Monoclonal antibody binding to microtubule-associated protein tau phosphorylated at threonine 217 and use thereof

Abstract

Disclosed in the present invention is a monoclonal antibody which specifically binds to microtubule-associated protein tau phosphorylated at threonine 217 (microtubule-associated protein tau with 217 Thr phosphorylation, pTau217). The present invention provides an amino acid sequence of a variable region of the monoclonal antibody and amino acid sequences of complementarity determining regions (CDRs) contained in same. The monoclonal antibody disclosed by the present invention has the capability of specifically recognizing and remarkably binding to pTau217, and can be used as a reagent raw material for the development and production of pTau217 clinical in-vitro diagnosis (IVD) medical instruments and products based on an immunological detection principle.

Description

Monoclonal antibody binding to microtubule-associated protein tau phosphorylated at threonine 217 and application thereof TECHNICAL FIELD

[0001] The present application relates to a monoclonal antibody specifically binding to microtubule-associated protein tau phosphorylated at threonine 217 (pTau217) and the application of the antibody in immunodetection technology and product development and production. BACKGROUND

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, and its characteristic pathological changes include neurofibrillary tangles and β-amyloid plaque deposition in neurons. Although tau is phosphorylated in a normal healthy brain, it is over-phosphorylated in neurodegenerative diseases including AD. Neurofibrillary tangles found in Alzheimer's disease are caused by over-phosphorylation of tau. Phosphorylation of tau can occur at many sites, usually caused by kinases GSK3, CDK5, and results in reduced ability to bind microtubules.

[0003] In AD patients, tau protein is abnormally phosphorylated at threonine 181, 217, 231, etc. (pTau181, pTau217, pTau231, etc.), and aggregates to form neurofibrillary tangles, leading to destruction of neuronal structure and loss of function. Studies have shown that the level of phosphorylated tau protein in the cerebrospinal fluid of AD patients is significantly increased, and the degree of increase is positively correlated with the degree of cognitive decline. Phosphorylated tau protein as a marker for the diagnosis of Alzheimer's disease provides strong support for early intervention and treatment.

[0004] Recent studies have shown that in the early stages of Alzheimer's disease, the tau protein phosphorylated at threonine 217 (pTau217) in the cerebrospinal fluid and plasma is elevated. In addition, compared with pTau181, a recognized AD biomarker, cerebrospinal fluid pTau217 is more closely related to tau deposition in the brain of AD. Another study found that in multiple cohorts, the ratio of pTau217 to non-phosphorylated tau in plasma (%pTau217) was compared with the FDA-approved cerebrospinal fluid (CSF) detection method, and the performance was equivalent or superior to the FDA-approved CSF detection method in detecting Alzheimer's disease pathology. Therefore, the detection method of plasma pTau217 is expected to make up for the shortcomings of PET imaging and cerebrospinal fluid detection, improve the accessibility of Alzheimer's disease detection and the detection rate of Alzheimer's disease, and enable more patients to receive specific treatment as soon as possible.

[0005] The core of the pTau217 immunodetection technology and product is a monoclonal antibody that specifically recognizes pTau217. The antigen binding sensitivity, specificity and pairing ability of the monoclonal antibody determine the quality of the detection kit to a great extent. Immunodiagnosis is a diagnostic method that uses the specific reaction between antigen and antibody to determine the concentration of disease markers in the body, thereby judging the physical condition of the human body. It is widely used in infectious diseases, heart diseases, tumors, pregnancy detection, etc.

[0006] pTau217 is extremely small in content in samples, and the antibody used for its detection must have extremely high affinity to ensure effective capture and recognition of the target molecule. At the same time, the specificity of the antibody, that is, its ability to accurately distinguish and tightly bind pTau217 rather than other proteins, is also essential. Since the key to pTau217 identification lies in recognizing a single phosphorylation site, this undoubtedly puts a very high demand on the specificity of the antibody. The current market has very limited choices of pTau217 monoclonal antibodies, especially those that do not cross-react with other important tau protein phosphorylation sites such as pTau181 and pTau231, and the supporting detection technology. There are widespread problems such as insufficient sensitivity, poor specificity, and detection results that do not match clinical pathological characteristics, which seriously restrict the practical application of pTau217 as a reliable biomarker. Therefore, high-quality anti-pTau217 monoclonal antibodies are a key condition for the development and production of pTau217 immunodetection kits. SUMMARY

[0007] To solve the above problems, the present application provides a monoclonal antibody that specifically recognizes and binds to pTau217 antigen (and has no binding activity to tau antigen (Tau217) that is not phosphorylated at the 217th threonine). The antibody not only binds to the phosphorylated threonine at the 217th site of the tau protein antigen, but also has dependent recognition of the specific key amino acids near the 217th threonine. Therefore, it has the advantages of high sensitivity and strong specificity.

