Monoclonal antibody against pTau217 protein phosphorylated at position 217 and its application

Abstract

This application provides a monoclonal antibody against phosphorylated pTau217 protein at position 217. The heavy chain CDR amino acid sequence is H-CDR1:GYTFRNYW, H-CDR2:IYPGGGYT, H-CDR3:VVSDY; the light chain CDR amino acid sequence is L-CDR1:ESVEYSGISL, L-CDR2:AAS, L-CDR3:QQSRKVPST. This antibody can be used to prepare kits for diagnosing Alzheimer's disease, providing good sensitivity and specificity.

Description

Technical Field

[0001] The present application belongs to the technical field of Alzheimer's disease diagnosis. Specifically, the present application provides a monoclonal antibody against pTau217 protein phosphorylated at position 217 and its application. Background Art

[0002] Alzheimer's disease (AD), the main cause of dementia, is a progressive neurodegenerative disease with an insidious onset.

[0003] It is generally believed that the cause of AD is mainly due to the excessive phosphorylation of Tau protein in the brain, which leads to the deposition of neurofibrillary tangles (NFTs) and β-amyloid protein (Aβ), which in turn leads to abnormal neuronal function. Tau protein belongs to the microtubule associated protein (MAPs) family. It is expressed in the central and peripheral systems and is most abundant in the axons of nerve cells. Tau protein in normal humans plays an important role in regulating and maintaining microtubule stability, assisting axonal transport and stabilizing the cytoskeleton. The full-length Tau protein can be divided into four functional regions, including the N-terminal region, the proline-rich domain (PRD), the microtubule binding domain (MBD) and the C-terminal region. 20% of the amino acids of Tau protein can be potential phosphorylation sites. Normally, the microtubule binding region of Tau protein is positively charged. When the microtubule binding region is highly phosphorylated, it detaches from tubulin and no longer has the ability to bind to and stabilize microtubules, resulting in the failure of tubulin. In addition, highly phosphorylated Tau protein aggregates and precipitates, which is toxic to nerve cells, which together cause nerve fiber degeneration and loss of function. In addition to phosphorylation, abnormal glycosylation and ubiquitination of Tau protein cause Tau protein to lose its stabilizing effect on microtubules, thereby leading to the decline of nerve cells. Because Tau protein has multiple phosphorylation sites and involves a large number of protein kinases and phosphatases in regulation, the pathogenesis of AD is more complicated.

[0004] Studies have shown that there are a large number of abnormal Tau proteins in the brains of AD patients. PET imaging of Tau protein neurofibrillary tangles in vivo can be used for the diagnosis, treatment and monitoring of Alzheimer's disease (AD). At the same time, abnormal tau proteins in cerebrospinal fluid (CSF) can also be used as early and specific indicators of AD pathology. Among them, p-tau181 in cerebrospinal fluid has been considered as a marker of NFT and is used to support the diagnosis of clinical AD and the prediction of mild cognitive impairment. In addition, p-tau at many phosphorylation sites, including p-tau181, p-tau217, and p-tau231, have shown that they have the potential to become AD diagnostic markers. However, compared with PET or CSF markers, the determination of plasma phosphorylated tau protein (p-tau) is less expensive and less invasive. In addition, studies have shown that plasma p-tau217 is elevated from preclinical to clinical disease stages, indicating that plasma p-tau217 has better early diagnostic capabilities. Therefore, establishing a sensitive and accurate p-tau217 detection method, especially the determination of plasma p-tau217, is low-cost, less invasive, and easy to carry out in the population, which is very suitable for the early screening and diagnosis of AD.

