Monoclonal antibody combined with 181-site threonine phosphorylated microtubule associated protein tau and application thereof

Abstract

The invention discloses two monoclonal antibodies which are specifically combined with a microtubule associated protein tau (microtubule associated protein with 181 Thrphorylation, pTau181) of which the 181 site is phosphorylated by threonine, and a preparation method of the monoclonal antibodies. The invention provides a variable domain amino acid sequence of the two monoclonal antibodies, and a complementary determining region (CDR) amino acid sequence contained in the variable domain amino acid sequence and the CDR amino acid sequence contained in the variable domain amino acid sequence and the CDR amino acid sequence. The two monoclonal antibodies provided by the invention both have the capabilities of specific recognition and remarkable combination with pTau181. The two antibodies are used as reagent raw materials and can be used for development and production of pTau181 clinical in vitro diagnosis (IVD) medical equipment products based on an immunological detection principle.

Description

Technical Field

[0001] This invention relates to two monoclonal antibodies that specifically bind to microtubule-associated protein tau (pTau181) phosphorylated at threonine position 181, and the application of these antibodies in immunoassay technology and product development and production. Background Technology

[0002] Tau, a microtubule-associated protein, is a protein that plays a supporting role in neuronal axons. Under normal conditions, it maintains the structure and function of neurons by regulating the stability of microtubules.

[0003] Alzheimer's disease (AD) is a common neurodegenerative disease characterized by neurofibrillary tangles and the deposition of β-amyloid plaques within neurons. In AD patients, tau protein undergoes aberrant phosphorylation of threonine residues at sites 181, 217, and 231 (pTau181, pTau 217, pTau 231, etc.), accumulating to form neurofibrillary tangles, leading to neuronal structural damage and functional loss. Studies have shown that phosphorylated tau protein levels are significantly elevated in the cerebrospinal fluid of AD patients, and the degree of increase is positively correlated with the degree of cognitive decline. Phosphorylated tau protein serves as a diagnostic biomarker for Alzheimer's disease, providing strong support for early intervention and treatment. Given that the levels of total Tau protein and phosphorylated Tau at threonine 181 (pTau181) in cerebrospinal fluid (CSF) are significantly elevated during the progression of Alzheimer's disease (AD), especially with pTau181 concentrations being 3 to 4 times higher than normal, and that plasma pTau181 concentrations are correlated with those in CSF, this provides a powerful biomarker for distinguishing AD from other non-AD neurodegenerative diseases.

[0004] The core of pTau181 immunoassay technology and products is a monoclonal antibody that specifically recognizes pTau181. The antigen-binding sensitivity, specificity, and pairing ability of this monoclonal antibody largely determine the quality of the test kit. Clinical testing for pTau181 primarily employs immunodiagnostic methods such as chemiluminescence and enzyme-linked immunosorbent assay (ELISA). Immunodiagnostics utilizes the specific reaction between antigens and antibodies to measure the concentration of disease markers in the body, thereby assessing the body's health status. It is widely used in infectious diseases, heart diseases, tumors, and pregnancy testing.

[0005] The low concentration of pTau181 in samples necessitates that antibodies used for its detection possess extremely high affinity to ensure effective capture and recognition of the target molecule. Simultaneously, antibody specificity—its ability to accurately distinguish and bind tightly to pTau181 rather than other proteins—is also essential. Given that the key to pTau181 identification lies in recognizing a single phosphorylation site, this undoubtedly places extremely high demands on antibody specificity. Currently, the available pTau181 monoclonal antibodies, especially those without cross-reactivity with phosphorylation sites of other important tau proteins such as pTau217 and pTau231, and their accompanying detection technologies, are extremely limited. Furthermore, they generally suffer from insufficient sensitivity, poor specificity, and discrepancies between detection results and clinicopathological characteristics, severely restricting the practical application of pTau181 as a reliable biomarker. Therefore, high-quality anti-pTau181 monoclonal antibodies are a crucial condition for the development and production of pTau181 immunoassay kits. Summary of the Invention

[0006] To address the aforementioned issues, this invention employs a strategy of simultaneous screening for immunorecognition of natural full-length phosphorylated tau protein and recognition of multiple epitopes (pTau181, Tau181, pTau217, pTau231) for antibody preparation. This strategy ensures both antibody recognition of the naturally active phosphorylated tau protein antigen and precise recognition of threonine phosphorylation at position 181. Using this strategy, a monoclonal antibody was prepared that specifically recognizes and significantly binds to the pTau181 antigen (without binding activity to the tau antigen (Tau181) without threonine phosphorylation at position 181). This antibody, while binding to threonine phosphorylation at position 181 of the tau protein antigen, also exhibits dependent recognition of specific key amino acids adjacent to threonine at position 181, thus possessing the advantages of high sensitivity and strong specificity. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the above-mentioned monoclonal antibody were obtained using molecular biology methods, and the complementarity-determining region (CDR) sequences contained therein were identified.