[0008] The present application uses molecular biology methods to obtain the heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences of the above-mentioned monoclonal antibody, and identifies the complementarity determining region (CDR) sequences contained therein.

[0009] Specifically, the present application uses the following technical solutions:

[0010] The monoclonal antibody or antigen-binding portion thereof that binds to the pTau217 antigen is named pTau217-1, and the protein sequence thereof comprises:

[0011] a heavy chain variable region complementarity determining region CDR1 with an amino acid sequence as set forth in SEQ ID NO 1,

[0012] a heavy chain variable region complementarity determining region CDR2 having an amino acid sequence as set forth in SEQ ID NO 2,

[0013] a heavy chain variable region complementarity determining region CDR3 having an amino acid sequence as set forth in SEQ ID NO 3,

[0014] a light chain variable region complementarity determining region CDR1 having an amino acid sequence as set forth in SEQ ID NO 4,

[0015] a light chain variable region complementarity determining region CDR2 having an amino acid sequence as set forth in SEQ ID NO 5,

[0016] a light chain variable region complementarity determining region CDR3 having an amino acid sequence as set forth in SEQ ID NO 6.

[0017] Further, the above-mentioned protein sequence comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO 7, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO 8.

[0018] The monoclonal antibody or antigen-binding portion thereof binding to the pTau217 antigen according to the present application can also be a protein sequence obtained by amino acid mutation or modification based on the pTau217-1 heavy chain and light chain variable region amino acid sequences mentioned above.

[0019] The present application also relates to an immunoglobulin or a genetic engineering product thereof of the above-mentioned antibody or antigen-binding portion thereof, an antibody conjugate comprising the above-mentioned antibody or antigen-binding portion thereof, a hybridoma cell secreting the above-mentioned antibody or antigen-binding portion thereof, a nucleic acid encoding the above-mentioned antibody or antigen-binding portion thereof, an expression vector comprising the above-mentioned nucleic acid, and a host cell comprising the above-mentioned expression vector.

[0020] The present application also relates to the use of the above-mentioned antibody or antigen-binding portion thereof in the preparation of a kit for detecting pTau217.

[0021] Compared with the prior art, the present application has the following beneficial effects: (1) innovative monoclonal antibody variable region and CDR region amino acid sequences useful for detecting pTau217 are disclosed. The monoclonal antibody (pTau217-1) has a half maximal effective concentration (EC 50) up to 0.21 pg / ml. (2) The monoclonal antibody (pTau217-1) of the application can distinguish the two equally important tau antigen phosphorylation sites, pTau181 and pTau231, by specifically recognizing the 217th phosphorylated threonine and some specific amino acids near it, and has no cross-reactivity with the 181st and 231st phosphorylated tau antigens (pTau181 and pTau231), thereby achieving specific and accurate detection of the pTau217 phosphorylation site. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1: ELISA detection of the recognition activity and cross-reactivity of the antibody to the antigen, "tauAg": phosphorylated tau antigen, "SPpT217": 217th phosphorylated threonine polypeptide (15 amino acids before and after the 217th threonine of the tau antigen), "SP T217": 217th non-phosphorylated threonine polypeptide (15 amino acids before and after the 217th threonine of the tau antigen), "SPpT181": 181st phosphorylated threonine polypeptide (15 amino acids before and after the 181st threonine of the tau antigen), "SPpT231": 231st phosphorylated threonine polypeptide (15 amino acids before and after the 231st threonine of the tau antigen).

