Anti-human phosphorylated Tau181 rabbit monoclonal antibody, anti-human phosphorylated Tau217 rabbit monoclonal antibody, preparation method and joint detection application

Abstract

The invention relates to the technical field of immunodetection, in particular to an anti-human phosphorylated Tau181 rabbit monoclonal antibody, an anti-human phosphorylated Tau217 rabbit monoclonal antibody, a preparation method and combined detection application, provides two anti-human phosphorylated Tau rabbit monoclonal antibodies, and provides preparation, application and a combined application method of the two antibodies. The antibody has good accuracy and sensitivity in detection of a phosphorylated Tau marker, can be applied to sandwich ELISA, indirect ELISA, immunochromatography, chemiluminescence or flow detection fields and the like, and is beneficial to discovery and screening of patients suffering from early Alzheimer's disease; the invention provides an antibody pair formed by applying the two antibodies to immunodetection and jointly applying the two antibodies to immunodetection, the advantages of high accuracy, high sensitivity and low background are shown in detection, the G122 (p-Tau217)-G69 (p-Tau181) antibody pair can detect two indexes of Tau181 and Tau217 at a time, and compared with a common two-step detection method, the G122 (p-Tau217)-G69 (p-Tau181) antibody pair is quicker and more convenient, and the accuracy is guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to an anti-human phosphorylated Tau181 rabbit monoclonal antibody, an anti-human phosphorylated Tau217 rabbit monoclonal antibody, a preparation method and a combined detection application. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease, commonly known as senile dementia. It has two main symptoms: one is the abnormal deposition of β-amyloid protein outside the neuronal cells to form senile plaques, and the other is the abnormal phosphorylation of Tau protein to form NFTs (neuronal fibrillary tangles). There are a large number of abnormal Tau proteins in the brains of AD patients. The abnormal modification and content changes of Tau protein play an important role in the clinical pathology of AD. Tau protein is a protein encoded by chromosome 17 of the human body. In the nervous system, this protein is the main component of microtubules. Recent studies have shown that the aggregation caused by abnormal phosphorylation of Tau protein is highly correlated with AD.

[0003] Currently, a variety of immunological methods have been developed for the detection of Tau protein in the plasma and cerebrospinal fluid (CSF) of AD patients, among which monoclonal antibodies are the core components of such detection technologies. These antibodies need to be highly specific to accurately identify phosphorylated Tau protein. It is worth noting that a number of studies have revealed that phosphorylated Tau protein in plasma, especially p-Tau181, p-Tau217, and p-Tau231, show excellent diagnostic accuracy for Alzheimer's disease, and its performance is comparable to that of CSF and neuroimaging markers, opening up new avenues for the application of blood tests in AD diagnosis. The "Guidelines for the Diagnosis and Treatment of Alzheimer's Disease Dementia in China (2020 Edition)" clearly defines p-Tau181 and p-Tau217 as important biomarkers for distinguishing AD from non-AD dementia.

[0004] However, although the existing technology can detect phosphorylated Tau protein through immunoassay techniques such as ELISA, the antibodies prepared by the existing technology have problems with low detection accuracy and sensitivity during detection. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-human phosphorylated Tau181 rabbit monoclonal antibody, an anti-human phosphorylated Tau217 rabbit monoclonal antibody, a preparation method and a combined detection application, aiming to solve the technical problem that the antibodies prepared in the prior art have low detection accuracy and sensitivity during detection.

[0006] To achieve the above object, the present invention adopts a preparation method comprising the following steps:

[0007] Full-length human Tau protein antigen, p-Tau181 polypeptide antigen and p-Tau217 polypeptide antigen were selected for use;

[0008] The full-length human Tau protein antigen, p-Tau181 polypeptide antigen, and p-Tau217 polypeptide antigen were mixed with complete Freund's adjuvant and emulsified, and New Zealand rabbits were immunized by subcutaneous injection.

[0009] 40 ml of peripheral blood of rabbits with three antigens was collected using a blood collection tube. The peripheral blood was mixed with an equal amount of PBS and then slowly added to the lymphocyte separation solution. The cells were centrifuged at room temperature at 400 × g for 30 minutes. The mononuclear cell layer was then aspirated. After washing with PBS to remove platelets and lymphocyte separation solution, rabbit PBMC cells with three antigens were obtained.

[0010] The above three antigens (Ag) were pre-labeled with AF488 fluorescence; 5x10 7 The rabbit PBMCs cells were reacted with biotin-labeled goat anti-rabbit CD4, CD8, and IgM for 15 minutes, and the CD4+, CD8+, and IgM+ cell populations were removed by magnetic bead sorting using Streptavidin pre-coupled magnetic beads, and the collected cells were reacted with the corresponding fluorescently labeled antigens and fluorescently labeled goat anti-rabbit IgG-AF647 for 15 minutes, and the cells were washed, and finally 7-AAD was added for reaction; the 7-AAD-, Ag+, and IgG+ cell populations were sorted out by flow cytometry;

[0011] The single-cell sequencing method was used to sequence the 7-AAD-, Ag+, and IgG+ cell populations collected above, and the single-cell immune repertoire 5' library construction process was used to construct a single-cell immune repertoire. 4 The number of cells was loaded separately, and the VDJ library and gene expression library were established respectively. After PE150 second-generation sequencing, the VDJ genes of heavy and light chain pairing were initially screened out through the bioinformatics analysis process; the genes were synthesized and plasmid vectors were constructed, and HEK293 cells were transfected with 48-well plates for expression, and the positive clones were finally determined by the indirect ELISA method; the positive clones finally obtained are as follows:

[0012] p-Tau181-positive clones: G69, G92;

[0013] p-Tau217 positive clones: G32, G95, G122;

[0014] Total-Tau positive clones: G70, G75;

[0015] The plasmid vector of the positive clone is expanded to transfect the production-scale HEK293 cells. After the transfected cells are cultured for 120-144 hours, the cell suspension is collected, centrifuged and the supernatant is obtained. Antibody purification is performed by Protein A affinity chromatography to obtain anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies.