[0007] Specifically, the present invention adopts the following technical solution:

[0008] A monoclonal antibody or its antigen-binding moiety that binds to the pTau181 antigen is named pTau181-1, and its protein sequence includes:

[0009] The amino acid sequence is as shown in SEQ ID NO 1, specifically the complementarity-determining region CDR1 of the heavy chain variable region.

[0010] The amino acid sequence is as shown in SEQ ID NO 2, specifically the complementarity-determining region (CDR2) of the heavy chain variable region.

[0011] The amino acid sequence is as shown in SEQ ID NO 3, specifically the complementarity-determining region (CDR3) of the heavy chain variable region.

[0012] The amino acid sequence is as shown in SEQ ID NO 4, specifically the complementarity-determining region CDR1 of the light chain variable region.

[0013] The amino acid sequence is as shown in SEQ ID NO 5, specifically the complementarity-determining region CDR2 of the light chain variable region.

[0014] The amino acid sequence is as shown in SEQ ID NO 6, specifically the complementarity-determining region CDR3 of the light chain variable region.

[0015] Furthermore, the above protein sequence contains the amino acid sequence of the heavy chain variable region as listed in SEQ ID NO 13, and the amino acid sequence of the light chain variable region as listed in SEQ ID NO 14.

[0016] A monoclonal antibody or its antigen-binding moiety that binds to the pTau181 antigen is named pTau181-2, and its protein sequence includes:

[0017] The amino acid sequence is as shown in SEQ ID NO 7, specifically the complementarity-determining region CDR1 of the heavy chain variable region.

[0018] The amino acid sequence is as shown in SEQ ID NO 8, specifically the complementarity-determining region CDR2 of the heavy chain variable region.

[0019] The amino acid sequence is as shown in SEQ ID NO 9, specifically the complementarity-determining region (CDR3) of the heavy chain variable region.

[0020] The amino acid sequence is as shown in SEQ ID NO 10, specifically the complementarity-determining region CDR1 of the light chain variable region.

[0021] The amino acid sequence is as shown in SEQ ID NO 11, specifically the complementarity-determining region CDR2 of the light chain variable region.

[0022] The amino acid sequence is as shown in SEQ ID NO 12, specifically the complementarity-determining region (CDR3) of the light chain variable region.

[0023] Furthermore, the above protein sequences contain amino acid sequences of the heavy chain variable region as listed in SEQ ID NO 15, and amino acid sequences of the light chain variable region as listed in SEQ ID NO 16.

[0024] The monoclonal antibody or its antigen-binding portion that binds to the pTau181 antigen described in this invention may also be a protein sequence obtained by amino acid mutation or modification using the aforementioned pTau181-1 heavy chain and light chain variable region amino acid sequences as template sequences.

[0025] The monoclonal antibody or its antigen-binding portion that binds to the pTau181 antigen described in this invention may also be a protein sequence obtained by amino acid mutation or modification using the aforementioned pTau181-2 heavy chain and light chain variable region amino acid sequences as template sequences.

[0026] The present invention also relates to immunoglobulins or their antigen-binding portions thereof, or genetically engineered derivatives thereof, antibody conjugates comprising the aforementioned antibodies or their antigen-binding portions, hybridoma cells secreting the aforementioned antibodies or their antigen-binding portions, nucleic acids encoding the aforementioned antibodies or their antigen-binding portions, expression vectors comprising the aforementioned nucleic acids, and host cells comprising the aforementioned expression vectors.