[0023] Figure 2: ELISA detection of the half maximal effective concentration (EC50) of the pTau217-1 antibody to antigen recognition. 50 ), coated antigen: SP pT217, 217th phosphorylated threonine polypeptide (15 amino acids before and after the 217th threonine of the tau antigen). DETAILED DESCRIPTION

[0024] The term "antibody" as used herein refers to any form of antibody or fragment thereof that is capable of displaying the desired biological activity. Thus, 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, so long as they display the desired biological activity.

[0025] The term "specifically binds" as used herein is a term of art well known in the art, and methods of determining such specific binding of an antibody to an antigen are well known in the art. For example, in some embodiments, "specifically binds" means that the antibody binds to the intended target, but does not significantly bind to other targets. The antibody binds to the intended target epitope with significantly increased affinity and / or for a longer duration as compared to binding to other epitopes.

[0026] 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.

[0027] 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).

[0028] 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.

[0029] The following is a detailed description of the preparation and testing process of this monoclonal antibody. The antibody was prepared using a strategy of simultaneous screening with immunization of the natural full-length phosphorylated tau protein and recognition of multiple epitopes (pTau217, Tau217, pTau181, pTau231). This strategy ensured that the antibody recognized the natural active phosphorylated tau protein antigen and also ensured its precise recognition of threonine phosphorylation at position 217.

[0030] 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.

[0031] Mouse myeloma cell line SP2 / 0 was purchased from the Cell Bank of Chinese Academy of Sciences, and experimental animals were purchased from Shanghai Slek Experimental Animal Co., Ltd.

[0032] Example 1 Antigen preparation

[0033] (1) The tau antigen coding gene sequence was searched in the NCBI database, and the tau antigen gene (protein N-terminal 6xHis tag) was synthesized by synthetic biology method (Shanghai Genechem Co., Ltd.) on pcDNA3.1 mammalian expression vector.

[0034] (2) The tau antigen protein was prepared by using mammalian cell transient transfection expression technology. The specific experimental operation method was performed according to the description related to ExpiCHO TM Expression System Kit (A29133, Thermo Fisher Scientific).

[0035] (3) The tau protein was affinity purified by Ni-IMAC magnetic beads (A50588, Thermo Fisher Scientific). The specific experimental operation was performed according to the description related to the product instruction.

[0036] (4) The purified tau protein was phosphorylated in vitro by using AMPK series kinase (purchased from Thermo Fisher Scientific). The specific experimental operation was performed according to the description related to the product instruction to prepare the phosphorylated tau antigen protein (tauAg).

[0037] (5) The antibody cross-reactivity screening was performed by using 217 threonine phosphorylated tau epitope polypeptide (SPpT217) and 217 threonine non-phosphorylated tau epitope polypeptide (SPT217). The polypeptide range consisted of 15 amino acids before and after 217Thr amino acid (Shanghai Genechem Co., Ltd.), and the purity was >90%. Meanwhile, the antibody cross-reactivity screening was performed by using 181 threonine phosphorylated tau epitope polypeptide (SPpT181) and 231 threonine phosphorylated tau epitope polypeptide (SP pT231). The two polypeptides were prepared by using the same design strategy and preparation method as SP pT217.

[0038] Example 2 Antibody preparation

[0039] 1. Animal immunization

[0040] (1) The experimental animals were Balb / c strain female 6-week-old mice. Clean level feeding.

[0041] (2) Immunization procedure: After mixing tauAg with Freund's complete adjuvant (purchased from Sigma-Aldrich) and emulsifying, the mice were immunized by subcutaneous injection at multiple sites. The dose of tauAg was 0.05-0.2 mg / time / mouse. The first immunization was performed with complete Freund's adjuvant, and the booster immunization was performed with incomplete Freund's adjuvant (purchased from Sigma-Aldrich). The interval between each immunization was 3 weeks, and the mice were immunized for a total of 3 times.

[0042] (3) After the third immunization, the mice were subjected to recall stimulation before cell fusion. 0.05-0.2 mg of antigen tauAg was dissolved in 0.5 ml of phosphate buffer solution (PBS) at pH 7.2, and intraperitoneal injection was performed.

[0043] (4) Three days after the recall stimulation, cell fusion and hybridoma construction were performed.