[0016] The preparation method of the full-length human Tau protein antigen is as follows:

[0017] The full-length gene sequence of human Tau (2N4R) protein was synthesized and expressed by transiently transfecting HEK293 cells. After culturing the transfected cells for 144 hours, the cell supernatant was collected and then purified by His tag to obtain the full-length human Tau protein.

[0018] As described above, the combined detection application of anti-human phosphorylated Tau181 rabbit monoclonal antibody and anti-human phosphorylated Tau217 rabbit monoclonal antibody,

[0019] Anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies were used to prepare immunoassay products for detecting human phosphorylated Tau protein;

[0020] The immunoassay product is a detection kit or a detection test strip or a raw material for an immunoassay reagent.

[0021] Wherein, the detection kit is a double antibody sandwich ELISA detection kit or a chemiluminescent immunoassay kit.

[0022] The present invention also provides a p-Tau181 rabbit monoclonal antibody, which is prepared by the preparation method as described above, wherein the p-Tau181 rabbit monoclonal antibody comprises a first heavy chain variable region VH sequence and a first light chain variable region VL sequence, wherein the first heavy chain variable region VH sequence is shown in SEQ ID NO1, and the first light chain variable region VL sequence is shown in SEQ ID NO2;

[0023] The first heavy chain variable region VH sequence includes three heavy chain CDR region sequences, namely HCDR1-3, and the sequences are shown in SEQ ID NOs 3-5;

[0024] The first light chain variable region VL sequence includes three light chain CDR region sequences, namely LCDR1-3, and the sequences are shown in SEQ ID NO6-8.

[0025] SEQ ID NO1:

[0026] QSLEESGGDLVKPGASLTLTCTASGFDLNSNYWMSWVRQAPGKGLEWIACIVTDDGRLYYTWAKGRFTISKTSSTTVTLQMTGLTAADTATYFCVRWYDLWGPGTLVTVSS

[0027] SEQ ID NO2:

[0028] AQVLTQTPSPVSAAVGGTVSISCQSSESVYSKNWLSWYQQKPGQPPKLLIYEASKLSSGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYNRYSDTYGFGGGTEVVVK

[0029] SEQ ID NO3:

[0030] GFDLNSNYW

[0031] SEQ ID NO4:

[0032] IVTDDGR

[0033] SEQ ID NO5:

[0034] VRW

[0035] SEQ ID NO6:

[0036] ESVYSKNW

[0037] SEQ ID NO7:

[0038] EAS

[0039] SEQ ID NO8:

[0040] AGGYNRYSDTYG

[0041] Among them, the first nucleic acid sequence encoding the first heavy chain variable region VH and the first light chain variable region VL is shown in SEQ ID NO9-10.

[0042] SEQ ID NO9:

[0043] CAGTCGTGTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCGACCTCAATAGTAACTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTGTTACTGATGATGGC AGGCTATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCGGTCTGACAGCCGCGGACACGGCCAGTATTTTTGTGTGAGATGGTATGACTTGTGGGGCCCCGGCACCCTGGTCACCGTCTCCTCA

[0044] SEQ ID NO10:

[0045] GCCCAAGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTGAGAGTGTTTATAGTAAGAACTGGTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTG TCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAATCGTTATAGTGATACCTATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0046] The present invention also provides an anti-human phosphorylated Tau217 rabbit monoclonal antibody, which is prepared by the preparation method described above.

[0047] The Tau217 rabbit monoclonal antibody comprises a second heavy chain variable region VH sequence and a second light chain variable region VL sequence, wherein the second heavy chain variable region VH sequence is shown in SEQ ID NO11, and the second light chain variable region VL sequence is shown in SEQ ID NO12;

[0048] The second heavy chain variable region VH sequence includes three heavy chain CDR region sequences, namely HCDR1-3, and the sequences are shown in SEQ ID NOs 13-15;

[0049] The second light chain variable region VL sequence includes three light chain CDR region sequences, namely LCDR1-3, and the sequences are shown in SEQ ID NOs 16-18.

[0050] SEQ ID NO11:

[0051] QEQLVESGGGLVQPEGSLTLTCTASGFSFSSSCYMCWVRQAPGKGLEWIGCIYTGSGSTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARKYSDSGYSPNFNLWGPGTLVTVSS

[0052] SEQ ID NO12:

[0053] AAVLTQTPASVSAAVGGTVSINCQSSQSVYDTNRLSWYQQKPGQPPKLLIYGASYLASGVPSRFKGSGSGTQFTLTITDLECDDAATYYCAGGYSVSSDRNAFGGGTEVVVK

[0054] SEQ ID NO13:

[0055] GFSFSSSCY

[0056] SEQ ID NO14:

[0057] IYTGSGS

[0058] SEQ ID NO15:

[0059] ARKYSDSGYSPNFNL

[0060] SEQ ID NO16:

[0061] QSVYDTNR

[0062] SEQ ID NO17:

[0063] GAS

[0064] SEQ ID NO18:

[0065] AGGYSVSSDRNA

[0066] Among them, the second nucleic acid sequence encoding the second heavy chain variable region VH and the second light chain variable region VL is shown in SEQ ID NOs 19-20.