[0027] Compared to existing technologies, the advantages of this invention are: (1) It discloses innovative amino acid sequences of the variable region and CDR region of monoclonal antibodies that can be used to detect pTau181. The monoclonal antibodies (pTau181-1 and pTau181-2) achieve a half-maximal effective concentration (EC50) for antigen recognition. 50 (1) The concentrations can reach 0.298 and 0.201 μg / ml, respectively. (2) The invented monoclonal antibodies (pTau181-1 and pTau181-2) accurately recognize phosphorylated threonine at position 181 and certain specific amino acids nearby, while having no cross-reactivity with tau antigen phosphorylated at position 217 (pTau217) and tau antigen phosphorylated at position 231 (pTau231). This allows them to distinguish between the two equally important tau antigen phosphorylation sites, pTau217 and pTau231, achieving specific and accurate detection of the pTau181 phosphorylation site. Attached Figure Description

[0028] Figure 1 ELISA detects the recognition activity and cross-reactivity of antibodies against antigens. "tau Ag": phosphorylated tau antigen; "SP pT181": phosphorylated polypeptide with threonine at position 181 (15 amino acids before and after threonine at position 181 of tau antigen); "SPT181": non-phosphorylated polypeptide with threonine at position 181 (15 amino acids before and after threonine at position 181 of tau antigen); "SP pT217": phosphorylated polypeptide with threonine at position 217 (15 amino acids before and after threonine at position 217 of tau antigen); "SP pT231": phosphorylated polypeptide with threonine at position 231 (15 amino acids before and after threonine at position 231 of tau antigen).

[0029] Figure 2: Half-maximal effect concentration (EC50) of antibody for antigen recognition as determined by ELISA 50 ), Coating antigen: SP pT181, phosphorylated polypeptide of threonine at position 181 (15 amino acids before and after threonine at position 181 of tau antigen). Figure 2A Detection of antigen recognition by monoclonal antibody pTau181-1 Figure 2B Detection of antigen recognition by monoclonal antibody pTau181-2. Detailed Implementation

[0030] As used in this invention, the term "antibody" refers to any form of antibody or fragment thereof capable of exhibiting the desired biological activity. Therefore, it is used in the broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity.

[0031] The term "specific binding" as used in this invention is a well-known term in the art, and methods for determining such specific binding of antibodies to antigens are also well-known in the art. For example, in some embodiments, "specific binding" means that the antibody binds to the intended target but does not bind significantly to other targets. Compared to binding to other epitopes, the antibody binds to the intended target epitope with significantly increased affinity and / or for a longer duration.

[0032] As used in this invention, the term "antigen-binding moiety" includes a segment or derivative of an antibody that substantially retains its binding activity. Therefore, the term "antigen-binding moiety" refers to a portion of a full-length antibody, typically its antigen-binding region or variable region. The antigen-binding moiety may also include conserved amino acid substitutions that do not substantially alter its binding activity.

[0033] The “CDR” used in this invention is in accordance with the IMGT numbering system “THE INTERNATIONAL IMMUNOGENETICS INFORMATION”. The numbering is done through (www.imgt.org). As is common knowledge in the field, "CDR" can also be labeled and defined in other ways known in the field, including but not limited to the Kabat numbering system and the Chothia numbering system, and the tools and websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php) and the abysis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0034] The term "EC" as used in this invention 50 "Collapse" refers to the concentration for 50% of the maximal effect (EC50). 50 ), refers to the concentration that can cause 50% of the maximum effect.

[0035] The following describes the specific preparation and testing process of this monoclonal antibody. Unless otherwise specified, all reagents and materials used in the experimental process described in this invention are conventional commercial reagents and materials, and the methods used are standard experimental methods (for details, please refer to "Antibody Technology Experimental Guide" (E. Harlow, Science Press, 2005) and "Antibody Preparation and Use Experimental Guide" (GC Howard, Science Press, 2010). Unless otherwise specified, all academic terms and their English abbreviations mentioned shall be used in accordance with the provisions of "Immunology Terminology" (Science Press, 2008) compiled by the National Committee for Terminology in Science and Technology.

[0036] The mouse myeloma cell line SP2 / 0 was purchased from the Cell Bank of the Chinese Academy of Sciences, and the experimental animals were purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0037] Example 1 Antigen Preparation

[0038] (1) The tau antigen coding gene sequence was found in the NCBI database, and the tau antigen gene (protein N-terminus with 6×His tag) was synthesized (Sangon Biotech) into the pcDNA3.1 mammalian expression vector using synthetic biology methods.

[0039] (2) Tau antigen protein was prepared using transient transfection expression technology in mammalian cells. Specific experimental procedures were performed according to the ExpiCHO protocol. TM The instructions for use with the expression system kit (A29133, Thermo Fisher Scientific) were followed.

[0040] (3) Tau protein was purified by affinity using Ni-IMAC magnetic beads (A50588, Thermo Fisher Scientific). The specific experimental procedures were performed in accordance with the relevant descriptions in the product manual.