[0005] At present, the detection methods for phosphorylated tau protein include liquid chromatography-mass spectrometry and some methods based on specific antigen-antibody reactions, such as enzyme-linked immunosorbent assay and immunoblotting, but they all generally face the problem of highly specific monoclonal antibodies and highly sensitive detection methods for identifying tau protein at specific sites. As an emerging technology, single-molecule detection technology has extremely high sensitivity, which can reach the fg / mL level compared with traditional immunological diagnosis. Its digital detection results show small variation and a wide dynamic range of detection, and should be widely used in more and more clinical tests. It can directly perform ultra-high sensitivity detection of biomarkers in serum, plasma, and cerebrospinal fluid, which is very suitable for the analysis of low-abundance markers. Summary of the invention

[0006] On the one hand, the present application provides a monoclonal antibody to the pTau217 protein phosphorylated at position 217, wherein the heavy chain CDR amino acid sequence of the monoclonal antibody to the pTau217 protein phosphorylated at position 217 is H-CDR1: GYTFRNYW, H-CDR2: IYPGGGYT, H-CDR3: VVSDY; the light chain CDR amino acid sequence of the monoclonal antibody to the pTau217 protein phosphorylated at position 217 is L-CDR1: ESVEYSGISL, L-CDR2: AAS, L-CDR3: QQSRKVPST.

[0007] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody against the phosphorylated pTau217 protein at position 217 is SEQ ID NO.15, and / or the light chain amino acid sequence of the monoclonal antibody against the phosphorylated pTau217 protein at position 217 is SEQ ID NO.20.

[0008] On the other hand, the present application provides the use of the above-mentioned monoclonal antibody against pTau217 protein phosphorylated at position 217 in the preparation of a kit or a single-molecule immunoassay platform for diagnosing Alzheimer's disease.

[0009] Furthermore, the kit is an ELISA kit.

[0010] Furthermore, the monoclonal antibody against pTau217 protein phosphorylated at position 217 in the single-molecule immunoassay platform is used as a detection antibody, and the monoclonal antibody against Tau protein is also used as a capture antibody in the single-molecule immunoassay platform.

[0011] Furthermore, the heavy chain CDR amino acid sequence of the Tau protein monoclonal antibody is H-CDR1: GYTFTSYN, H-CDR2: IYPGNGGT, H-CDR3: ARSYGTSYVGAMDY; the light chain CDR amino acid sequence of the Tau protein monoclonal antibody is L-CDR1: QEISGY, L-CDR2: AAS, L-CDR3: LQYTIYPPT.

[0012] On the other hand, the present application provides a Tau protein monoclonal antibody, wherein the heavy chain CDR amino acid sequence of the Tau protein monoclonal antibody is H-CDR1: GYTFTSYN, H-CDR2: IYPGNGGT, H-CDR3: ARSYGTSYVGAMDY; the light chain CDR amino acid sequence of the Tau protein monoclonal antibody is L-CDR1: QEISGY, L-CDR2: AAS, L-CDR3: LQYTIYPPT.

[0013] Furthermore, the heavy chain amino acid sequence of the Tau protein monoclonal antibody is SEQ ID NO.5, and / or the light chain amino acid sequence of the Tau protein monoclonal antibody is SEQ ID NO.10.

[0014] A vector comprising the nucleic acid shown in SEQ ID NO.4 and SEQ ID NO.9, or the vector comprising the nucleic acid shown in SEQ ID NO.14 and SEQ ID NO.19

[0015] On the other hand, the present application provides a method for producing the above-mentioned pTau217 protein monoclonal antibody phosphorylated at position 217 or Tau protein monoclonal antibody, the method comprising culturing a host cell, wherein the host cell comprises the above-mentioned vector.

[0016] The host cell can be selected from various yeasts, Escherichia coli, Bacillus subtilis and cell-free expression systems, etc. Those skilled in the art can also select a suitable commercial vector according to the host and perform insertion and other operations according to the operating instructions.

[0017] When the antibody CDR region or variable region is known, those skilled in the art can routinely design the framework region and other steps, and adjust the corresponding gene sequence according to the host preference.

[0018] The present invention provides a highly sensitive and highly specific monoclonal antibody for phosphorylated Tau protein p-tau217 and a method for screening and identifying the same, and further provides the application of the monoclonal antibody in a single molecule immunoassay platform, providing a tool for the detection of plasma markers of AD and the early prediction and diagnosis of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of affinity test of 25 screened monoclonal antibodies to recombinant non-phosphorylated Tau protein (1 μg / mL concentration).

