weigh EDC (Shanghai Yubo Biotechnology Co., Ltd., catalog number ybC-0026), add morpholine ethanesulfonic acid to dissolve it, so that the concentration reaches 80mg / mL, mix thoroughly, take 25.6μL and add it to the above magnetic beads, mix well for 15min.

[0168] 4) Discard the liquid by magnetic separation, add an equal volume of morpholine ethanesulfonic acid and 16 μg of p-tau-289 antibody, and mix by roller for 2 hours.

[0169] 5) Magnetic separation of the discarded liquid, add 160 μL of morpholine ethanesulfonic acid and 16 μL of 10% BSA, and mix by roller for 1 hour.

[0170] 6) Discard the liquid after magnetic separation, and add 160 μL of magnetic bead diluent. The main components of the magnetic bead diluent are 50 mM tris(hydroxymethyl)aminomethane (Suzhou Yake Technology Co., Ltd., catalog number S0002), 0.9% sodium chloride (Shanghai Test, 10019318), 5% bovine serum albumin (Bio-Lapis, catalog number SNM380), and 0.1% Tween 20 (Sigma, catalog number P7949). Mix thoroughly, discard the liquid after magnetic separation, and repeat the washing process three times.

[0171] 7) Add magnetic bead diluent to dilute the magnetic beads to 0.4 mg / mL.

[0172] 2.2 Antibody-labeled acridine ester

[0173] 1) Take 1 mg of detection antibody (Fangyuan Standard Products Co., Ltd., catalog number KSDM001-06), add acridinium ester (Helison Company, catalog number 11015005) solution, so that the molar ratio of acridinium ester to antibody is 1:10, and mix well in the dark for 30 min.

[0174] 2) Add 1% of the total volume of 1M tris(hydroxymethyl)aminomethane (Suzhou Yake Technology Co., Ltd., item number S0002) and react in the dark for 15 min.

[0175] 3) After the reaction is complete, transfer the reactants to a 30kd ultrafiltration tube, centrifuge at 12000g for 10min, discard the filtrate, and repeat once.

[0176] 4) Invert the reactants in the ultrafiltration tube into a new centrifuge tube and centrifuge at 2000g for 1 min.

[0177] The OD280 and OD370 of the reactants were tested using a nanodrop, and the reactant concentration was calculated using the formula: concentration = (OD280 - 0.17 × OD370) / 1.36.

[0178] 5) Dilute it to 1 μg / mL with the detection antibody dilution solution and store it for later use.

[0179] 2.3 Detection

[0180] The antibodies coated on magnetic beads and the antibodies labeled with acridinium ester were combined to form a detection reagent, and clinical samples were tested using a Cosmetic fully automated chemiluminescence analyzer.

[0181] 2.4 Test Results

[0182] Test results by Figure 6 and Figure 7 It is known that the p-tau-289 antibody provided in this application has a good ability to distinguish between positive and negative samples of Alzheimer's disease, with an AUC of 0.97, and the signal values ​​of positive and negative samples are significantly different.

[0183] Example 3

[0184] This embodiment uses a single-molecule platform: the p-tau-289 antibody provided in this application is coated on magnetic beads and tested in combination with a commercially available tau-terminated antibody labeled with biotin.

[0185] 3.1 Magnetic bead coating

[0186] 1) Sonicate the magnetic bead stock solution for 10 seconds, then vortex for 10 seconds to suspend the magnetic beads evenly. Transfer 100 μL of the magnetic bead stock solution to a coated tube; transfer 0.2 mL of the activation solution (50 mM morpholine ethanesulfonic acid) to the coated tube, sonicate for 10 seconds, vortex for 10 seconds, resuspend the magnetic beads, and pipette the supernatant; repeat the operation twice.

[0187] 1) Activation of magnetic beads: Weigh appropriate amounts of EDC and NHS (Shanghai Yubo Biotechnology Co., Ltd., catalog number ybC-0026). Dissolve the weighed NHS in DMSO (MCE Company, catalog number HY-Y0320C) until the NHS solution concentration is 250 mg / mL; dissolve the weighed EDC in DMSO until the EDC solution concentration is 30 mg / mL; take V1 / 199 (mL) of the dissolved EDC solution and add it to the NHS solution, vortex and mix for 5 seconds to form a mixture of EDC and NHS for later use.

