Tau recombinant antigen, recombinant antibody targeting Tau protein and kit for detecting Tau protein

Abstract

The present application discloses a recombinant antibody targeting Tau protein, a nucleic acid molecule encoding the recombinant antibody, a nucleic acid molecule encoding the recombinant antibody, a recombinant antigen of Tau protein, a nucleic acid molecule encoding the recombinant antigen, the use of the recombinant antigen or nucleic acid molecule in obtaining the recombinant antibody, a kit for detecting Tau protein, and the use of the recombinant antibody or nucleic acid molecule in preparing a kit for diagnosing Alzheimer's disease. The recombinant antibody of the present application has high affinity, high sensitivity, and significantly shortened production cycle, and is more suitable as a core raw material for application in the field of in vitro diagnostic reagents.

Description

Technical Field

[0001] The present application belongs to the field of immunoassay technology, and relates to a recombinant antibody targeting Tau protein, a nucleic acid molecule encoding the recombinant antibody, a recombinant antigen of Tau protein, a nucleic acid molecule encoding the recombinant antigen, a kit for detecting Tau protein, and uses thereof. 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 current Tau antibody preparation technology has the following two main disadvantages:

[0006] (1) Insufficient antibody sensitivity

[0007] In the prior art, the affinity of the Tau mouse monoclonal antibody of Xiao Jian et al. is only 280nM (Western Blot determination), the affinity of the Tau humanized antibody invented by M. Novak et al. is only 10nM (BIAcore determination), and the affinity of the Tau recombinant antibody invented by Paul H. Weinreb et al. is 2.4-6.6nM (Elisa determination). Data from other similar inventions show that the sensitivity of their Tau antibodies is 0.1-1000nM. Insufficient antibody sensitivity will directly affect the sensitivity of the detection reagent, while reducing the specificity and anti-interference of the detection, and ultimately affecting the reliability of the detection results.

[0008] (2) Traditional monoclonal preparation technology has inherent deficiencies

[0009] During the research and development and production of in vitro diagnostic reagents, the batch-to-batch reproducibility of the products must be controlled. As one of the core raw materials, the performance difference between batches of antibodies will be directly reflected in the batch-to-batch reproducibility of the test kit. During the preparation of mouse monoclonal antibodies, the antibody titer is easily affected by the individual differences of animals. At the same time, the volume of mouse ascites is generally less than 5mL. Although the monoclonal antibodies obtained in this way may have stronger affinity, it is difficult to prepare large quantities of antibodies with stable batch-to-batch performance through traditional monoclonal antibody preparation processes. Summary of the invention

[0010] 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 the treatment effect are limited. In order to overcome the deficiencies in the prior art, the present application provides a recombinant antibody targeting Tau protein, a recombinant antigen for obtaining the recombinant antibody, and a nucleic acid molecule encoding the recombinant antigen. The recombinant antibody of the present application has higher sensitivity and a significantly shortened production cycle, and the nucleic acid molecule encoding the recombinant antigen of the present application can significantly increase the expression level of the recombinant antibody of the present application.

[0011] The specific technical solutions of this application are as follows:

[0012] 1. A recombinant antibody targeting Tau protein, wherein the recombinant antibody 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:

[0013] The amino acid sequence of CDR-H1 is shown in SEQ ID No: 1 or SEQ ID No: 2;

[0014] The amino acid sequence of CDR-H2 is shown in SEQ ID No: 3 or SEQ ID No: 4;

[0015] The amino acid sequence of CDR-H3 is shown in SEQ ID No: 5 or SEQ ID No: 6;

[0016] The amino acid sequence of CDR-L1 is shown in SEQ ID No: 7 or SEQ ID No: 8;

[0017] The amino acid sequence of CDR-L2 is shown in SEQ ID No: 9 or SEQ ID No: 10;

[0018] The amino acid sequence of CDR-L3 is shown in SEQ ID No:11 or SEQ ID No:12.