[0044] 2. Construction of hybridoma cell lines

[0045] (1) Preparation of feeder cell suspension one day before cell fusion: 2 ml of incomplete culture medium (RPMI1640, purchased from Thermo Fisher Scientific) was injected into the abdominal cavity of the mouse using a syringe, and then it was sucked up. The supernatant was discarded after centrifugation at 1000 rpm for 5 minutes. The abdominal cavity cells were resuspended with complete culture medium (RPMI1640+10% FBS) containing fetal bovine serum, and the cell concentration was adjusted to 5×10 5 / ml. Then, 50 μl / well was added to a 96-well cell culture plate, and the plate was incubated in a 37°C, 5% CO2 incubator.

[0046] (2) The logarithmically growing myeloma cells SP2 / 0 were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in incomplete culture medium (RPMI1640), and the number of cells was counted. The desired number of cells was obtained, and the cells were washed twice with incomplete culture medium.

[0047] (3) The spleen of the recall stimulated mouse after immunization was crushed, and the large pieces of tissue were removed to prepare an immune spleen cell suspension. The suspension was washed twice with incomplete culture medium.

[0048] (4) The myeloma cells and spleen cells were mixed together at a ratio of 1:10 or 1:5 in a 50 ml plastic centrifuge tube, and washed once with incomplete culture medium at 1200 rpm for 8 minutes.

[0049] (5) The supernatant was discarded, and the residual liquid was sucked up with a dropper to avoid affecting the concentration of polyethylene glycol (PEG).

[0050] (6) The bottom of the centrifuge tube was gently tapped to slightly loosen the cell sediment.

[0051] (7) Fusion: Add 1 ml of 50% PEG (Hybri-Max, Sigma-Aldrich, MW 1450) pre-warmed at 37°C, while stirring. Incubate for 90 seconds. TM

[0052] (8) Slowly add incomplete medium pre-warmed at 37°C to stop the PEG action. Centrifuge at 800 rpm for 6 minutes.

[0053] (9) Discard the supernatant and suspend the cells in about 6 ml of complete medium.

[0054] (10) Add complete medium according to the number of 96-well plates used. Adjust the cell density to 5 x 10 5 / ml according to the number of spleen cells.

[0055] (11) Add the cell suspension after fusion to the 96-well plate containing feeder cells, 50 μl / well, and incubate in a 37°C, 5% CO2 incubator.

[0056] (12) After 24 hours, add complete medium containing 2 × HAT (purchased from Sigma-Aldrich) to each well, 100 μl / well. Incubate in a 37°C, 5% CO2 incubator for 3-7 days.

[0057] 3. Positive monoclonal screening

[0058] (1) Prepare a feeder cell suspension: Inject 2 ml of incomplete medium (RPMI 1640) into the abdominal cavity of a mouse using a syringe, then aspirate, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in complete medium (RPMI 1640 + 10% FBS) with fetal bovine serum to obtain abdominal cavity cells, and adjust the cell concentration to 5 x 10 5 / ml.

[0059] (2) Count the positive well cells and adjust the cell number to 1-5 x 10 3 / ml

[0060] (3) Take 130 cells and place them in 6.5 ml of complete medium containing feeder cells, i.e. 20 cells / ml, 100 μl / well, add 2 cells / well to rows A, B, and C. Add 2.9 ml of complete medium containing feeder cells to the remaining 2.9 ml of cell suspension, adjust the cell number to 10 cells / ml, 100 μl / well, add 1 cell / well to rows D, E, and F. Add 2.2 ml of complete medium containing feeder cells to the remaining 2.2 ml of cell suspension, adjust the cell number to 5 cells / ml, 100 μl / well, and add 0.5 cells / well to rows G and H.

[0061] ​(4) After 4-5 days of culture, small cell clones can be observed under an inverted microscope. Add 200 μl / well of complete medium.

[0062] (5) On day 8-9, cell clones can be observed by naked eye. Antibody detection should be performed in time. Note: The primary cloned hybridoma cells need to be supplemented with HT (purchased from Sigma-Aldrich) in the complete culture medium.

[0063] The antibody detection steps are as follows:

[0064] (a) Antigen coating. Take the antigen (tauAg or the above-mentioned phosphorylated / non-phosphorylated polypeptide: SPpT217, SP T217, SPpT181, SPpT231) and prepare a coating solution of 10 μg / ml with 50 mM carbonate buffer at pH 9.6, and mix well. Add 100 μl / well to a 96-well ELISA detection plate. Place in a 4°C refrigerator overnight.