[0188] 2. Vortex mix the cleaned and resuspended magnetic beads for 5 seconds; add 20 μL of the mixture of EDC and NHS to the magnetic bead-coated tube and immediately vortex mix for 10 seconds.

[0189] 3) Activation and incubation: Place the coated tube on a roller mixer and incubate at 25°C for 30 min at 80 rpm. Centrifuge the activated magnetic bead coated tube at low speed for 2 s in a handheld centrifuge, then quickly place it on a magnetic rack and let it stand for 2 min. Use a pipette to remove the supernatant. Add 0.2 mL of coating solution to the magnetic bead coated tube, remove the coated tube from the magnetic rack, sonicate for 10 s, vortex for 10 s, resuspend the magnetic beads, and repeat the washing twice.

[0190] 4) Add 40 μg of capture antibody to the magnetic bead-coated tube and immediately vortex for 5 seconds to mix. Place the coated tube on a roller mixer and incubate at 25°C for 2.5 hours at 80 rpm.

[0191] 5) Magnetic separation of the coupled coated tube; add 0.2 mL PBST to the magnetic bead coated tube, sonicate for 2 s, vortex mix for 10 s, resuspend the magnetic bead, and magnetic separation again; repeat the operation once.

[0192] 6) Blocking after coupling: Add 0.2 mL of blocking solution (10% bovine serum albumin) to the coated tube, sonicate for 2 s, vortex for 10 s, and resuspend; place the coated tube on a roller mixer and incubate at 37°C for 2 h at 80 rpm; magnetically separate the coated tube; repeat the operation once.

[0193] 7) Storage: Separate the sealed magnetic beads magnetically, add 0.2 mL of blocking solution (10% bovine serum albumin), sonicate for 2 seconds, vortex mix for 10 seconds, and resuspend; repeat the operation once; store the coated magnetic beads at 4°C.

[0194] 3.2 Antibody-labeled biotin

[0195] 1) Dilute the antibody to be biotinylated to 1 mg / mL with 0.5 M sodium bicarbonate buffer (pH 8.0) (Maclean's, catalog number S885288); transfer the 1 mg / mL antibody to a 10 kDa ultrafiltration tube, equilibrate with a similar ultrafiltration tube, centrifuge at 13000 g for 20 min at 4 °C, and discard the filtrate; replenish the liquid in the inner tube with sodium bicarbonate buffer, invert the tube into a clean microcentrifuge tube, place it in a centrifuge with the open cap facing the center of the rotor, equilibrate with a similar ultrafiltration tube, and centrifuge at 1000 g for 2 min at 4 °C; transfer the concentrated antibody from the ultrafiltration tube to a collection tube and store at 4 °C for subsequent use.

[0196] 2) Preparation of NHS-Biotin solution: Dissolve 4 mg of biotin succinimide ester (NHS-Biotin) (MCE Company, catalog number HY-D0802) in 1 mL of DMSO (concentration of 4 mg / mL), aliquot and store at -20℃.

[0197] 3) Antibody-Biotin binding: Add 1 mg of antibody solution to 360 μg of NHS-Biotin solution, mix well on a vortex mixer, and then continue mixing at room temperature (rotary mixer) for 4 hours; add 50 μL of 1M ammonium chloride solution (Shanghai Zeye Biotechnology Co., Ltd., catalog number ZY-25-00360), and mix at room temperature (rotary mixer) for 10 min.

[0198] 4) Centrifugation and ultrafiltration: Transfer the biotinylated antibody system to an ultrafiltration tube and centrifuge at 13000g for 20 min at 4°C. Discard the filtrate. Invert the inner tube into a clean microcentrifuge tube and place it in a centrifuge with the open cap facing the center of the rotor. Equilibrate with a similar ultrafiltration tube and centrifuge at 1000g for 2 min at 4°C. Collect the concentrated biotinylated antibody.

[0199] 5) Preservation: Add preservation solution to 1 mL in the collection tube. The main components of the preservation solution are 50 mM tris(hydroxymethyl)aminomethane, 0.9% sodium chloride, 5% bovine serum albumin, and 0.1% Tween-20. Store at 4°C protected from light.