[0041] (4) The purified tau protein was phosphorylated in vitro using AMPK series kinases (purchased from Thermo Fisher Scientific). The specific experimental procedures were carried out in accordance with the relevant descriptions in the product manual to prepare phosphorylated tau antigen protein (tauAg).

[0042] (5) Antibody cross-reactivity screening was performed using a 181-threonine phosphorylated tau epitope peptide (SP pTau181) and a 181-threonine non-phosphorylated tau epitope peptide (SP Tau181). The peptides consisted of 15 amino acids before and after the 181Thr amino acid (Sangon Biotech), with a purity >90%. Simultaneously, antibody cross-reactivity screening was performed using a 217-threonine phosphorylated tau epitope peptide (SP pTau217) and a 231-threonine phosphorylated tau epitope peptide (SP pTau231). Both peptides were prepared using the same design strategy and preparation method as SP pTau181.

[0043] Example 2 Antibody Preparation

[0044] 1. Animal immunization

[0045] (1) The experimental animals were 6-week-old female Balb / c mice. They were housed in a clean environment.

[0046] (2) Immunization schedule: TauAg was mixed with Freund's complete adjuvant (purchased from Sigma-Aldrich), emulsified, and then administered via subcutaneous injection at multiple sites. The dose of tauAg was 0.05-0.2 mg / time / animal. The first immunization used complete Freund's adjuvant, and booster immunizations used incomplete Freund's adjuvant (purchased from Sigma-Aldrich). The interval between each immunization was 3 weeks, for a total of 3 immunizations.

[0047] (3) After the third immunization and before cell fusion, mice were given a recall stimulus. 0.05-0.2 mg of antigen tauAg was dissolved in 0.5 ml of pH 7.2 phosphate buffer (PBS) and injected intraperitoneally.

[0048] (4) Three days after recalling the stimulus, cell fusion and hybridoma construction were carried out.

[0049] 2. Construction of hybridoma cell lines

[0050] (1) Preparation of feeder cell suspension one day before cell fusion: Inject approximately 2 ml of incomplete culture medium (RPMI 1640, purchased from Thermo Fisher Scientific) into the peritoneal cavity of mice using a syringe, then aspirate, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in complete culture medium (RPMI 1640 + 10% FBS) with added fetal bovine serum to obtain peritoneal cells, and adjust the cell concentration to 5 × 10⁶ cells / mL. 5 Add 50 μl / well to a 96-well cell culture plate and incubate at 37°C in a 5% CO2 incubator.

[0051] (2) Take logarithmically growing myeloma cells SP2 / 0, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in incomplete culture medium (RPMI 1640) and count them. Take the required number of cells and wash them twice with incomplete culture medium.

[0052] (3) Take the spleen of the mouse after immunization recall stimulation, break it up, remove large pieces of tissue, prepare an immune spleen cell suspension, and wash it twice with incomplete culture medium.

[0053] (4) Mix myeloma cells and spleen cells at a ratio of 1:10 or 1:5, wash once with incomplete culture medium in a 50ml plastic centrifuge tube at 1200rpm for 8 minutes.

[0054] (5) Discard the supernatant and use a dropper to remove the residual liquid to avoid affecting the concentration of polyethylene glycol (PEG).

[0055] (6) Gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly.

[0056] (7) Fusion: Add 1 ml of 50% PEG (Hybri-Max) preheated at 37°C. TM Add Sigma-Aldrich (molecular weight 1450) while stirring. Allow to react for 90 seconds.

[0057] (8) Slowly add the incomplete culture medium preheated to 37°C to terminate the PEG reaction. Centrifuge at 800 rpm for 6 minutes.

[0058] (9) Discard the supernatant and gently suspend the culture in about 6 ml of complete culture medium.

[0059] (10) Add complete culture medium according to the number of 96-well culture plates used. Adjust the cell density to 5 × 10⁶ cells / well based on the number of spleen cells. 5 / ml.

[0060] (11) Add the fused cell suspension to a 96-well plate containing feeder cells, 50 μl / well, and incubate at 37°C in a 5% CO2 incubator.

[0061] (12) After 24 hours, add 100 μl of complete culture medium containing 2×HAT (purchased from Sigma-Aldrich) per well. Incubate at 37°C in a 5% CO2 incubator for 3-7 days.

[0062] 3. Screening for positive monoclonal antibodies

[0063] (1) Preparation of feeder cell suspension: Inject approximately 2 ml of incomplete culture medium (RPMI 1640) into the peritoneal cavity of mice using a syringe, then aspirate, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in complete culture medium (RPMI 1640 + 10% FBS) with added fetal bovine serum to obtain peritoneal cells. Adjust the cell concentration to 5 × 10⁻⁶ cells / mL. 5 / ml.