[0020] Figure 2 The test results of two pairs of healthy subjects and AD patients were combined.

[0021] Figure 3 This is the AUC curve of the detection effect of combining two pairs of healthy subjects and AD patient samples. DETAILED DESCRIPTION

[0022] Example 1 Materials and Methods

[0023] Peptide synthesis:

[0024] The peptide synthesis was completed by Wuhan Baiyixin Biotechnology. According to the amino acid sequence of Tau protein (P10636-8), the 210-223aa peptide CSRTPSLP was designed T PPTREP (SEQ ID NO.1) was synthesized and coupled to KLH and BSA carriers, wherein the threonine at position 217 was phosphorylated. Cysteine ​​C was added to the N-terminus of the synthetic sequence for coupling, and the specific amino acid sequence of the synthetic polypeptide was:

[0025] SP1, KLH-CSRTPSLP T PPTREP( T is the phosphorylation site)

[0026] SP2, BSA-CSRTPSLP T PPTREP ( T is the phosphorylation site)

[0027] SP3, SRTPSLP T PPTREP naked peptide ( T is the phosphorylation site)

[0028] SP4, BSA-CSRTPSLPTPPTREP

[0029] Among them, SP1 / 2 / 3 polypeptides are 217 phosphorylated and modified, and SP4 is not phosphorylated and modified.

[0030] Preparation of recombinant Tau protein:

[0031] The tau protein N segment 1-124aa was recombinantly expressed using a prokaryotic expression system. The gene was synthesized by Anhui General Biotechnology and cloned into the pET32a expression vector. The specific nucleotide sequence is:

[0032] ATGGCAGAACCGCGCCAGGAATTTGAAGTTATGGAAGATCATGCAGGTACCTATGGTCTGGGTGACCGCAAAGATCAGGGTGGCTATACCATGCATCAGGATCAGGAAGGCGATACCGATGCCGGTCTGAAAGAAAGCCCGCTGCAGACCCCGACCGAAGATGGCAGTGAAGAACCGGGCAGTGAAAC CAGTGATGCCAAAAGCACCCCGACCGCAGAAGATGTTACCGCCCCGCTGGTGGATGAAGGCGCACCGGGTAAACAGGCCGCCGCCCAACCGCATACCGAAATTCCGGAAGGTACCACCGCCGAAGAAGCCGGCATTGGTGACACCCCGAGCCTGGAAGATGAAGCCGCAGGCCATGTGACCCAG(SEQ ID NO.2)

[0033] The amino acid sequence is:

[0034] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQ(SEQ ID NO.3)

[0035] The synthesized plasmid was operated according to the molecular cloning manual, transformed into BL21 (DE3) competent cells, the correct clone was picked, cultured and induced for expression with IPTG, the collected bacteria were broken and subjected to nickel column affinity chromatography and ion exchange chromatography to obtain high-purity Tau protein.

[0036] Example 2 Immunization of mice

[0037] After 20ug of SP1 polypeptide was emulsified and mixed with an equal volume of Freund's complete adjuvant, 6-week-old female Balb / c mice were immunized with multiple subcutaneous injections. At 2 weeks and 4 weeks, the immunization was boosted with Freund's incomplete adjuvant according to the above method. At week 6, mouse serum was taken to detect the antibody titers of SP1 and SP2 polypeptides respectively. The mouse with the highest titer was selected and boosted with 20ug of SP1 polypeptide through the tail vein. After 3 days, the spleen of the mouse was taken for the preparation of hybridoma cells. The immunization method of Tau protein is the same as above.

[0038] Example 3 Screening of pTau217 mAb

[0039] All spleen cells of immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and placed in HAT medium for screening and culture. When the fused cells grew to 1 / 2 of the bottom of the well, clones that reacted positively with polypeptide SP1 and negatively with polypeptide SP4 were screened by indirect ELISA. The screening results were then retested by competition with polypeptide SP3 to further screen clones that had competitive inhibition reactions with naked peptides. The hybridoma cells were reduced to a monoclonal state by limiting dilution, and the cell lines were expanded and frozen.