[0067] SEQ ID NO19:

[0068] CAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCTCCTTCAGTAGCAGCTGCTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTATACTGGTAGTGGTAGCACTTACTAC GCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAAATATTCTGATAGTGGTTATTCCCCCAATTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0069] SEQ ID NO20:

[0070] GCCGCCGTGCTGACCCAGACACCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAATTGCCAGTCCAGTCAGAGTGTTTATGATACCAACCGCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCTATCTA GCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCACCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGTGAGTAGTGATAGAAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0071] The present invention also provides a recombinant expression vector, comprising the first nucleic acid sequence and the second nucleic acid sequence or nucleic acid sequences encoding anti-human phosphorylated p-Tau181 rabbit monoclonal antibody and anti-human phosphorylated Tau217 rabbit monoclonal antibody.

[0072] The present invention also provides a recombinant expression cell, comprising the recombinant expression vector as described above.

[0073] As described above, the combined detection application of anti-human phosphorylated Tau181 rabbit monoclonal antibody and anti-human phosphorylated Tau217 rabbit monoclonal antibody,

[0074] Anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies were used to prepare immunoassay products for detecting human phosphorylated Tau protein;

[0075] The immunoassay product is a detection kit or a detection test strip or a raw material for an immunoassay reagent.

[0076] Wherein, the detection kit is a double antibody sandwich ELISA detection kit or a chemiluminescent immunoassay kit.

[0077] The present invention discloses an anti-human phosphorylated Tau181 rabbit monoclonal antibody, an anti-human phosphorylated Tau217 rabbit monoclonal antibody, a preparation method and a combined detection application. The present invention provides two anti-human phosphorylated Tau rabbit monoclonal antibodies, namely p-Tau181 and p-Tau217. Both rabbit monoclonal antibodies include two chains, heavy chain and light chain, the heavy chain variable region (V region) includes HCDR1, 2, 3, and the light chain variable region (V region) includes LCDR1, 2, 3. The present application provides methods for preparing, applying and combining these two antibodies. The antibodies have good accuracy and sensitivity in detecting phosphorylated Tau markers, and can be applied to detection fields such as sandwich ELISA, indirect ELISA, immunochromatography, chemiluminescence or flow cytometry, which is beneficial to the discovery and screening of patients with early Alzheimer's disease. The present application also provides nucleic acid sequences encoding these antibodies. The present application provides antibody pairs for applying these two antibodies to immunodetection and combining them for immunodetection, which show the advantages of high accuracy, high sensitivity and low background in detection. Among them, the G122 (p-Tau217)-G69 (p-Tau181) antibody pair can detect Tau181 and Tau217 at one time, which is faster and more convenient than the ordinary two-step detection method and guarantees accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0079] Figure 1It is a schematic diagram of the G69 clone-specific indirect ELISA identification method of the present invention.

[0080] Figure 2 It is a schematic diagram of the G70 clone-specific indirect ELISA identification method of the present invention.

[0081] Figure 3 It is a schematic diagram of the G122 clone-specific indirect ELISA identification method of the present invention.

[0082] Figure 4 It is a schematic diagram of the specific WB identification of G69 clone of the present invention.

[0083] Figure 5 It is a schematic diagram of the specific WB identification of G122 clone of the present invention.

[0084] Figure 6 Schematic diagram of the standard curve of the G69-G70 antibody pair of the present invention.

[0085] Figure 7 It is a schematic diagram of the standard curve of the G122-G70 antibody pair of the present invention.

[0086] Figure 8 Schematic diagram of the standard curve of the G122-G69 antibody pair of the present invention. DETAILED DESCRIPTION

[0087] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0088] The present invention provides a preparation method.

[0089] The steps include:

[0090] S100, preparation of immune antigens;

[0091] For this specific embodiment, the immune antigen is designed according to the 1-441aa sequence of Tau (2N4R) protein (NP_005901.2):

[0092] Full-length human Tau protein antigen: The full-length gene sequence of human Tau (2N4R) protein was synthesized and expressed by transient transfection of HEK293 cells. After culturing the transfected cells for 144 hours, the cell supernatant was collected and then purified by His tag to obtain the full-length human Tau protein.

[0093] p-Tau181 polypeptide antigen: IPAKTPPAPKT(p)PPSSGEPPKS, aa171-191, purity>=95%, N-terminal Cys, C-terminal amidation modification.

[0094] p-Tau217 peptide antigen: TPGSRSRTPSLPT(p)PPTREPKKVA, aa205-227, purity>=95%, N-terminal Cys added, C-terminal amidation modification.

[0095] The peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd. The two synthesized phosphorylated peptides were coupled with KLH as antigens. At the same time, two corresponding non-phosphorylated peptides were synthesized as detection antigens.

[0096] S200, the full-length human Tau protein antigen, p-Tau181 polypeptide antigen and p-Tau217 polypeptide antigen were mixed and emulsified with complete Freund's adjuvant, and New Zealand rabbits were immunized by subcutaneous injection;

[0097] For this specific embodiment, the initial immunization dose is 400ug / rabbit, and after an interval of two weeks, incomplete Freund's adjuvant emulsification is used for booster immunization, and the immunization dose is 300ug / rabbit. After three immunizations, peripheral blood samples are taken through the ear vein, and the serum titer is determined by indirect ELISA. When the ELISA titer is greater than or equal to 640,000, it is considered qualified, and the rabbit with the highest titer is selected for PBMCs collection.