[0064] (2) Count the cells in the positive wells and adjust the cell count to 1-5 × 10⁻⁵. 3 / ml

[0065] (3) Take 130 cells and place them in 6.5 ml of complete culture medium containing feeder cells, i.e., 20 cells / ml, 100 μl / well, and add them in three rows (A, B, C) to make 2 cells per well. Add another 2.9 ml of complete culture medium containing feeder cells to the remaining 2.9 ml of cell suspension, resulting in 10 cells / ml, and add them in three rows (D, E, F) to make 1 cell per well. Add another 2.2 ml of complete culture medium containing feeder cells to the remaining 2.2 ml of cell suspension, resulting in 5 cells / ml, and add them in two rows (G, H) to make 0.5 cells per well.

[0066] (4) After culturing for 4-5 days, small cell clones can be seen under an inverted microscope. Add 200 μl of complete culture medium per well.

[0067] (5) On days 8-9, cell clones are visible to the naked eye, and antibody testing should be performed promptly. Note: Initially cloned hybridoma cells need to be added to complete culture medium with HT (purchased from Sigma-Aldrich).

[0068] The antibody testing steps are as follows:

[0069] (a) Antigen coating. Prepare a 10 μg / ml coating solution by dissolving the antigen (tauAg or the above phosphorylated / non-phosphorylated peptides: SPpTau181, SPTau181, SP pTau217, SP pTau231) in 50 mM carbonate buffer (pH 9.6). Mix well. Add 100 μl to each well of a 96-well ELISA plate. Incubate overnight at 4°C.

[0070] (b) Blocking. Wash the plate. Add blocking buffer (0.5% m / v bovine serum albumin BSA dissolved in PBS), 200 μl / well. Incubate at 37°C for 2 hours.

[0071] (c) Add primary antibody (sample to be tested). Wash plate. Add 100 μl of the diluted primary antibody solution (cell culture supernatant from the hybridoma screening process) to each well. Incubate at 37°C for 2 hours.

[0072] (d) Add secondary antibody. Wash the plate. Add the diluted secondary antibody (ab6789, abcam, 1:2000 dilution) to the wells. 100 μl / well. Incubate at 37°C for 1 hour.

[0073] (e) Color development. Wash the plate. Prepare the TMB substrate and add it to each well (100 μl / well). Develop the color for 5-10 minutes. Stop the reaction by adding 100 μl / well of 2M sulfuric acid.

[0074] (f) Detection. The absorbance (OD) value of the solution at 450 nm was measured using an ELISA reader.

[0075] Based on the absorbance value, cells that are positive only for the pTau181 peptide were selected, and the clones were further dispersed by limiting dilution until single-well monoclonal positive cells were selected.

[0076] (6) The titer of the monoclonal positive cell supernatant was determined (diluted 5 times, 25 times, and 125 times respectively), and the monoclonal cells that could produce antiphosphorylated pTau181 antibodies with high antigen binding capacity were finally screened and labeled as C-pTau181-1 and C-pTau181-2 respectively.

[0077] 4. Sequencing of the variable region genes of C-pTau181-1 and C-pTau181-2 antibodies from positive clones.

[0078] (1) RNA extraction: Trypsin digestion to suspend hybridoma cells, centrifuge at 1000 rpm for 5 minutes, wash twice with PBS, and use a hemocytometer to identify cells up to 10⁻⁶. 7 Add 1 ml of TRI REAGENT (purchased from Sigma-Aldrich) for lysis, mix well by pipetting, and after 5 min at room temperature, add 200 μl of chloroform (shake vigorously, incubate at room temperature for 5 min, then on ice for 5 min); centrifuge at 12000 g for 15 min at 4 °C, then take 600 μl of the aqueous phase and add an equal volume of isopropanol (incubate at room temperature for 5 min, then on ice for 5 min). Immediately after adding the isopropanol, invert the container 5-10 times to mix well; centrifuge at 12000 g for 15 min at 4 °C, discard the supernatant, and aspirate dry with a pipette; wash once with 1 ml of 75% ethanol, mix well by pipetting, centrifuge at 7500 g for 5 min at 4 °C, and discard the supernatant; dry in air at room temperature for 20 min, then add 50 μl of ddH2O (DEPC treated); measure the OD value, and perform 1% Agarose electrophoresis to determine the quality and concentration of RNA.