[0040] Screening of positive clones by indirect ELISA:

[0041] Peptides SP2 and SP4 were coated in microplates (coating buffer carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate, fixed to 1L pure water), with a coating concentration of 1μg / mL, overnight at 4℃; 1% gelatin blocking (BSA-containing blocking solution cannot be used), 150 μL per well, blocked at 37℃ for 2 hours, washed once with washing solution, patted dry; 50μL cell culture supernatant was added, reacted at 37℃ for 30min. The liquid in the wells was thrown out, the plate was washed 4 times with PBST washing solution, patted dry, 50μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:10000 in PBS) was added, reacted at 37℃ for 30min, washed again 4 times, patted dry, 50μL / well of TMB colorimetric solution was added for color development at room temperature for 10min, and finally 0.5M sulfuric acid was added to terminate the reaction, and the OD450nm value was measured with an enzyme reader. The immune antigen was KLH-coupled polypeptide SP1, so BSA-coupled polypeptide SP2 was used for screening to eliminate background interference from the carrier. At the same time, non-phosphorylated polypeptide SP4 was used for detection to select the cell supernatant that only reacted with polypeptide SP2 but not with polypeptide SP4 for the next step of identification.

[0042] Identification of positive cell lines by competitive method:

[0043] Uncoupled peptide SP3 is a small molecule hapten, which can produce competitive inhibition with coupled peptides when detecting the reaction with antibodies. When there is a phosphorylation site-specific antibody in the detection supernatant, the free naked peptide will bind to it, resulting in a weaker reaction between the coated solid phase antigen and the antibody, which is manifested as a decrease in the detection reading. The lower the reading, the more antibodies there are in the supernatant or the higher the inhibitory titer. Specifically: coated peptide SP2 (coating buffer carbonate buffer: sodium carbonate 1.59g, sodium bicarbonate 2.93g, fixed to 1L pure water), coating concentration of 1μg / mL, 4℃ overnight; 1% gelatin blocking (BSA-containing blocking solution cannot be used), 150 microliters per well, 37℃ blocking for 2 hours, wash the plate once with washing solution, and pat dry; add 50μL of cell culture supernatant and naked peptide SP3 (1ug / ml) mixture, and react at 37℃ for 30min. Shake out the liquid in the wells, wash the plate 4 times with PBST, pat dry, add 50μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:10000 in PBS), react at 37℃ for 30min, wash the plate 4 times, pat dry, add 50μL / well of TMB colorimetric solution for color development at room temperature for 10min, finally add 0.5M sulfuric acid to terminate the reaction, and measure the OD450nm value with an enzyme reader. Select the cell wells with a reading lower than the positive control, which are the monoclonal antibody cells specific to the phosphorylation site.

[0044] A total of 51 monoclonal antibodies were screened that reacted with BSA-conjugated pTau217, but did not react with BSA-conjugated non-phosphorylated pTau217 or other BSA-conjugated peptides (as shown in Table 1).

[0045] Table 1 Affinity test values ​​of purified monoclonal antibodies at 1ug / ml

[0046]

[0047]

[0048] Example 4 Screening of Tau protein monoclonal antibodies:

[0049] The hybridoma cell fusion method was the same as above. The screening test was indirect ELISA. 1ug / ml of recombinant Tau protein was coated and the same concentration of pET32a carrier protein was used for counter-screening. Finally, the cell line that reacted with the recombinant Tau protein but not with the carrier protein was selected as the positive clone, and then continuous limiting dilution, expansion culture and ascites preparation were performed.

[0050] Preparation of monoclonal antibody ascites:

[0051] After the selected monoclonal cell lines were expanded and cultured, 0.2 ml (containing 2.5×10 6 Cells) were pretreated with Freund's incomplete adjuvant in female BALB / c mice. About 10 days later, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000r / min for 10 minutes to collect the middle layer.

