Monoclonal antibody combined with 217-site threonine phosphorylated microtubule associated protein tau and application thereof

Abstract

The invention discloses a monoclonal antibody which is specifically combined with a microtubule associated protein tau (microtubule associated protein with 217 Thrphorylation, pTau217) of which the 217 site is phosphorylated by threonine, and a preparation method of the monoclonal antibody. The invention provides an amino acid sequence of a variable domain of the monoclonal antibody, and an amino acid sequence of a complementary determining region (CDR) contained in the amino acid sequence of the variable domain of the monoclonal antibody. The monoclonal antibody disclosed by the invention has the capability of specifically recognizing and remarkably combining pTau217, and can be used for developing and producing pTau217 clinical in vitro diagnosis (IVD) medical equipment products based on an immunological detection principle as a reagent raw material.

Description

TECHNICAL FIELD

[0001] The present application relates to a monoclonal antibody specifically binding to the 217th threonine phosphorylated microtubule-associated protein tau (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 in neurons and deposition of β-amyloid plaques. Although tau is phosphorylated in the 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, pTau 231, 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 217th threonine phosphorylated tau protein (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, i.e. 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 with non-phosphorylated threonine at position 217 (Tau217)). The antibody has a high sensitivity and strong specificity, as it not only binds to the phosphorylated threonine at position 217 of the tau protein antigen, but also has dependent recognition of the specific key amino acids near the threonine at position 217.

[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 pTau217 antigen is named pTau217-1, and its protein sequence 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 precisely recognizing the phosphorylated threonine at position 217 and some specific amino acids near it, and has no cross-reactivity with the tau antigens phosphorylated at threonine 181 (pTau181) and threonine 231 (pTau231), achieving specific and accurate detection of the pTau217 phosphorylation site. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 : ELISA detection of antibody recognition activity and cross-reactivity to antigen, "tauAg": phosphorylated tau antigen, "SP pT217": 217 threonine phosphorylated polypeptide (15 amino acids before and after the 217 threonine of tau antigen), "SPT217": 217 threonine non-phosphorylated polypeptide (15 amino acids before and after the 217 threonine of tau antigen), "SP pT181": 181 threonine phosphorylated polypeptide (15 amino acids before and after the 181 threonine of tau antigen), "SP pT231": 231 threonine phosphorylated polypeptide (15 amino acids before and after the 231 threonine of tau antigen).

[0023] Figure 2 : ELISA detection of the half maximal effective concentration (EC 50 ) of pTau217-1 antibody recognition of antigen, coating antigen: SP pT217, 217 threonine phosphorylated polypeptide (15 amino acids before and after the 217 threonine of 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] The term "antigen binding portion" of the application includes fragments or derivatives of antibodies that substantially retain their binding activity. Thus, the term "antigen binding portion" refers to a portion of a full-length antibody, typically the antigen binding or variable region thereof. An antigen binding portion can also include conservative amino acid substitutions that do not substantially alter its binding activity.

[0027] The "CDRs" used in the application are numbered according to the IMGT numbering system "THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM " (www.imgt.org). As a matter of common general knowledge in the art, "CDRs" can also be annotated and defined by other ways well known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, and the tools websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0028] The term "EC 50 " used in the application refers to the concentration for 50% of maximal effect (EC 50 ), which refers to the concentration that can cause 50% of the maximum effect.

[0029] The following is the specific preparation and testing process of the monoclonal antibody. The strategy of natural full-length phosphorylated tau protein immunization and multi-epitope (pTau217, Tau217, pTau181, pTau231) recognition synchronous screening is used for antibody preparation, which ensures that the antibody recognizes natural active phosphorylated tau protein antigen and accurately recognizes the 217 th threonine phosphorylation.

[0030] Unless otherwise specified, the reagents and materials used in the experimental process described in the application are conventional commercial reagents and materials, and the methods used are standard experimental methods (for specific details, refer to "Guidelines for Antibody Technology Experiments" (E. Harlow, Science Press 2005) and "Guidelines for Antibody Preparation and Use Experiments" G.C. Howard, Science Press 2010), and the academic special terms mentioned and their English abbreviations are used according to the provisions of "Immunology Terms" (Science Press 2008) compiled by the National Scientific and Technical Terminology Approval Committee unless otherwise specified.

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

[0032] Example 1 Antigen preparation

[0033] (1) Search for the sequence of the tau antigen coding gene in the NCBI database, and synthesize the tau antigen gene (protein N-terminal 6xHis tag) using synthetic biology method (Shanghai Generbiotech Co., Ltd.) on the pcDNA3.1 mammalian expression vector.

[0034] (2) Use mammalian cell transient transfection expression technology to prepare tau antigen protein. The specific experimental operation method is described in the ExpiCHO TM Expression System Kit (A29133, Thermo Fisher Scientific) manual.

[0035] (3) The tau protein is purified by Ni-IMAC magnetic beads (A50588, Thermo Fisher Scientific). The specific experimental operation is described in the product manual.

[0036] (4) The purified tau protein is phosphorylated in vitro using the AMPK series of kinases (purchased from Thermo Fisher Scientific). The specific experimental operation is described in the product manual. Phosphorylated tau antigen protein (tauAg) is prepared.