[0019] 2. The recombinant antibody according to item 1, wherein

[0020] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1;

[0021] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 3;

[0022] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 5;

[0023] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 7,

[0024] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 9;

[0025] The amino acid sequence of the CDR-L3 is shown in SEQ ID No:11.

[0026] 3. The recombinant antibody according to item 1, wherein

[0027] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 2;

[0028] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 4;

[0029] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 6;

[0030] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 8;

[0031] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 10;

[0032] The amino acid sequence of the CDR-L3 is shown in SEQ ID No:12.

[0033] 4. The recombinant antibody according to any one of items 1 to 3, wherein the recombinant antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0034] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13 or SEQ ID No: 14, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13 or SEQ ID No: 14;

[0035] The amino acid sequence of the light chain variable region is as shown in SEQ ID No:15 or SEQ ID No:16, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:15 or SEQ ID No:16.

[0036] 5. The recombinant antibody according to item 4, wherein

[0037] 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;

[0038] The amino acid sequence of the light chain variable region is as shown in SEQ ID No:15, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:15.

[0039] 6. The recombinant antibody according to item 4, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 14;

[0040] The amino acid sequence of the light chain variable region is as shown in SEQ ID No:16, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:16.

[0041] 7. A nucleic acid molecule encoding the recombinant antibody according to any one of items 1 to 6.

[0042] 8. A recombinant Tau protein antigen, wherein the amino acid sequence of the recombinant antigen is shown in SEQ ID No: 17.

[0043] 9. A nucleic acid molecule encoding the Tau protein recombinant antigen as described in item 8, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No: 18.

[0044] 10. Use of the recombinant Tau protein antigen according to item 8 or the nucleic acid molecule according to item 9 in obtaining the recombinant antibody according to any one of items 1 to 6.

[0045] 11. A kit for detecting Tau protein, wherein the kit comprises:

[0046] Magnetic beads coated with a primary antibody targeting Tau protein; and

[0047] A secondary antibody targeting Tau protein labeled with a chemiluminescent agent;

[0048] The first antibody and the second antibody targeting the Tau protein are the recombinant antibodies described in any one of Items 1 to 6.

[0049] 12. The kit according to item 11, wherein it is used for diagnosing Alzheimer's disease.

[0050] 13. Use of the recombinant antibody according to any one of items 1 to 6, or the nucleic acid molecule according to item 7, in the preparation of a kit for diagnosing Alzheimer's disease.

[0051] Effects of the Invention

[0052] The present application provides a new recombinant antibody targeting Tau protein, a nucleic acid molecule encoding a recombinant antigen for obtaining the antibody, and a kit containing the recombinant antibody. The nucleic acid molecule encoding the recombinant antigen of the present application can significantly increase the expression level of the recombinant antibody of the present application. The total Tau recombinant antibody of the present application obtained by using the recombinant antigen of the present application as an immunogen has excellent and stable performance, high affinity, and high sensitivity. At the same time, the production cycle of the total Tau recombinant antibody of the present application is significantly shortened, and it is more suitable for use as a core raw material in the field of in vitro diagnostic reagents, which can bring significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] 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

[0054] 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.

[0055] 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.

[0056] 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.

[0057] In some embodiments, the Tau protein of the present application is the full-length Tau protein 2N4R / Tau-441.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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 ways to define 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] In the first aspect, the present application provides a recombinant antibody targeting Tau protein, which 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 CDR-H1 is as shown in SEQ ID No: 1 or SEQ ID No: 2; the amino acid sequence of CDR-H2 is as shown in SEQ ID No: 3 or SEQ ID No: 4; the amino acid sequence of CDR-H3 is as shown in SEQ ID No: 5 or SEQ ID No: 6; the amino acid sequence of CDR-L1 is as shown in SEQ ID No: 7 or SEQ ID No: 8; the amino acid sequence of CDR-L2 is as shown in SEQ ID No: 9 or SEQ ID No: 10; the amino acid sequence of CDR-L3 is as shown in SEQ ID No: 11 or SEQ ID No: 12.

[0076] In some embodiments, 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).

[0077] In some embodiments, 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).