[0200] 3.3 Detection

[0201] The reagent was prepared by combining the antibodies coated on magnetic beads and the biotin-labeled antibodies with signal amplification components purchased from Yuce Biotechnology, and then using a single-molecule detection instrument to test clinical samples.

[0202] Test results by Figure 8 and Figure 9 It is evident that the p-tau-289 antibody provided in this application has excellent ability to distinguish between positive and negative Alzheimer's disease samples, with an AUC of 0.96, and the signal values ​​of positive and negative samples show significant differences.

[0203] Example 4

[0204] This embodiment uses a flow cytometry platform to extract exosomes from clinical samples and detect them using the p-tau-289 antibody provided in this application.

[0205] 4.1 Sample Processing

[0206] 1) Centrifuge the sample at 18000×g for 30 min at 4℃ using a high-speed centrifuge and collect the supernatant.

[0207] 4.2 Exosome Extraction

[0208] 1) Take 10 μL of plasma, add 70 μL of PBS (filtered through a 0.22 μm membrane) and 20 μL of 40% PEG8000, mix thoroughly by pipetting 10 times, let stand at room temperature for 60 min, centrifuge at 18000g, 4℃ for 60 min. Discard the supernatant, add 100 μL of PBS (filtered through a 0.22 μm membrane) to resuspend, mix thoroughly by pipetting 100 times, aliquot into 10 μL / vial, and store at -80℃ for later use.

[0209] 4.3 Antibody labeling

[0210] 1) EV blocking: Take 10 μL of EV, add 30 μL of 2% BSA, mix by pipetting 5 times, block at 26℃ for 1 h, and then add 10-30 μL of PBS to stop the blocking.

[0211] 2) Add 15 μg of p-tau-289 antibody labeled with fluorescent dye (Thermo Scientific, catalog number Z25308) and mix thoroughly by pipetting 5 times. When preparing the antibody, label the p-tau-289 antibody (AF488) provided in this application with the fluorescent dye. Incubate at 4°C overnight (not less than 16 h).

[0212] 4.4 Probe Detection

[0213] 1) Add 30 μL of lipid probe to the sample, mix by pipetting 5 times, and incubate at 4°C in the dark for 60 min.

[0214] 2) Add 400 μL of 4% PFA (Waltman, catalog number 161-20141), mix thoroughly by pipetting 3 times, and fix at 26°C (room temperature) for 60 min.

[0215] 3) Add 200μL PBS and mix by pipetting 3 times. Dilute and load onto the instrument. Count 50,000 particles under the fixed PE gate and collect at a low speed (control the number of particles per second to below 10,000, 2-3,000 / second is optimal).

[0216] 4) Statistical analysis of antibody labeling rates under the PE category.

[0217] The results are as follows Figures 10-11 As shown, by Figure 10 and Figure 11 It is known that the p-tau-289 antibody provided in this application has a good ability to distinguish exosomes extracted from positive and negative samples of Alzheimer's disease, with an AUC of 0.96, and the signal values ​​of positive and negative samples are significantly different.

[0218] Example 5

[0219] The concentration of phosphorylated tau-289 protein (p-tau-289) in the sample was detected using magnetic microparticle chemiluminescence method, double antibody sandwich method, combined with ruthenium tripyridine.

[0220] Step 1: The test sample, magnetically microparticle-labeled monoclonal antibody, and acridine ester-labeled antibody are mixed thoroughly to form a solid-phase antibody-antigen-antibody-acidine ester sandwich complex. Unbound acridine ester-labeled antibody and other substances are removed through washing.

[0221] Step 2: Add excitation solution and pre-excitation solution to the reaction mixture. The acridinium ester on the immune complex on the surface of the magnetic microparticles emits photons after excitation. The luminescence intensity is proportional to the concentration of p-tau-289 in the analyte.

[0222] 5.1 p-tau-289 antibody obtained by coating with magnetic microparticles

[0223] 1) Take 32μL of M300 / Carboxyl 10% magnetic beads, and discard the liquid after magnetic separation.