[0098] S300, PBMCs separation:

[0099] For this specific embodiment, 40 ml of peripheral blood of rabbits with three antigens were collected through the rabbit ear artery using a blood collection tube, and the peripheral blood was mixed with an equal amount of PBS and slowly added to the lymphocyte separation solution, centrifuged at room temperature 400×g for 30 minutes, and then the mononuclear cell layer was carefully aspirated. After washing with PBS to remove platelets and lymphocyte separation solution, rabbit PBMCs with three antigens were obtained.

[0100] S400, specific B cell sorting:

[0101] In this specific embodiment, the above three antigens (Ag) were pre-labeled with AF488 fluorescence; 5x10 7 The rabbit PBMCs cells were reacted with biotin-labeled goat anti-rabbit CD4, CD8, and IgM for 15 minutes, and the CD4+, CD8+, and IgM+ cell populations were removed by magnetic bead sorting using Streptavidin pre-coupled magnetic beads, and the collected cells were reacted with the corresponding fluorescently labeled antigens and fluorescently labeled goat anti-rabbit IgG-AF647 for 15 minutes, and the cells were washed, and finally 7-AAD was added for reaction; the 7-AAD-, Ag+, and IgG+ cell populations were sorted out by flow cytometry;

[0102] S500, positive clones obtained:

[0103] In this specific embodiment, the single-cell sequencing method was used to sequence the 7-AAD-, Ag+, and IgG+ cell populations collected above, and the single-cell immune repertoire 5' library construction process was used to construct the library. 4 The number of cells was loaded separately, and the VDJ library and gene expression library were established respectively. After PE150 second-generation sequencing, the VDJ genes of heavy and light chain pairing were initially screened out through the bioinformatics analysis process; the genes were synthesized and plasmid vectors were constructed, and HEK293 cells were transfected with 48-well plates for expression, and the positive clones were finally determined by the indirect ELISA method; the positive clones finally obtained are as follows:

[0104] p-Tau181-positive clones: G69, G92;

[0105] p-Tau217 positive clones: G32, G95, G122;

[0106] Total-Tau positive clones: G70, G75;

[0107] Preparation and purification of S600 monoclonal antibodies:

[0108] According to this specific embodiment, the plasmid vector of the positive clone is expanded to transfect the production-scale HEK293 cells. After the transfected cells are cultured for 120-144 hours, the cell suspension is collected, centrifuged to obtain the supernatant, and the antibody is purified by Protein A affinity chromatography to obtain anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies.

[0109] The present invention also provides an anti-human phosphorylated Tau181 rabbit monoclonal antibody, wherein the p-Tau181 rabbit monoclonal antibody comprises a first heavy chain variable region VH sequence and a first light chain variable region VL sequence, wherein the first heavy chain variable region VH sequence is shown as SEQ ID NO1, and the first light chain variable region VL sequence is shown as SEQ ID NO2;

[0110] The first heavy chain variable region VH sequence includes three heavy chain CDR region sequences, namely HCDR1-3, and the sequences are shown in SEQ ID NOs 3-5;

[0111] The first light chain variable region VL sequence includes three light chain CDR region sequences, namely LCDR1-3, and the sequences are shown in SEQ ID NO6-8.

[0112] SEQ ID NO1:

[0113] QSLEESGGDLVKPGASLTLTCTASGFDLNSNYWMSWVRQAPGKGLEWIACIVTDDGRLYYTWAKGRFTISKTSSTTVTLQMTGLTAADTATYFCVRWYDLWGPGTLVTVSS

[0114] SEQ ID NO2:

[0115] AQVLTQTPSPVSAAVGGTVSISCQSSESVYSKNWLSWYQQKPGQPPKLLIYEASKLSSGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYNRYSDTYGFGGGTEVVVK

[0116] SEQ ID NO3:

[0117] GFDLNSNYW

[0118] SEQ ID NO4:

[0119] IVTDDGR

[0120] SEQ ID NO5:

[0121] VRW

[0122] SEQ ID NO6:

[0123] ESVYSKNW

[0124] SEQ ID NO7:

[0125] EAS

[0126] SEQ ID NO8:

[0127] AGGYNRYSDTYG

[0128] Among them, the first nucleic acid sequence encoding the first heavy chain variable region VH and the first light chain variable region VL is shown in SEQ ID NO9-10.

[0129] SEQ ID NO9:

[0130] CAGTCGTGTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCGACCTCAATAGTAACTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTGTTACTGATGATGGC AGGCTATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCGGTCTGACAGCCGCGGACACGGCCAGTATTTTTGTGTGAGATGGTATGACTTGTGGGGCCCCGGCACCCTGGTCACCGTCTCCTCA

[0131] SEQ ID NO10:

[0132] GCCCAAGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTGAGAGTGTTTATAGTAAGAACTGGTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTG TCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAATCGTTATAGTGATACCTATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0133] The present invention also provides an anti-human phosphorylated Tau217 rabbit monoclonal antibody.

[0134] The Tau217 rabbit monoclonal antibody comprises a second heavy chain variable region VH sequence and a second light chain variable region VL sequence, wherein the second heavy chain variable region VH sequence is shown in SEQ ID NO11, and the second light chain variable region VL sequence is shown in SEQ ID NO12;

[0135] The second heavy chain variable region VH sequence includes three heavy chain CDR region sequences, namely HCDR1-3, and the sequences are shown in SEQ ID NOs 13-15;

[0136] The second light chain variable region VL sequence includes three light chain CDR region sequences, namely LCDR1-3, and the sequences are shown in SEQ ID NOs 16-18.