[0079] (2) RT-PCR: After mixing the reverse transcription primers (Random Primer) and RNA in the specified ratio, incubate at 70°C for 10 min, then on ice for 2-3 min. Next, add the other reagents for the reverse transcription system (purchased from Thermo Fisher Scientific), incubate at 37°C for 1.5 h, then at 95°C for 5 min, on ice, and freeze at -20°C. After reverse transcription, perform polymerase chain reaction (PCR) using housekeeping gene HPRT primers to detect the quality of the cDNA obtained from RNA reverse transcription.

[0080]

[0081] (3) 5′race PCR: Utilizing the terminal transferase activity of TdT enzyme, 3-5 dG residues are automatically added when reverse transcription reaches the 5′ end of the first strand. Then, using PolyC (Universal Primer), a universal primer containing partial adapter sequences, as the upstream primer and a gene-specific primer as the downstream primer, PCR is performed using G-cDNA as a template to amplify the cDNA fragment at the 5′ end of the target gene. After obtaining the cDNA, 1 μl of cDNA is added to 3 μl of dGTP and 2 μl of TdT enzyme (purchased from Promega), incubated at 37°C for 15 minutes, and then quickly placed in a 70°C incubator for 10 minutes to obtain G-tailed cDNA. In a 50 μl PCR reaction system, 1 μl of G-tailed cDNA template, 1 μl of PolyC, and specific primers (Table 1) are added, and amplification and extension are performed using 0.5 μl of Taq Platium (purchased from Qiagen).

[0082] Table 2 Primer sequences for amplifying the variable region gene of the antibody

[0083]

[0084] The reaction conditions are:

[0085]

[0086] (4) Enzyme digestion, ligation, and transformation: The PCR product was recovered from the gel. An A tail was added to the 3' end of the smoothed DNA fragment, and then ligated with a pGEM-T Vector (purchased from Promega). Ligation was performed at 16°C for at least 3 hours for transformation, or overnight at 4°C. After ligation, competent DH5α cells were removed and placed at room temperature until partially thawed. 5 μl of recombinant plasmid was added and gently stirred until mixed. The cells were incubated on ice for 30 minutes, subjected to a heat shock at 42°C for 45-50 seconds, and then placed on ice for 2-3 minutes. 500 μl of Amp-LB liquid medium was added, and the cells were incubated at 37°C on a shaker for 20-30 minutes. 300 μl of the bacterial culture was plated and incubated upside down at 37°C for 12-16 hours. After obtaining a series of colonies, positive clones were selected using a blue-white screening system, and their positivity was verified again using PCR and enzyme digestion systems.

[0087]

[0088] (5) Sequencing and sequence analysis: After obtaining positive bacteria, plasmid sequencing was performed (Shanghai Sangon Biotech). Following the sequencing results, the IMGT database V gene alignment tool was used to analyze the sequencing results, determining the amino acid sequences of the antibody's heavy and light chain variable regions and their complementary determinant regions as follows:

[0089] The full-length amino acid sequence of the H chain (heavy chain) variable region of pTau181-1 is shown in SEQ ID NO 13, and the full-length amino acid sequence of the L chain (light chain) variable region is shown in SEQ ID NO 14; among which,

[0090] SEQ ID NO 13—

[0091] QVKLEQSGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTADYSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCARESLRREGFDYWGQGTTLTVSSSEQ ID NO 14—

[0092] DIVLTQTPSSLSVSAGAKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPFTFGAGTKLELK

[0093] Heavy chain variable region complementarity-determining region CDR1 SEQ ID NO 1 GFTFTDYY Heavy chain variable region complementarity-determining region CDR2 SEQ ID NO 2 IRNKANGYTA Heavy chain variable region complementarity-determining region CDR3 SEQ ID NO 3 ARESLRREGFDY Light chain variable region complementarity-determining region CDR1 SEQ ID NO 4 QSLLNSGNQKNY Light chain variable region complementarity-determining region CDR2 SEQ ID NO 5 GAS Light chain variable region complementarity-determining region CDR3 SEQ ID NO 6 QNDHSYPFT

[0094] The full-length amino acid sequence of the H chain (heavy chain) variable region of pTau181-2 is shown in SEQ ID NO 15, and the full-length amino acid sequence of the L chain (light chain) variable region is shown in SEQ ID NO 16; among which,