[0052] Purification of monoclonal antibodies (Protein L affinity chromatography)

[0053] The ascites was centrifuged at 12000r / min for 5 minutes, the supernatant was diluted 10 times with binding buffer (20mM PBS, 150mM NaCl, pH7.4), filtered through a 0.22um filter, and the filtered sample was pumped into the Protein L purification column (Hangzhou New Dragon Bio) equilibrated with binding buffer at a low speed through a peristaltic pump, connected to an AKTA purifier, washed with binding buffer for 5-10 column volumes until the UV absorption peak was washed flat, and then eluted with elution buffer (0.1M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1M Tris-HCl, pH 9, and placed in a dialysis bag (MW: 8000~14000), and dialyzed in 0.01M PBS pH 7.4 solution at 2~8℃ for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The purified monoclonal antibody was subjected to SDS-PAGE electrophoresis, and its concentration was determined by a micro-ultraviolet spectrophotometer, and then stored in aliquots.

[0054] like Figure 1As shown, 25 monoclonal antibodies were screened out, which could bind to recombinant non-phosphorylated Tau protein with high affinity.

[0055] Example 5 Identification and pairing screening of monoclonal antibodies

[0056] Affinity identification of monoclonal antibodies:

[0057] SP2, SP4 and other peptides coupled with BSA (BSA-P non-tau protein peptides) were used to coat and detect the specificity and binding activity of pTau217 monoclonal antibody, and pET32a-Tau and carrier protein were used to coat and detect the specificity and binding activity of Tau protein monoclonal antibody. The specific coating steps refer to the above-mentioned indirect ELISA, wherein the antibody to be tested is diluted according to a concentration gradient of 10ug / ml, 1ug / ml, 100ng / ml, 10ng / ml, 1ng / ml, reacted at 37°C for 30min, 50μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted with PBS at 1:10000) was added, reacted at 37°C for 30min, and after washing and patting dry, 50μL / well of TMB color developing solution was added for color development at room temperature for 10min, and finally 0.5M sulfuric acid was added to terminate the reaction, and the OD450nm value was measured with an enzyme marker (the results have been shown in Examples 3 and 4).

[0058] Paired screening of monoclonal antibodies:

[0059] The monoclonal antibodies to be paired can be divided into two groups: monoclonal antibodies targeting phosphorylated Tau protein 217 and monoclonal antibodies targeting non-phosphorylated Tau protein 1-124aa. The two groups of monoclonal antibodies are coated and labeled with HRP respectively. Antibody HRP labeling: The screened monoclonal antibodies are labeled according to the instructions of G-Biosciences' HOOK™ HRP PLUS Labeling Kit (Cat#: 786-313). Finally, the labeled antigens are dialyzed overnight in 0.01M PBS, pH 7.4 buffer, glycerol is added at a volume of 1:1, and the cells are stored at -20°C.

[0060] The paired ELISA selected the phosphorylated full-length p-Tau protein as the screening antigen, and the concentration of the fixed coated antibody was 1ug / ml. The HRP-labeled antibody was diluted 1000 times, and the double antibody sandwich ELISA experiment was performed on the phosphorylated full-length Tau antigen. Finally, the top 5 combinations with higher paired detection signals were screened out, and the single-molecule immune platform was used to verify the detection of clinical plasma samples (healthy people and AD patients, determined by neurologists based on clinical symptoms and examination results) of the antibody pairing.

[0061] The monoclonal antibody targeting phosphorylated Tau protein 217 and the monoclonal antibody targeting non-phosphorylated Tau protein 1-124aa were coated or HRP-labeled, and the gradient-diluted phosphorylated modified full-length Tau protein was detected. Five pairs of combinations with strong detection values ​​at 10pg / ml were selected (see the table below) for downstream single-molecule immune platform testing. From the results, it can be seen that the screened paired combinations are all coated with monoclonal antibodies against non-phosphorylated Tau protein.