[0078] In some embodiments, the recombinant antibody 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 SEQ ID No: 14, 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 or SEQ ID No: 14; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 15 or SEQ ID No: 16, 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 or SEQ ID No: 14. No:16 has an amino acid sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

[0079] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13 (EVMLQESGGGLVQPGSSLRLTCATSGFTFTDYYMSWVRQPPGKAL EWLGVIRNKANGYTTEYSASVKGRFTISRDNTQGILYLHMNTLSAEDSAT YYCAREEDYRHGFPYWGQGTLVTVSA), 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: 13; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 15 (DIIMTQSPSSLSVSVGEKVTMNCNSSQSLLNTGNQKNYLAWYQQK PGQTPKLLIHGALTRESGIPDRFTGRGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGTGTKLELRRAD), 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.

[0080] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14 (EVKLIESGGGLMQPGSSLRLSCASSGFVFIDYYMSWVRQPPGKALE WLGLVRNKANGYRTEYSSTVKGRFTISRDNSQGILYLHMNTLSAEDSATY YCAREGSYPYGFPDWGQGTLVTVSA), 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: 14; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 16 (DIVMTQSPSSLSASVGERVTMNCKSSQSLLHSGNQKNYLAWYQQK 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID No:16.

[0081] In a second aspect, the present application provides a nucleic acid molecule encoding any of the aforementioned recombinant antibodies.

[0082] In a third aspect, the present application provides a Tau protein recombinant antigen, whose amino acid sequence is shown in SEQ ID No: 17 (MHHHHHHLAEAKVLANRELDKYGVSDYHKNLINNAKTVEGVKD ).

[0083] In some embodiments, the preparation method of the recombinant antigen of the present application is: confirm the ABP tag amino acid sequence (UniPort, P19909) and Tau-F amino acid sequence (UniPort, P10636-8) (2N4R / Tau-441 form) with the help of literature and database, and construct them into pET30a vector in series after codon optimization. After the vector is sequenced correctly, it is transformed into Escherichia coli BL21 (DE3) competent state and the recombinant antigen is prepared and purified. The recombinant antigen is purified by Ni-NTA Agarose (QIAGEN, Cat. No. 30210).

[0084] Albumin Binding Protein (ABP) derived from Streptococcus protein G is a protein of about 15 kDa, which can be used as a folding tool in protein engineering. Existing Tau recombinant antigens are often prepared by E. coli expression systems. This application can obtain antigens with stronger stability, immunogenicity and solubility with the help of ABP tags, and thus can obtain antibodies with higher affinity.

[0085]

[0086] In a fifth aspect, the present application also provides the use of the Tau protein recombinant antigen described in the third aspect or the nucleic acid molecule encoding the Tau protein recombinant antigen described in the fourth aspect in obtaining the recombinant antibody targeting Tau protein of the present application.

[0087] In a sixth aspect, the present application also provides a kit for detecting Tau protein, 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 first antibody and the second antibody targeting Tau protein are respectively the recombinant antibodies described in any one of the aforementioned first aspects.

[0088] In some embodiments, the kit further comprises a sample standard to be tested, a substrate, a coating buffer, a washing solution, a blocking solution, and a sample diluent, wherein the coating buffer provides a high pH environment for magnetic bead coating to improve the coating efficiency; the blocking solution is used to block uncoupled vacancies to prevent non-specific binding in the later stage. In some embodiments, the substrate comprises a luminescent excitation solution A and a luminescent excitation solution B, wherein the luminescent excitation solution A is an aqueous solution of nitric acid and hydrogen peroxide, the luminescent excitation solution B is an aqueous solution of sodium hydroxide, the coating buffer is a TBS-T buffer, the washing solution is PBS and Tween-20; the blocking solution is a BSA blocking solution, and the sample diluent is a PBS buffer.

[0089] 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 marker that is not bound to the antibody. In some embodiments, the storage buffer is TBS-T buffer, and the quenching buffer is 5% DL-lysine.

[0090] The kit of the present application can be used to diagnose Alzheimer's disease.