[0095] SEQ ID NO 15—

[0096] QVQLEESGSELVRPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGNIYPGSGNTIYDEKFKNRATTLTVDTSSNTAYMHLTSLTSEDSAVYYCTRGEIRLRDYAMAYWGQGTSVTVSSSEQ ID NO 16—

[0097] DIVLTQTPLSLPVSLGDQASISCRSRQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYTFGGGTKLEIK

[0098] Heavy chain variable region complementarity-determining region CDR1 SEQ ID NO 7 GYTFTNYW Heavy chain variable region complementarity-determining region CDR2 SEQ ID NO 8 IYPGSGNT Heavy chain variable region complementarity-determining region CDR3 SEQ ID NO 9 TRGEIRLRDYAMAY Light chain variable region complementarity-determining region CDR1 SEQ ID NO 10 QSIVHSNGNTY Light chain variable region complementarity-determining region CDR2 SEQ ID NO 11 KVS Light chain variable region complementarity-determining region CDR3 SEQ ID NO 12 FQGSHVPYT

[0099] The above sequence adopts "THE INTERNATIONAL IMMUNOGENETICS INFORMATION" The database-related tools (www.imgt.org) were used for analysis and identification.

[0100] 5. Antibody production and purification

[0101] (1) Recovery of positive clones C-pTau181-1 and C-pTau181-2: Incubate at 37℃ and 5% CO2 for 2-3 days. Then, culture the cells in serum-free SFM medium (12045084, Thermo Fisher Scientific) for 3 days. Collect the culture supernatant, centrifuge, and freeze at -20℃.

[0102] (2) Dilute the culture supernatant 4 times with pre-cooled phosphate buffer (PBS), centrifuge at 2000 rpm for 5 minutes, and remove the precipitate. Slowly add saturated ammonium sulfate solution dropwise to the supernatant at 4°C while stirring, until the final solution has a concentration of 50% ammonium sulfate.

[0103] (3) Place this solution on ice for 30-60 minutes, then centrifuge at 5000 rpm for 10 minutes and discard the supernatant.

[0104] (4) Dissolve the precipitate in Tris-HCl buffer (40mM NaCl).

[0105] (5) Place the bag into a dialysis bag and dialyze to remove salt in Tris-HCl buffer (20mM NaCl).

[0106] (6) Centrifuge to remove precipitate.

[0107] (7) After diluting the solution (1:100 or higher), the protein content was measured at 280 nm.

[0108] (8) DEAE-cellulose column: Equilibrate with 20 mM NaCl Tris buffer. Dilute the dialyz sample equally with Tris buffer. Enter the sample into the column at a rate of 1-2 ml / min, eluting with a linear NaCl gradient. Elute the monoclonal antibody with 40 mM and 80 mM NaCl. Collect the protein peak at OD280 nm, aliquot the monoclonal antibody, and store at -20°C.

[0109] Example 3: Detection of antibody recognition and cross-reactivity with antigens (ELISA)

[0110] (1) Antigen coating. Prepare a 10 μg / ml coating solution by dissolving the antigen (tauAg from Example 1 and the above phosphorylated / non-phosphorylated peptides) in 50 mM carbonate buffer at pH 9.6. Add 100 μl to each well of a 96-well ELISA plate. Incubate overnight at 4°C.

[0111] (2) Blocking. Wash the plate. Add blocking buffer (0.5% m / v bovine serum albumin BSA dissolved in PBS), 200 μl / well. Incubate at 37°C for 2 hours.

[0112] (3) Add primary antibody (sample to be tested). Wash plate. Add the diluted primary antibody solution (monoclonal antibody purified in Example 2) to the wells, 100 μl / well. Incubate at 37°C for 2 hours.

[0113] (4) Add secondary antibody. Wash the plate. Add the diluted secondary antibody (ab6789, abcam, 1:2000 dilution) to the wells. 100 μl / well. 37℃, 1 hour.

[0114] (5) Color development. Wash the plate. Prepare the TMB substrate and add it to the wells, 100 μl / well. Develop the color for 5-10 minutes. Stop the reaction by adding 100 μl / well of 2M sulfuric acid.

[0115] (6) Detection. The absorbance (OD) value of the solution at 450 nm was measured using an ELISA reader. Figure 1As shown, pTau181-1 and pTau181-2 exhibit significant antigen recognition specificity for pTau181, and show no cross-reactivity with Tau181, pTau217, and pTau231. As controls, some hybridoma clone-secreted antibodies and some existing commercially available antibodies obtained simultaneously during the antibody screening process in Example 2 showed varying degrees of cross-reactivity with Tau181 and / or pTau217 and / or pTau231 in the cross-reactivity experiment.