[0224] 2) Add 160 μL of 50 mM morpholine ethanesulfonic acid, mix thoroughly, discard the liquid after magnetic separation, repeat 3 times, and finally add 160 μL of MES.

[0225] 3) Weigh EDC, add morpholine ethanesulfonic acid to dissolve it, so that the concentration reaches 80 mg / mL, mix thoroughly, take 25.6 μL and add it to the above magnetic beads, mix well for 15 min.

[0226] 4) Discard the liquid by magnetic separation, add an equal volume of morpholine ethanesulfonic acid and 16 μg of p-tau-289 antibody, and mix by roller for 2 hours.

[0227] 5) After magnetic separation, discard the liquid, add 160 μL of morpholine ethanesulfonic acid and 16 μL of 2% CE510, and mix thoroughly by roller for 1 hour.

[0228] 6) Discard the liquid after magnetic separation, and add 160 μL of magnetic bead diluent. The main components of the magnetic bead diluent are 50 mM tris(hydroxymethyl)aminomethane, 0.9% sodium chloride, 5% bovine serum albumin, and 0.1% Tween 20. Mix thoroughly, discard the liquid after magnetic separation, and repeat the washing process three times.

[0229] 7) Add magnetic bead diluent to dilute the magnetic beads to 0.4 mg / mL.

[0230] 5.2 Antibody-labeled triple pyridine ruthenium

[0231] 1) Dilute ruthenium tripyridine (Shenzhen Jinsaitu Biotechnology Co., Ltd., catalog number 161698-59-5) with DMF to 4 mg / mL.

[0232] 2) Take 1 mg of detection antibody and add ruthenium tripyridine solution to make the molar ratio of ruthenium tripyridine to antibody 1:10. Mix well in the dark for 30 min.

[0233] 3) Add 1% of the total volume of 1M tris(hydroxymethyl)aminomethane and react in the dark for 15 min.

[0234] 4) After the reaction is complete, transfer the reactants to a 30kd ultrafiltration tube, centrifuge at 12000g for 10min, discard the filtrate, and repeat once.

[0235] 5) Invert the reactants in the ultrafiltration tube into a new centrifuge tube and centrifuge at 2000g for 1 min.

[0236] 6) Dilute it to 2 μg / mL with the detection antibody diluent and store it for later use.

[0237] 5.3 Detection

[0238] The antibodies coated on magnetic beads and the antibodies labeled with ruthenium tripyridine were combined to form a detection reagent, and clinical samples were tested using a Pumen automated electrochemiluminescence analyzer.

[0239] Test results by Figure 12 and Figure 13 It is known that the p-tau-289 antibody provided in this application has a good ability to distinguish between positive and negative samples of Alzheimer's disease, with an AUC of 0.97, and the signal values ​​of positive and negative samples are significantly different.

[0240] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A monoclonal antibody that specifically binds to a p-tau-289 phosphorylated protein, characterized in that, The Tau protein gene sequence is shown in SEQ ID NO.40, the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.38, and the light chain variable region is shown in SEQ ID NO.

39.

2. The monoclonal antibody that specifically binds to p-tau-289 phosphorylated protein according to claim 1, characterized in that, The monoclonal antibody also includes a heavy chain constant region and a light chain constant region.

3. The monoclonal antibody that specifically binds to p-tau-289 phosphorylated protein according to claim 2, characterized in that, The monoclonal antibody satisfies one or more of the following conditions (1) and (2): (1) The monoclonal antibody has a sequence comprising a constant region of any one of IgG, IgA, IgM, IgE, and IgD; and, (2) The heavy chain constant region and the light chain constant region are selected from any one of the following species: human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

4. A nucleic acid molecule, characterized in that, Encoding the monoclonal antibody as described in any one of claims 1 to 3.

5. A carrier, characterized in that, Includes the nucleic acid molecule as described in claim 4.

6. A cell, characterized in that, It includes the nucleic acid molecule as described in claim 4 or the vector as described in claim 5.

7. A method for preparing the cell according to claim 6, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

8. A method for preparing a monoclonal antibody that specifically binds to p-tau-289 phosphorylated protein according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: Cultivating the cells according to claim 6; and Monoclonal antibodies that specifically bind to p-tau-289 phosphorylated protein were isolated from the resulting cultures.

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