[0137] SEQ ID NO11:

[0138] QEQLVESGGGLVQPEGSLTLTCTASGFSFSSSCYMCWVRQAPGKGLEWIGCIYTGSGSTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARKYSDSGYSPNFNLWGPGTLVTVSS

[0139] SEQ ID NO12:

[0140] AAVLTQTPASVSAAVGGTVSINCQSSQSVYDTNRLSWYQQKPGQPPKLLIYGASYLASGVPSRFKGSGSGTQFTLTITDLECDDAATYYCAGGYSVSSDRNAFGGGTEVVVK

[0141] SEQ ID NO13:

[0142] GFSFSSSCY

[0143] SEQ ID NO14:

[0144] IYTGSGS

[0145] SEQ ID NO15:

[0146] ARKYSDSGYSPNFNL

[0147] SEQ ID NO16:

[0148] QSVYDTNR

[0149] SEQ ID NO17:

[0150] GAS

[0151] SEQ ID NO18:

[0152] AGGYSVSSDRNA

[0153] Among them, the second nucleic acid sequence encoding the second heavy chain variable region VH and the second light chain variable region VL is shown in SEQ ID NOs 19-20.

[0154] SEQ ID NO19:

[0155] CAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCTCCTTCAGTAGCAGCTGCTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTATACTGGTAGTGGTAGCACTTACTAC GCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAAATATTCTGATAGTGGTTATTCCCCCAATTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0156] SEQ ID NO20:

[0157] GCCGCCGTGCTGACCCAGACACCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAATTGCCAGTCCAGTCAGAGTGTTTATGATACCAACCGCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCTATCTA GCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCACCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGTGAGTAGTGATAGAAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0158] The present invention also provides a recombinant expression vector, comprising the first nucleic acid sequence and the second nucleic acid sequence or nucleic acid sequences encoding anti-human phosphorylated p-Tau181 rabbit monoclonal antibody and anti-human phosphorylated Tau217 rabbit monoclonal antibody.

[0159] The present invention also provides a recombinant expression cell, comprising the recombinant expression vector as described above.

[0160] See also Figures 1 to 8 , Figure 1 This is a schematic diagram of the G69 clone-specific indirect ELISA identification method of the present invention, Figure 2 This is a schematic diagram of the indirect ELISA method for identifying the G70 clone specificity of the present invention. Figure 3 Schematic diagram of the indirect ELISA method for G122 clone specificity identification of the present invention, Figure 4 This is a schematic diagram of the specific WB method identification of G69 clones of the present invention. Figure 5 This is a schematic diagram of the specific WB method identification of G122 clone of the present invention, Figure 6 is a schematic diagram of the standard curve of the G69-G70 antibody pair of the present invention, Figure 7 is a schematic diagram of the standard curve of the G122-G70 antibody pair of the present invention, Figure 8 Schematic diagram of the standard curve of the G122-G69 antibody pair of the present invention.

[0161] Identification of the prepared rabbit monoclonal antibody:

[0162] 1. Indirect ELSA method

[0163] Coating: 96-well ELISA plates were coated with human phosphorylated Tau protein, non-phosphorylated Tau protein (Tau protein treated with phosphatase), phosphorylated Tau181 polypeptide, non-phosphorylated Tau181 polypeptide, phosphorylated Tau217 polypeptide, and non-phosphorylated Tau217 polypeptide at 1ug / ml, 100ul per well, overnight at 4°C, and then blocked with 1% BSA in PBST.

[0164] Determination: Add the screened positive clone antibody at a concentration of 0.1ug / ml, react at 37℃ for 60min, wash the plate 3 times with PBST, add HRP-goat anti-rabbit IgG secondary antibody, react at 37℃ for 60min, wash the plate 5 times with PBST, add TMB for color development for 10min, add stop solution, and measure the OD450 value on a microplate reader.

[0165] Figure 1 The results showed that the G69 clone was specific to phosphorylated Tau protein and phosphorylated Tau181 peptide, and was negative for non-phosphorylated Tau protein and non-phosphorylated Tau181 peptide.

[0166] Figure 2 The results showed that the G70 clone was specific for both phosphorylated and non-phosphorylated Tau proteins.

[0167] Figure 3 The results showed that the G122 clone was specific to phosphorylated Tau protein and phosphorylated Tau217 peptide, and negative to non-phosphorylated Tau protein and non-phosphorylated Tau217 peptide.

[0168] The results showed that the G69 clone can be used for the detection of Tau181 with a very low background value. The G122 clone can be used for the detection of Tau217 with a very low background value. The G70 clone can be used for the detection of Total Tau or as a component of an antibody pair.

[0169] 2. Western Blot (WB)

[0170] HEK293 cells transfected with Tau (2NR4) protein were used to prepare cell lysate, and 1ug / well was loaded. After immunoblotting, the membrane was divided into two parts: one treated with phosphatase and the other not. G69 and G122 clone antibodies were reacted with the membrane at a concentration of 0.5ug / ml for 60 minutes at 37°C, and the plate was washed 3 times with PBST, and HRP-goat anti-rabbit IgG secondary antibody was added, reacted at 37°C for 60 minutes, and the plate was washed 5 times with PBST, and chemiluminescent substrate was added for exposure determination.

[0171] Figure 4 The results showed that the G69 clone was specific for phosphorylated Tau protein and negative for non-phosphorylated Tau protein.

[0172] Figure 5 The results showed that the G122 clone was specific for phosphorylated Tau protein and negative for non-phosphorylated Tau protein.