[0116] (7) Plot an antibody concentration-OD value curve with antibody dilution on the x-axis and corresponding OD value on the y-axis. Use a curve fitting tool (such as a four-parameter logistic curve fitting) to determine EC. 50 The values, as shown in Figure 2, represent the half-maximal effective concentrations (EC50) of the monoclonal antibodies (pTau181-1 and pTau181-2) for antigen recognition. 50 The concentrations were 0.298 and 0.201 μg / ml, respectively.

[0117] Example 4: Verification of Genetically Engineered Antibody Expression

[0118] The antibody heavy and light chain variable regions were constructed on a mouse IgG1 / κ expression plasmid (InvivoGen) and analyzed using ExpiCHO. TM Cells were transfected using the expression system kit (A29133, Thermo Fisher Scientific), and the culture supernatant was harvested. Antibody purification was then performed using the method described in Example 2. The obtained antibody, like the antibody expressed on hybridoma cells, showed significant recognition of tau Ag (ELISA), demonstrating that the obtained antibody sequence was the correct variable region sequence.

Claims

1. Monoclonal antibodies or their antigen-binding moieties that bind to the pTau181 antigen. The characteristic is that the protein sequence contains: The amino acid sequence is as shown in SEQ ID NO 1, specifically the complementarity-determining region CDR1 of the heavy chain variable region. The amino acid sequence is as shown in SEQ ID NO 2, specifically the complementarity-determining region (CDR2) of the heavy chain variable region. The amino acid sequence is as shown in SEQ ID NO 3, specifically the complementarity-determining region (CDR3) of the heavy chain variable region. The amino acid sequence is as shown in SEQ ID NO 4, specifically the complementarity-determining region CDR1 of the light chain variable region. The amino acid sequence is as shown in SEQ ID NO 5, specifically the complementarity-determining region CDR2 of the light chain variable region. The amino acid sequence is as shown in SEQ ID NO 6, specifically the complementarity-determining region CDR3 of the light chain variable region.

2. The monoclonal antibody or its antigen-binding portion according to claim 1, characterized in that the protein sequence includes the amino acid sequence of the heavy chain variable region as listed in SEQ ID NO 13, and the amino acid sequence of the light chain variable region as listed in SEQ ID NO 14.

3. Monoclonal antibodies or their antigen-binding moieties that bind to the pTau181 antigen. The characteristic is that the protein sequence contains: The amino acid sequence is as shown in SEQ ID NO 7, specifically the complementarity-determining region CDR1 of the heavy chain variable region. The amino acid sequence is as shown in SEQ ID NO 8, specifically the complementarity-determining region CDR2 of the heavy chain variable region. The amino acid sequence is as shown in SEQ ID NO 9, specifically the complementarity-determining region (CDR3) of the heavy chain variable region. The amino acid sequence is as shown in SEQ ID NO 10, specifically the complementarity-determining region CDR1 of the light chain variable region. The amino acid sequence is as shown in SEQ ID NO 11, specifically the complementarity-determining region CDR2 of the light chain variable region. The amino acid sequence is as shown in SEQ ID NO 12, specifically the complementarity-determining region (CDR3) of the light chain variable region.

4. The monoclonal antibody or its antigen-binding portion according to claim 3, characterized in that the protein sequence includes the amino acid sequence of the heavy chain variable region as listed in SEQ ID NO 15, and the amino acid sequence of the light chain variable region as listed in SEQ ID NO 16.

5. A monoclonal antibody or its antigen-binding moiety that binds to the pTau181 antigen, characterized in that it comprises a protein sequence obtained by amino acid mutation or modification using the heavy chain and light chain variable region amino acid sequences of claim 2 as template sequences.

6. A monoclonal antibody or its antigen-binding moiety that binds to the pTau181 antigen, characterized in that it comprises a protein sequence obtained by amino acid mutation or modification using the heavy chain and light chain variable region amino acid sequences of claim 4 as template sequences.

7. An immunoglobulin or its genetically engineered derivative comprising the antibody or antigen-binding portion thereof as described in claims 1-6.

8. An antibody conjugate comprising the antibody or its antigen-binding portion as described in claims 1-6.

9. A nucleic acid encoding the antibody or its antigen-binding portion as described in claims 1-6.

10. An expression vector comprising the nucleic acid of claim 9.

11. A host cell comprising the expression vector of claim 10.

12. Hybridoma cells that secrete the antibodies of claims 1-6.

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