[0091] In a seventh aspect, the present application also provides a use of any of the aforementioned recombinant antibodies or any of the aforementioned nucleic acid molecules in the preparation of a kit for diagnosing Alzheimer's disease.

[0092] In some embodiments, the kit may be a magnetic particle chemiluminescence kit, a magnetic particle electrochemiluminescence kit, an electrochemiluminescence kit, an ELISA kit, a POCT detection kit, a SERS detection kit, a mass spectrometry kit, a microfluidics kit and a photochemical kit, etc., preferably a magnetic particle chemiluminescence kit.

[0093] Example

[0094] 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.

[0095] Example 1 Preparation of recombinant antigen Tau-ABP

[0096] 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) 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.

[0097] Example 2 Preparation of recombinant antibodies rTau-A and rTau-B

[0098] The preparation method of the recombinant antibody in this embodiment is as follows: mice are immunized with the recombinant antigen Tau-ABP prepared in the embodiment, and the mouse monoclonal antibody cell line combination with the highest sensitivity is obtained, followed by antibody variable region sequence sequencing, and chimeric vector construction is completed according to the variable region sequence 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). Both vectors are commercial vectors with their own constant region sequences and do not require additional processing; after the recombinant antibodies are expressed in the Hek293T cell line, purified recombinant antibodies rTau-A and rTau-B are obtained via Protein A 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.

[0099] Example 3 Performance Verification of Recombinant Antibodies rTau-A and rTau-B

[0100] (1) Determination of recombinant antibody concentration

[0101] 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 antibody. 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.

[0102] Table 1

[0103] Concentration (mg / mL) 20220801 20220802 rTau-A 12.3 13.2 rTau-B 10.9 12.1

[0104] (2) Determination of recombinant antibody concentration

[0105] 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.

[0106] 20220801 batch rTau-A:

[0107] Table 2

[0108]

[0109] 20220802 batch rTau-A:

[0110] Table 3

[0111]

[0112] 20220801 batch rTau-B:

[0113] Table 4

[0114]

[0115] 20220802 batch rTau-B:

[0116] Table 5

[0117]

[0118] 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.

[0119] Table 6

[0120]

[0121] Example 4 Preparation of a kit for detecting Tau protein and detection process

[0122] (1) Preparation of Tau protein antibody coated magnetic beads working solution: Magnetic beads selected were tosyl-activated magnetic beads (ThermoDynabeads TM M-280Tosylactivated, Cat#30110D), the original concentration is 100 mg / mL. Vortex and mix well, then sonicate for 5 minutes and fully resuspend. Use a pipette to draw 100 μL (about 10 mg magnetic beads) of the fully resuspended magnetic bead mother solution, and remove the liquid by magnetic separation for 5 minutes. Add 165 μL of 0.1M borate buffer (pH 9.5) and 200 μg of rTau-A recombinant antibody, mix well and incubate overnight at 37°C. After the incubation, remove the liquid by magnetic separation for 5 minutes. Add 10% BSA and block at 37°C for 3h. After the blocking is completed, remove the liquid by magnetic separation for 5 minutes. 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°C for use. The mass ratio of recombinant antibody to magnetic beads in this embodiment is 1:50. The coated mother solution needs to be diluted 20 times with TBS-T buffer to prepare the working solution.

[0123] (2) Preparation of Tau protein labeled antibody working solution: 0.25 mg of rTau-B recombinant antibody was taken and mixed 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 embodiment, the molar ratio of the recombinant antibody to NSP-SA-NHS was 1:20. The labeled mother solution needs to be diluted 4000 times with TBS-T buffer to prepare the working solution for use.

[0124] (3) Sample traceability: G1200 platform is used according to the instruction manual GTotal Tau (Fujirebio, Catalog No. 231302) was used to trace the origin of Tau antigen.

[0125] (4) Packaging of calibrators and quality controls: Tau calibrators and quality controls are prepared 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.

[0126] (5) Kit assembly: The three batches of coated antibodies and labeled antibodies produced in pilot tests were packaged in the reagent compartment and assembled into a kit together with the calibrators and quality control products. The specific information is shown in Table 7 below:

[0127] Table 7

[0128]

[0129] (6) On-machine testing

[0130] 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.