[0173] 3. Determination of affinity constant of monoclonal antibodies

[0174] The affinity constant (Kd) of the antibody was determined by non-competitive ELISA. The antigen concentration was 1, 0.5, 0.25, 0.125, 0.0625ug / mL, 100uL / well was added to the 96-well ELISA plate for coating, incubated at 4°C for 24h, and then blocked with 1% BSA in PBST. Wash the plate 3 times with PBST, dilute the monoclonal antibody to be tested starting from 10ug / mL, add 100uL to each well, react at 37°C for 2h, wash the plate 3 times, and then add 100uL of 1:10000 HRP-goat anti-rabbit IgG secondary antibody to each well, react at 37°C for 60min, wash the plate 5 times with PBST, add TMB for color development for 10min, add stop solution, and measure the OD450 value on the ELISA instrument. Then, draw an S-shaped curve with the logarithm of the antibody concentration as the horizontal axis and the OD value as the vertical axis to calculate the affinity constant of the tested antibody. The affinity constants of each antibody measured are as follows:

[0175] G69: Kd = 7.69 × 10-11 mol / L

[0176] G122: Kd = 5.65 × 10-11 mol / L

[0177] The prepared rabbit monoclonal antibody was tested:

[0178] Coating: Coat the ELISA plate with the tested antibody at a concentration of 5ug / ml and 100ul / well, and block with 1% BSA solution after overnight at 4℃.

[0179] Determination: Add 100ul of human phosphorylated Tau full-length protein to each well at a concentration of 10ng / ml, react at 37℃ for 60 minutes, wash 3 times with PBST, then add 100ul / well of biotin-labeled detection antibody at a concentration of 0.1ug / ml, react at 37℃ for 60 minutes, wash 3 times with PBST. Add HRP-labeled streptavidin secondary antibody, react at 37℃ for 30min, wash 5 times with PBST. Add TMB for color development for 10min, add stop solution, and measure OD450 value on an enzyme reader. The most ideal antibody pair selected by the experiment is as follows:

[0180] Coating antibody Detection antibodies G69 G70 G122 G70 G122 G69

[0181] Table 1 Antibody pairing experimental results

[0182] The results showed that the G69-G70 antibody pair can be used as an indicator for detecting p-Tau181, and the G122-G70 antibody pair can be used as an indicator for detecting p-Tau217. It is worth noting that the G122-G69 antibody pair can be used as a dual indicator for detecting p-Tau181 and p-Tau217.

[0183] Determine the detection parameters of the prepared rabbit monoclonal antibody:

[0184] Coating: The above three antibodies were coated on the ELISA plate at a concentration of 5ug / ml and 100ul / well, respectively, and then blocked with 1% BSA solution after overnight at 4°C.

[0185] Determination: Dilute the full-length human phosphorylated Tau protein in multiples starting at a concentration of 1ng / ml, add 100ul / well to the ELISA plate, react at 37℃ for 60 minutes, wash 3 times with PBST, then add 100ul / well of biotin-labeled detection antibody at a concentration of 0.1ug / ml, react at 37℃ for 60 minutes, wash 3 times with PBST. Add HRP-labeled streptavidin secondary antibody, react at 37℃ for 30min, wash 5 times with PBST. Add TMB for color development for 10min, add stop solution, and measure OD450 value on an ELISA reader.

[0186] Parameter determination: Draw a standard curve based on OD450 value and antigen concentration and calculate the R2 value and detection sensitivity.

[0187] Figure 6 The results showed that the detection standard curve of the G69-G70 antibody pair had a detection range of 0-1000 pg / mL, an R2 value of 0.9988, and a detection sensitivity of less than 35 pg / ml.

[0188] Figure 7 The results showed that the detection standard curve of the G122-G70 antibody pair had a detection range of 0-1000pg / mL, an R2 value of 0.9992, and a detection sensitivity of less than 30pg / ml.

[0189] Figure 8 The results showed that the detection standard curve of the G122-G69 antibody pair had a detection range of 0-1000 pg / mL, an R2 value of 0.9991, and a detection sensitivity of less than 45 pg / ml.

[0190] Among the above antibody pairs, the G69-G70 antibody pair can be used to detect the p-Tau181 index alone, or the G122-G70 antibody pair can be used to detect the p-Tau217 index. The G69-G70 and G122-G70 antibody pairs can also be combined to simultaneously detect the p-Tau181 and p-Tau217 indexes.

[0191] It is worth noting that the G122-G69 antibody pair can be used to simultaneously detect both p-Tau181 and p-Tau217, which is fast, convenient and accurate.

[0192] A combined detection application of anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies, using the anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies as described above,

[0193] Anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies were used to prepare immunoassay products for detecting human phosphorylated Tau protein;

[0194] The immunoassay product is a test kit or a test strip or a raw material used as an immunoassay reagent, and the immunoassay product uses the p-Tau181 rabbit monoclonal antibody and the Tau217 rabbit monoclonal antibody in combination;

[0195] Meanwhile, the immunoassay product may use the p-Tau181 rabbit monoclonal antibody or the p-Tau217 rabbit monoclonal antibody alone.

[0196] Wherein, the detection kit is a double antibody sandwich ELISA detection kit or a chemiluminescent immunoassay kit.

[0197] The detection kit development process:

[0198] 1. Coating strips: Dilute the coating antibody in the above antibody pair to 5ug / ml with PBS buffer and coat it on the microwell plate.

[0199] 100 μl was added to each well, incubated overnight at 4°C, washed 3 times with PBST, and added with 1% BSA, 5% sucrose, 0.05%

[0200] After the Proclin300 PBS was reacted at 37℃ for 2h, the blocking solution in the wells was discarded and the wells were dried; the coated plate was placed at 37℃ to dry

[0201] After 2 hours, the coating is completed, and the samples are sealed in an aluminum foil bag and stored at 4°C for future use.