[0065] (b) Blocking. Wash the plate. Add blocking solution (0.5% m / v bovine serum albumin BSA dissolved in PBS), 200 μl / well. Incubate in a 37°C incubator for 2 hours.

[0066] (c) Add primary antibody (sample to be tested). Wash the plate. Add the diluted primary antibody solution (cell culture supernatant during hybridoma screening) to the wells, 100 μl / well. 37°C, 2 hours.

[0067] (d) Add secondary antibody. Wash the plate. Add the diluted secondary antibody (ab6789, abcam, 1:2000 dilution for use) to the wells. 100 μl / well. 37°C, 1 hour.

[0068] (e) Color development. Wash the plate. Prepare TMB substrate and add to the wells, 100 μl / well. Color development for 5-10 minutes. Add 2M sulfuric acid 100 μl / well to stop the reaction.

[0069] (f) Detection. Detect the absorbance OD value of the solution at 450 nm with an enzyme marker.

[0070] According to the absorbance value, select cells that produce a positive reaction only to the pTau217 polypeptide, and continue to perform limited dilution and dispersion cloning until single clone positive cells are selected in a single well.

[0071] (6) Perform titer determination on the supernatant of the single clone positive cells (diluted by 5 times, 25 times, and 125 times, respectively), and finally screen the single clone cells that can produce high antigen binding force of the anti-phosphorylated pTau217 antibody, marked as C-pTau217-1.

[0072] 4. Sequencing of the variable region gene of the C-pTau217-1 antibody of the positive clone

[0073] (1) RNA extraction: trypsin digestion to hybridoma cell suspension, centrifugation at 1000 rpm for 5 minutes (min), PBS washing twice, cell counting board to determine cells to 10 7 ; add 1 ml TRI REAGENT (purchased from Sigma-Aldrich) lysis, mix with micropipette, add 200 μl chloroform (vigorously shake, room temperature for 5 min, 5 min on ice) after 5 min; high-speed centrifugation 12000g, 15 min, 4℃, take the water phase (600 μl) and add an equal volume of isopropanol (room temperature for 5 min, 5 min on ice), immediately after adding isopropanol, mix well by turning up and down 5-10 times; high-speed centrifugation 12000g, 15 min, 4℃, pour off the supernatant and dry with a pipette; 1 ml 75% ethanol washing once, mix evenly by blowing, centrifugation 7500g, 5 min, 4℃ to remove the supernatant; dry at room temperature for 20 min, add 50 μl ddH2O (DEPC treated); detect OD value, 1% agarose electrophoresis to determine the quality and concentration of RNA.

[0074] (2) RT-PCR: mix the reverse transcription primer Random Primer and RNA in proportion, then 70℃ water bath for 10 min, ice bath for 2-3 min; then add other reagents of reverse transcription system (purchased from Thermo Fisher Scientific), 37℃ water bath for 1.5 h, 95℃ water bath for 5 min, ice bath, -20℃ freezing. After reverse transcription, use housekeeping gene HPRT primer for polymerase chain reaction (PCR) to detect the quality of cDNA obtained by RNA reverse transcription.

[0075] (3) 5' race PCR: use the terminal transferase activity of TdT enzyme to automatically add 3-5 dG residues at the 5' end of the first strand after reverse transcription; then use universal primer Poly C containing part of the adapter sequence as the upstream primer, use gene-specific primer as the downstream primer, and use G-cDNA as the template for PCR to amplify the 5' end cDNA fragment of the target gene. After obtaining the cDNA, add 1 μl of cDNA to 3 μl of dGTP and 2 μl of TdT enzyme (purchased from Promega), and then quickly put it into 70℃ water bath for 10 min after 37℃ water bath for 15 min, to obtain the G-tailed cDNA. In a 50 μl PCR reaction system, add 1 μl of G-tailed cDNA template, 1 μl of Poly C, and specific primer (Table 1), and perform amplification and extension under the action of 0.5 μl of Taq Platium (purchased from Qiagen);

[0076] Table 2 antibody variable region gene primer sequence

[0077] The reaction conditions are as follows:

[0078] (4) Enzymatic digestion, ligation, transformation: the PCR product is recovered, an A tail is added to the 3' end of the end-smoothed DNA segment, and then ligated with pGEM-T Vector (purchased from Promega) at 16°C for more than 3 hours for transformation, or can be placed at 4°C for overnight ligation. After ligation, the competent bacteria DH5a are taken out and placed at room temperature to semi-melt. 5 μl of recombinant plasmid is added, and gently stirred until mixed. Ice bath for 30 min, 42°C heat shock for 45-50 s, and placed on ice for 2-3 min. Add 500 μl of Amp-LB liquid medium, and incubate at 37°C for 20-30 min. Take 300 μl of bacterial solution and plate, and incubate at 37°C for 12-16 h. After obtaining a series of colonies, positive clones are selected by blue-white spot screening system, and whether they are positive bacteria is verified again by PCR and enzyme digestion system.

[0079] (5) Sequencing and sequence analysis: after obtaining the positive bacteria, plasmid sequencing is performed (Shanghai SunGene). After obtaining the sequencing results, the sequencing results are analyzed using the IMGT database V gene alignment tool to determine the amino acid sequences of the heavy chain and light chain variable regions and the complementary determining regions contained therein as follows:

[0080] The full-length amino acid sequence of the H chain (heavy chain) variable region of pTau217-1 is SEQ ID NO 7-QVKLQESGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENDNTRYDPKFQGKASIAADTSSNTAYLQLSSLTSEDTAVYYCAPTGTGFGMDYWGQGTSVTVSS, and the full-length amino acid sequence of the L chain (light chain) variable region is SEQ ID NO 8-DIVMTQTTLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK; wherein,

[0081] The heavy chain variable region complementary determining region CDR1 is SEQ ID NO 1-GFNIKDYY;

[0082] The heavy chain variable region complementary determining region CDR2 is SEQ ID NO 2-IDPENDNT;

[0083] Heavy chain variable region complementarity determining region CDR3: SEQ ID NO 3 - APTGTGFGMDY;

[0084] Light chain variable region complementarity determining region CDR1: SEQ ID NO 4 - QSIVHRNGNTY;

[0085] Light chain variable region complementarity determining region CDR2: SEQ ID NO 5 - KVS;

[0086] Light chain variable region complementarity determining region CDR3: SEQ ID NO 6 - FQGSHVPYT;

[0087] The above sequences were identified by analysis using tools associated with the database of “THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM” (www.imgt.org).

[0088] 5. Antibody production and purification

[0089] (1) Positive clone C-pTau217-1 resuscitation: 37°C, 5% CO2 incubator for 2-3 days. Then use hybridoma special SFM serum-free medium (12045084, Thermo Fisher Scientific) for cell culture for 3 days. Collect the culture supernatant, centrifuge, and store at -20°C.

[0090] (2) Dilute the culture supernatant 4 times with pre-cooled phosphate buffer solution (PBS), centrifuge at 2000 rpm for 5 minutes, and remove the precipitate. Slowly add saturated ammonium sulfate solution to the supernatant at 4°C, stirring while adding, so that the final solution is 50% ammonium sulfate concentration.

[0091] (3) This solution is placed in ice for 30-60 minutes, then centrifuged at 5000 rpm for 10 minutes to remove the supernatant.

[0092] (4) Dissolve the precipitate in Tris-HCl buffer (40mM NaCl).

[0093] (5) Load the dialysis bag into Tris-HCl buffer (20mM NaCl) for desalting.

[0094] (6) Centrifuge to remove the precipitate.

[0095] (7) After dilution (1:100 or higher), measure the protein content at 280 nm.

[0096] ​(8) DEAE-cellulose column: equilibrated with 20 mM NaCl Tris buffer. The sample was dialyzed against Tris buffer. The sample was applied to the column bed at a flow rate of 1-2 ml / min, and eluted with a linear gradient of NaCl. The monoclonal antibody was eluted at 40 mM and 80 mM NaCl. The protein peak was collected by measuring OD280nm, and the monoclonal antibody was aliquoted and stored at -20 °C.

[0097] Example 3 Detection of the recognition of the antibody to the antigen and cross-reactivity (ELISA)

[0098] (1) Antigen coating. The antigen (tauAg of Example 1, the above-mentioned phosphorylated / non-phosphorylated polypeptide) was prepared into a coating solution of 10 μg / ml with 50 mM carbonate buffer at pH 9.6, and mixed well. 100 μl / well was added to a 96-well ELISA assay plate, and placed in a 4 °C refrigerator overnight.