[0131] 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 performs magnetic separation after incubation at 37°C for 20 minutes. After discarding the supernatant, add the immunoassay instrument's supporting washing solution (PBS and Tween-20), and repeat the washing 3 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, and the results are shown in Table 8 below.

[0132] Table 8

[0133] Antigen concentration pg / mL Mean deviation 5 6.661 33.22％ 10 10.244 2.44％ 20 19.461 -2.70％ 50 51.6 3.20％ 100 98.92 -1.08％ 200 196.768 -1.62％ 500 511.728 2.35％ 1000 958.845 -4.12％ 1500 1528.521 1.90％ 2000 1919.408 -4.03％ 3000 3146.019 4.87％ 4000 3436.819 -14.08％ 5000 3990.926 -20.18％

[0134] It can be seen from Table 8 above that, compared with the kits of the prior art, the kit of the present application has a lower detection limit and high detection sensitivity, and the detection value deviation within the antigen concentration range of 10 to 3000 pg / mL is within ±5%.

[0135] Example 5 Kit Performance Evaluation

[0136] (1) Precision

[0137] 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 9 below:

[0138] Table 9

[0139]

[0140] 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 10%, indicating that the above-mentioned kit meets the requirements for precision performance evaluation.

[0141] (2) Linear range

[0142] One sample of cerebrospinal fluid supplemented with Tau-ABP antigen was selected, and 12 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 10 below (concentration units are pg / mL).

[0143] Table 10

[0144]

[0145]

[0146] Table 10 above shows that the correlation coefficient r of the Tau kit is not less than 0.9900 in the range of [10-3000] pg / mL and there is no outlier. Therefore, the linear range of the Tau kit is [10-3000] pg / mL.

[0147] (3) Sensitivity

[0148] The blank sample was tested with the above kit and repeated 20 times to determine the sensitivity. The Tau kit test results are shown in Table 11 below (concentration units are pg / mL):

[0149] Table 11

[0150] project parameter Mean 627.4 SD 27.0524 Mean+2SD 681.505 Corresponding concentration value 6.951pg / mL

[0151] The average luminescence value of the blank samples measured by the Tau kit was 627.4. Substituting this value into the reaction curve, the sensitivity of the total Tau kit was 6.951 pg / mL.

[0152] (4) Sample test results

[0153] Test 14 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 = 1.0366x-38.861 and R 2 =0.8724, which proves that this product has good consistency with the commercial product.

[0154] 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 [10-3000] pg / mL, which is much wider than the linear range of the prior art. The upper limit of detection of the kit is at least 6 times that of the prior art. At the same time, the kit of the present application has good consistency with commercial products.

[0155] 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 recombinant antibody targeting Tau protein, wherein: The recombinant antibody 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; or 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 recombinant antibody according to claim 1, wherein The recombinant antibody 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%, or 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%, or 99% identity with SEQ ID No: 15; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 14, or is 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 shown in SEQ ID No: 16, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:

16.

3. A nucleic acid molecule encoding the recombinant antibody according to claim 1 or 2.

4. A recombinant Tau protein antigen, wherein: The amino acid sequence of the recombinant antigen is shown in SEQ ID No:

17.

5. A nucleic acid molecule encoding the Tau protein recombinant antigen according to claim 4, wherein: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No:

18.

6. Use of the Tau protein recombinant antigen according to claim 4 or the nucleic acid molecule according to claim 5 in obtaining the recombinant antibody according to claim 1 or 2.

7. A kit for detecting Tau protein, wherein: 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 first antibody and the second antibody targeting Tau protein are the recombinant antibodies according to claim 1 or 2. The kit for detecting Tau protein according to claim 7 , which is used for diagnosing Alzheimer's disease.

9. Use of the recombinant antibody according to claim 1 or 2, or the nucleic acid molecule according to claim 3, in the preparation of a kit for diagnosing Alzheimer's disease.

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