[0202] 2. Reagent Preparation

[0203] 2.1 Detection antibody: Use biotin labeling method to label the detection antibody in the above antibody pair and prepare it into a 10x concentrated solution of 1ug / mL.

[0204] 2.2 The washing buffer is conventional PBST at pH 7.4, containing 0.05% Proclin 300, prepared into a 20-fold concentrated solution.

[0205] 2.3 The enzyme conjugate is HRP-labeled streptavidin and prepared into a 10x concentrated solution of 1 ug / mL.

[0206] 2.4 Enzyme substrate solution (color developing solution): single-component TMB (purchased from SolarBio).

[0207] 2.5 The dilution solution is PBS containing 1% BSA, 5% glycerol, and 0.05% Proclin 300, which is sterilized by filtration.

[0208] 2.6 Use 1N HCl as the stop solution.

[0209] 2.7 Standard: Full-length human phosphorylated Tau protein, diluted to 10ug / ml with PBS containing 1% BSA, 5% sucrose, 10% glycerol, and 0.05% Proclin300, filtered for sterilization, and freeze-dried for aseptic packaging.

[0210] 3. The above components are packaged in a special box for the kit to obtain the finished kit.

[0211] Using an anti-human phosphorylated Tau181 rabbit monoclonal antibody, an anti-human phosphorylated Tau217 rabbit monoclonal antibody, a preparation method and a combined detection application of this embodiment, the present invention provides two rabbit monoclonal antibodies against human phosphorylated Tau, namely p-Tau181 and p-Tau217. Both rabbit monoclonal antibodies include two chains, heavy chain and light chain, the heavy chain variable region (V region) includes HCDR1, 2, 3, and the light chain variable region (V region) includes LCDR1, 2, 3. The present application provides methods for preparing, applying and combining these two antibodies. The antibodies have good accuracy and sensitivity in detecting phosphorylated Tau markers, and can be applied to detection fields such as sandwich ELISA, indirect ELISA, immunochromatography, chemiluminescence or flow cytometry, which is conducive to the discovery and screening of patients with early Alzheimer's disease. The present application also provides nucleic acid sequences encoding these antibodies. The present application provides antibody pairs for the application of these two antibodies to immunodetection and combined use in immunodetection, showing the advantages of high accuracy, high sensitivity and low background in detection. Among them, the G122 (p-Tau217)-G69 (p-Tau181) antibody pair can detect Tau181 and Tau217 at one time, which is faster and more convenient than the ordinary two-step detection method and guarantees accuracy.

[0212] Sequence Listing:

[0213] SEQ ID NO1:

[0214] QSLEESGGDLVKPGASLTLTCTASGFDLNSNYWMSWVRQAPGKGLEWIACIVTDDGRLYYTWAKGRFTISKTSSTTVTLQMTGLTAADTATYFCVRWYDLWGPGTLVTVSS

[0215] SEQ ID NO2:

[0216] AQVLTQTPSPVSAAVGGTVSISCQSSESVYSKNWLSWYQQKPGQPPKLLIYEASKLSSGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYNRYSDTYGFGGGTEVVVK

[0217] SEQ ID NO3:

[0218] GFDLNSNYW

[0219] SEQ ID NO4:

[0220] IVTDDGR

[0221] SEQ ID NO5:

[0222] VRWYDL

[0223] SEQ ID NO6:

[0224] ESVYSKNW

[0225] SEQ ID NO7:

[0226] EAS

[0227] SEQ ID NO8:

[0228] AGGYNRYSDTYG

[0229] SEQ ID NO9:

[0230] CAGTCGTTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCGACCTCAATAGTAACTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTGTTACTGATGATGGCAGGCTATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCGGTCTGACAGCCGCGGACACGGCCACGTATTTTTGTGTGAGATGGTATGACTTGTGGGGCCCCGGCACCCTGGTCACCGTCTCCTCA

[0231] SEQ ID NO10:

[0232] GCCCAAGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTGAGAGTGTTTATAGTAAGAACTGGTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTGTCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAATCGTTATAGTGATACCTATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0233] SEQ ID NO11:

[0234] QEQLVESGGGLVQPEGSLTLTCTASGFSFSSSCYMCWVRQAPGKGLEWIGCIYTGSGSTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARKYSDSGYSPNFNLWGPGTLVTVSS

[0235] SEQ ID NO12:

[0236] AAVLTQTPASVSAAVGGTVSINCQSSQSVYDTNRLSWYQQKPGQPPKLLIYGASYLASGVPSRFKGSGSGTQFTLTITDLECDDAATYYCAGGYSVSSDRNAFGGGTEVVVK

[0237] SEQ ID NO13:

[0238] GFSFSSSCY

[0239] SEQ ID NO14:

[0240] IYTGSGS

[0241] SEQ ID NO15:

[0242] ARKYSDSGYSPNFNL

[0243] SEQ ID NO16:

[0244] QSVYDTNR

[0245] SEQ ID NO17:

[0246] GAS

[0247] SEQ ID NO18:

[0248] AGGYSVSSDRNA

[0249] SEQ ID NO19:

[0250] CAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCTCCTTCAGTAGCAGCTGCTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTATACTGGTAGTGGTAGCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAAATATTCTGATAGTGGTTATTCCCCCAATTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0251] SEQ ID NO20:

[0252] GCCGCCGTGCTGACCCAGACACCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAATTGCCAGTCCAGTCAGAGTGTTTATGATACCAACCGCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCTATCTAGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCACCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGTGAGTAGTGATAGAAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0253] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A preparation method, characterized in that: The steps include: Full-length human Tau protein antigen, p-Tau181 polypeptide antigen and p-Tau217 polypeptide antigen were selected for use; The full-length human Tau protein antigen, p-Tau181 polypeptide antigen, and p-Tau217 polypeptide antigen were mixed with complete Freund's adjuvant and emulsified, and New Zealand rabbits were immunized by subcutaneous injection. 40 ml of peripheral blood of rabbits with three antigens was collected using blood collection tubes. The peripheral blood was mixed with an equal amount of PBS and then slowly added to the lymphocyte separation solution. The cells were centrifuged at room temperature at 400 × g for 30 minutes. The mononuclear cell layer was then aspirated. The platelets and lymphocyte separation solution were removed by washing with PBS to obtain the rabbit PBMCs with three antigens. The above three antigens (Ag) were pre-labeled with AF488 fluorescence; 5x10 7 The rabbit PBMCs cells were reacted with biotin-labeled goat anti-rabbit CD4, CD8, and IgM for 15 minutes, and the CD4+, CD8+, and IgM+ cell populations were removed by magnetic bead sorting using Streptavidin pre-coupled magnetic beads, and the collected cells were reacted with the corresponding fluorescently labeled antigens and fluorescently labeled goat anti-rabbit IgG-AF647 for 15 minutes, and the cells were washed, and finally 7-AAD was added for reaction; the 7-AAD-, Ag+, and IgG+ cell populations were sorted out by flow cytometry; The single-cell sequencing method was used to sequence the 7-AAD-, Ag+, and IgG+ cell populations collected above, and the single-cell immune repertoire 5' library construction process was used to construct a single-cell immune repertoire. 4 The number of cells was loaded separately, and the VDJ library and gene expression library were established respectively. After PE150 second-generation sequencing, the VDJ genes of heavy and light chain pairing were initially screened out through the bioinformatics analysis process; the genes were synthesized and plasmid vectors were constructed, and HEK293 cells were transfected with 48-well plates for expression, and the positive clones were finally determined by the indirect ELISA method; the positive clones finally obtained are as follows: p-Tau181-positive clones: G69, G92; p-Tau217 positive clones: G32, G95, G122; Total-Tau positive clones: G70, G75; The plasmid vector of the positive clone is expanded to transfect the production-scale HEK293 cells. After the transfected cells are cultured for 120-144 hours, the cell suspension is collected, centrifuged and the supernatant is obtained. Antibody purification is performed by Protein A affinity chromatography to obtain anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies.

2. The preparation method according to claim 1, characterized in that The preparation method of full-length human Tau protein antigen is as follows: The full-length gene sequence of human Tau (2N4R) protein was synthesized and expressed by transiently transfecting HEK293 cells. After culturing the transfected cells for 144 hours, the cell supernatant was collected and then purified by His tag to obtain the full-length human Tau protein.

3. An anti-human phosphorylated Tau181 rabbit monoclonal antibody prepared by the preparation method according to claim 2, characterized in that: The p-Tau181 rabbit monoclonal antibody comprises a first heavy chain variable region VH sequence and a first light chain variable region VL sequence, wherein the first heavy chain variable region VH sequence is shown in SEQ ID NO1, and the first light chain variable region VL sequence is shown in SEQ ID NO2; The first heavy chain variable region VH sequence includes three heavy chain CDR region sequences, namely HCDR1-3, and the sequences are shown in SEQ ID NOs 3-5; The first light chain variable region VL sequence includes, characterized by comprising three light chain CDR region sequences, namely LCDR1-3, and the sequences are shown in SEQ ID NO6-8.

4. The anti-human phosphorylated Tau181 rabbit monoclonal antibody according to claim 3, characterized in that: The first nucleic acid sequence encoding the first heavy chain variable region VH and the first light chain variable region VL is shown in SEQ ID NOs 9-10.

5. An anti-human phosphorylated Tau217 rabbit monoclonal antibody prepared by the preparation method according to claim 2, characterized in that: The Tau217 rabbit monoclonal antibody comprises a second heavy chain variable region VH sequence and a second light chain variable region VL sequence, wherein the second heavy chain variable region VH sequence is shown in SEQ ID NO11, and the second light chain variable region VL sequence is shown in SEQ ID NO12; The second heavy chain variable region VH sequence includes three heavy chain CDR region sequences, namely HCDR1-3, and the sequences are shown in SEQ ID NOs 13-15; The second light chain variable region VL sequence includes three light chain CDR region sequences, namely LCDR1-3, and the sequences are shown in SEQ ID NOs 16-18.

6. The anti-human phosphorylated Tau217 rabbit monoclonal antibody according to claim 5, characterized in that: The second nucleic acid sequence encoding the second heavy chain variable region VH and the second light chain variable region VL is shown in SEQ ID NOs 19-20.

7. The combined detection application of the anti-human phosphorylated Tau181 rabbit monoclonal antibody and the anti-human phosphorylated Tau217 rabbit monoclonal antibody prepared by the preparation method according to claim 2, characterized in that: Anti-human phosphorylated Tau181 and Tau217 rabbit monoclonal antibodies were used to prepare immunoassay products for detecting human phosphorylated Tau protein.

8. The combined detection application according to claim 7, characterized in that: The immunoassay product is a detection kit or a detection test strip or a raw material for an immunoassay reagent.

9. The combined detection application according to claim 8, characterized in that: The detection kit is a double antibody sandwich ELISA detection kit or a chemiluminescent immunoassay kit.

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