[0062] Table 2 Five pairs of combinations with strong readings at 10 pg / ml

[0063]

[0064] Example 6 Detection of phosphorylated pTau217 protein in plasma of healthy subjects and AD patients using a single molecule immunoassay platform

[0065] The paired combinations screened by the ELISA sandwich method were tested on the single molecule immune platform. The plasma samples of 40 healthy people and 40 AD patients were tested. It was found that combination 2, Tau124-3 as the capture antibody and pTau217-32 as the detection antibody, can effectively distinguish AD patients from healthy people on the single molecule immune platform. The specific visualization effect of the distinction is shown in Figure 2 The AUC of the ROC curve for this method is 0.852, which can basically meet the early warning diagnosis of AD. The AUC result of the ROC curve can be seen Figure 3 .

[0066] Monoclonal antibody gene sequence

[0067] Tau124-3 mAb VH:

[0068] Nucleotide sequence:

[0069] Cagcgtgagctgcagcagtctggggctgaactggtgaggtctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatttccactgggtaaagcagacacctggacagggcctggaatggattggatgtatttatcctggaaatggtggtactaactacagtcagaagttcaggggcaaggccacattgactgcagacacatcatccagcacagcctacatgcagatcagcagcctgacatctgaagactctgcggtctatttctgtgcaagaagctacggtactagttacgtgggtgctatggactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO.4)

[0070] Amino acid sequence

[0071] QRELQQSGAELVRSGASVKMSCKASGYTFTSYNFHWVKQTPGQGLEWIGCIYPGNGGTNYSQKFRGKATLTADTSSSTAYMQISSLTSEDSAVYFCARSYGTSYVGAMDYWGQGTSVTVSS (SEQ ID NO.5)

[0072] CDR amino acid sequence

[0073] CDR1: GYTFTSYN (SEQ ID NO.6)

[0074] CDR2: IYPGNGGT (SEQ ID NO.7)

[0075] CDR3: ARSYGTSYVGAMDY (SEQ ID NO.8)

[0076] Tau124 - 3 monoclonal antibody VK:

[0077] Nucleotide sequence:

[0078] Gacatccagatgacacaatctccatcctccttatctgcctctctgggagaaagagtcagtctcacttgtcgggcaagtcaggaaattagtggttacttaagctggcttcagcagaaaccagatggaactattaaacgcctgatctacgccgcatccactttagattc tggtgtcccaaaaaggttcagtggcagtaggtctgggtcagattattctctcaccatcagcagccttgagtctgaagattttgcagactattactgtctacaatatactatttatcctcccacgttcggaggggggaccaagctggaaataaaacgtacggtg (seq ID NO.9)

[0079] Amino acid sequence

[0080] DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYTIYPPTFGGGTKLEIKRTV (SEQ ID NO.10)

[0081] CDR amino acid sequences

[0082] CDR1: QEISGY (SEQ ID NO.11)

[0083] CDR2: AAS (Due to WIPO sequence annotation, SEQ ID NO.12 is AAST)

[0084] CDR3: LQYTIYPPT(SEQ ID NO.13)

[0085] pTau217-32 mAb VH:

[0086] Nucleotide sequence

[0087] Gaggtgcagctggtggagtctggagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggctacaccttcagaaactactggctaggttgggtaaagcagaggcctggacatggacttgagtggattggagatatttaccctggaggtggttatactaactacaatgagaagttcaagggcaaggccacactgactgcagacacatcctccagcactgcctacatgcagctcaggagcctgacatctgaggactctgctgtctatttctgtgtagtaagtgactactggggccaaggcgccactctcaccgtctcctca (SEQ ID NO.14)

[0088] Amino acid sequence

[0089] EVQLVESGAELVRPGTSVKISCKASGYTFRNYWLGWVKQRPGHGLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLRSLTSEDSAVYFCVVSDYWGQGATLTVSS (SEQ ID NO.15)

[0090] CDR amino acid sequence

[0091] CDR1: GYTFRNYW (SEQ ID NO.16)

[0092] CDR2: IYPGGGYT (SEQ ID NO.17)

[0093] CDR3: VVSDY (SEQ ID NO.18)

[0094] pTau217-32 monoclonal antibody VK

[0095] Nucleotide sequence:

[0096] Gacatcctgatgacccaatctccagcttctttggctgtgtctctaggacagagagtcaccatctcctgcagagccagtgaaagcgttgaatattctggcataagtttgctgcagtggtaccaacagaaaccaggacagtcacccaaactcctcatctatgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaccttccatcctgtggaggaggatgatattgcaatgtatttctgtcaacaaagtaggaaggttccttcgacattcggtggaggcaccaagctggagctgaaa(SEQ ID NO.19)

[0097] Amino acid sequence

[0098] DILMTQSPASLAVSLGQRVTISCRASESVEYSGISLLQWYQQKPGQSPKLLIYAASNVESGVPARFSGSGSGTDFSLTFHPVEEDDIAMYFCQQSRKVPSTFGGGTKLELK(SEQ ID NO.20)

[0099] CDR amino acid sequence

[0100] CDR1: ESVEYSGISL(SEQ ID NO.21)

[0101] CDR2: AAS (Due to the annotation of WIPO sequence, SEQ ID NO.22 is AASN)

[0102] CDR3: QQSRKVPST(SEQ ID NO.23)。

Claims

1. A monoclonal antibody against pTau217 protein phosphorylated at position 217, characterized in that: The heavy chain CDR amino acid sequence of the monoclonal antibody against the pTau217 protein phosphorylated at position 217 is H-CDR1: GYTFRNYW, H-CDR2: IYPGGGYT, H-CDR3: VVSDY; the light chain CDR amino acid sequence of the monoclonal antibody against the pTau217 protein phosphorylated at position 217 is L-CDR1: ESVEYSGISL, L-CDR2: AAS, L-CDR3: QQSRKVPST.

2. The monoclonal antibody against pTau217 phosphorylated at position 217 according to claim 1, wherein the heavy chain amino acid sequence of the monoclonal antibody against pTau217 phosphorylated at position 217 is SEQ ID NO.15, and / or the light chain amino acid sequence of the monoclonal antibody against pTau217 phosphorylated at position 217 is SEQ ID NO.

20.

3. Use of the monoclonal antibody against pTau217 phosphorylated at position 217 according to claim 1 or 2 in the preparation of a kit for diagnosing Alzheimer's disease or a single molecule immunoassay platform.

4. The use according to claim 3, wherein the kit is an ELISA kit.

5. The use according to claim 4, wherein the monoclonal antibody against pTau217 protein phosphorylated at position 217 in the single molecule immunoassay platform is a detection antibody, and the single molecule immunoassay platform further comprises a monoclonal antibody against Tau protein as a capture antibody.

6. The use according to claim 5, wherein the heavy chain CDR amino acid sequence of the Tau protein monoclonal antibody is H-CDR1: GYTFTSYN, H-CDR2: IYPGNGGT, H-CDR3: ARSYGTSYVGAMDY; the light chain CDR amino acid sequence of the Tau protein monoclonal antibody is L-CDR1: QEISGY, L-CDR2: AAS, L-CDR3: LQYTIYPPT.

7. A Tau protein monoclonal antibody, characterized in that: The heavy chain CDR amino acid sequence of the Tau protein monoclonal antibody is H-CDR1: GYTFTSYN, H-CDR2: IYPGNGGT, H-CDR3: ARSYGTSYVGAMDY; the light chain CDR amino acid sequence of the Tau protein monoclonal antibody is L-CDR1: QEISGY, L-CDR2: AAS, L-CDR3: LQYTIYPPT.

8. The Tau protein monoclonal antibody according to claim 7, wherein the heavy chain amino acid sequence of the Tau protein monoclonal antibody is SEQ ID NO.5, and / or the light chain amino acid sequence of the Tau protein monoclonal antibody is SEQ ID NO.

10.

9. A carrier, characterized in that The vector comprises the nucleic acid shown in SEQ ID NO.4 and SEQ ID NO.9, or the vector comprises the nucleic acid shown in SEQ ID NO.14 and SEQ ID NO.

19.

10. A method for producing a monoclonal antibody against pTau217 protein phosphorylated at position 217 according to claim 1 or 2 or a monoclonal antibody against Tau protein according to claim 7 or 8, the method comprising culturing a host cell, wherein the host cell comprises the vector according to claim 9.

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