[0099] (2) Blocking. The plate was washed. Blocking solution (0.5% m / v bovine serum albumin BSA dissolved in PBS) was added, 200 μl / well. Incubated at 37 °C for 2 hours.

[0100] (3) Addition of primary antibody (sample to be tested). The plate was washed. The diluted primary antibody solution (monoclonal antibody purified in Example 2) was added to the wells, 100 μl / well. 37 °C, 2 hours.

[0101] (4) Addition of secondary antibody. The plate was washed. The diluted secondary antibody (ab6789, abcam, 1:2000 dilution for use) was added to the wells. 100 μl / well. 37 °C, 1 hour.

[0102] (5) Color development. The plate was washed. TMB substrate was prepared and added to the wells, 100 μl / well. Color development for 5-10 minutes. 2 M sulfuric acid 100 μl / well was added to stop the reaction.

[0103] (6) Detection. The absorbance OD value of the solution at 450 nm was detected by a microplate reader. As shown in Figure 1, pTau217-1 has significant antigen recognition specificity for pTau217, and has no cross-reactivity with Tau217, pTau181 and pTau231. As a control, some hybridoma clones secreted antibodies and some existing commercial antibodies obtained during the antibody screening process in Example 2 showed different degrees of cross-recognition reactivity with Tau217 and / or pTau181 and / or pTau231 in the cross-reactivity experiment.

[0104] (7) The antibody concentration-OD value curve was plotted with the antibody dilution as the abscissa and the corresponding OD value as the ordinate. The EC50 value was determined using a curve fitting tool (such as a four-parameter logistic curve fitting). 50The half maximal effective concentration (EC50) of the monoclonal antibody (pTau217-1) for antigen recognition was 0.21 μg / ml, as shown in Figure 2. 50 ) can reach 0.21 μg / ml.

[0105] Example 4 Verification of genetically engineered antibody expression

[0106] The antibody heavy and light chain variable regions were constructed on a mouse IgGl / Kappa expression plasmid (InvivoGen) and transfected into cells using the ExpiCHO TM Expression System Kit (A29133, Thermo Fisher Scientific), the culture supernatant was harvested, and then the antibodies were purified using the methods listed in Example 2. The obtained antibodies had the same significant recognition of tauAg (ELISA) as the hybridoma cell expressed antibodies, proving that the obtained antibody sequences were correct variable region sequences.

Claims

The monoclonal antibody or antigen-binding portion thereof binding to the pTau217 antigen, characterized in that the protein sequence comprises: a heavy chain variable region complementarity determining region CDR1 having an amino acid sequence as set forth in SEQ ID NO 1, a heavy chain variable region complementarity determining region CDR2 having an amino acid sequence as set forth in SEQ ID NO 2, a heavy chain variable region complementarity determining region CDR3 having an amino acid sequence as set forth in SEQ ID NO 3, a light chain variable region complementarity determining region CDR1 having an amino acid sequence as set forth in SEQ ID NO 4, a light chain variable region complementarity determining region CDR2 having an amino acid sequence as set forth in SEQ ID NO 5, a light chain variable region complementarity determining region CDR3 having an amino acid sequence as set forth in SEQ ID NO 6. The monoclonal antibody or antigen-binding portion thereof according to claim 1, characterized in that the protein sequence comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO 7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO 8. The monoclonal antibody or antigen-binding portion thereof binding to the pTau217 antigen, characterized in that it comprises a protein sequence obtained by amino acid mutation or modification based on the heavy chain and light chain variable region amino acid sequences as set forth in claim 2. An immunoglobulin or a genetically engineered product thereof comprising the antibody or antigen-binding portion thereof according to claims 1-3. An antibody conjugate comprising the antibody or antigen-binding portion thereof according to claims 1-3. A nucleic acid encoding the antibody or antigen-binding portion thereof according to claims 1-3. An expression vector comprising the nucleic acid according to claim 6. A host cell comprising the expression vector according to claim 7. A hybridoma cell secreting the antibody according to claims 1-3. Use of the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-3 in the preparation of a kit for detecting pTau217.

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