[0037] (5) Use 217 threonine phosphorylated tau epitope polypeptide (SPpT217) and 217 threonine non-phosphorylated tau epitope polypeptide (SP T217) for antibody cross-reactivity screening. The polypeptide range consists of 15 amino acids before and after 217Thr amino acid (Shanghai Generbiotech Co., Ltd.), with a purity of >90%. At the same time, 181 threonine phosphorylated tau epitope polypeptide (SPpT181) and 231 threonine phosphorylated tau epitope polypeptide (SP pT231) are used for antibody cross-reactivity screening. The two polypeptides are prepared using the same design strategy and preparation method as SPpT217.

[0038] Example 2 Antibody preparation

[0039] 1. Animal immunization

[0040] (1) The experimental animals are 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. TM Act for 90 seconds.

[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 x HAT (purchased from Sigma-Aldrich), 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 about 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 min, PBS washing twice, cell counting with a hemocytometer to 10 7 ; add 1 ml TRI REAGENT (purchased from Sigma-Aldrich) for lysis, mix well with a micropipette, add 200 μl chloroform after 5 min at room temperature (shake vigorously, 5 min at room temperature, 5 min on ice); high-speed centrifugation at 12000 g for 15 min at 4°C, then take the water phase (600 μl) and add an equal volume of isopropanol (5 min at room temperature, 5 min on ice); immediately mix well after adding isopropanol; high-speed centrifugation at 12000 g for 15 min at 4°C, pour off the supernatant and dry with a pipette; wash once with 1 ml 75% ethanol, mix well, centrifuge at 7500 g for 5 min at 4°C to remove the supernatant; dry at room temperature for 20 min in the air, 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°C water bath for 10 min, ice bath for 2-3 min; then add other reagents of the reverse transcription system (purchased from Thermo Fisher Scientific), 37°C water bath for 1.5 h, 95°C water bath for 5 min, ice bath, -20°C storage. After reverse transcription, use the housekeeping gene HPRT primer to perform polymerase chain reaction (PCR) to detect the quality of cDNA obtained by RNA reverse transcription.

[0075]

[0076]

[0077] (3) 5'race PCR: using the terminal transferase activity of TdT enzyme, 3-5 dG residues are automatically added to the 5' end of the first strand when reverse transcription is completed; then using the universal primer Poly C containing part of the adapter sequence as the upstream primer and the gene-specific primer as the downstream primer, the cDNA fragment of the 5' end of the target gene is amplified by PCR using the G-cDNA as the template. After obtaining the cDNA, 1 μl of the cDNA is added to 3 μl of dGTP and 2 μl of TdT enzyme (purchased from Promega), and after incubation at 37°C for 15 minutes, it is quickly incubated at 70°C for 10 minutes to obtain the G-tailed cDNA. In a 50 μl PCR reaction system, 1 μl of the G-tailed cDNA template, 1 μl of Poly C and the specific primer (Table 1) are added, and amplification and extension are carried out under the action of 0.5 μl of TaqPlatium (purchased from Qiagen);

[0078] Table 2 primer sequences for amplifying antibody variable region genes

[0079]

[0080] The reaction conditions are as follows:

[0081]

[0082] (4) enzyme digestion, ligation and transformation: the PCR product is recovered by gel recovery, an A tail is added to the 3' end of the smooth DNA fragment, and then the pGEM-T Vector (purchased from Promega) is ligated, and the ligation is carried out at 16°C for more than 3 hours for transformation, or the ligation is carried out at 4°C overnight. After ligation, the competent bacteria DH5a are taken out and placed at room temperature to a semi-melted state. 5 μl of the recombinant plasmid is added, and the mixture is gently stirred to be mixed. Ice bath for 30 min, 42°C heat shock for 45-50 s, and then placed on ice for 2-3 min. 500 μl of Amp-LB liquid medium is added, and the culture is carried out at 37°C for 20-30 min. 300 μl of the bacterial solution is taken and plated, and the culture is carried out at 37°C for 12-16 h. After a series of colonies are obtained, the positive clones are picked by blue-white spot screening system, and whether they are positive bacteria is verified again by PCR and enzyme digestion system.

[0083]

[0084]

[0085] (5) sequencing and sequence analysis: after obtaining the positive bacteria, plasmid sequencing is carried out (Shanghai SunGene). After obtaining the sequencing results, the IMGT database V gene alignment tool is used to analyze the sequencing results to determine the amino acid sequences of the heavy chain and light chain variable regions and the complementarity determining regions of the antibody as follows:

[0086] The full-length amino acid sequence of the H chain (heavy chain) variable region of pTau217-1 is as shown in SEQ ID NO 7 - QVKLQESGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLEWIGWIDPEN DNTRYDPKFQGKASIAADTSSNTAYLQLSSLTSEDTAVYYCAPTGTGFGMDYWGQ GTSVTVSS, and the full-length amino acid sequence of the L chain (light chain) variable region is as shown in SEQ ID NO 8 - DIVMTQTTLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLQKPGQSPKLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK; wherein,

[0087] Complementarity determining region CDR1 of the heavy chain variable region: SEQ ID NO 1 - GFNIKDYY;

[0088] Complementarity determining region CDR2 of the heavy chain variable region: SEQ ID NO 2 - IDPENDNT;

[0089] Complementarity determining region CDR3 of the heavy chain variable region: SEQ ID NO 3 - APTGTGFGMDY;

[0090] Complementarity determining region CDR1 of the light chain variable region: SEQ ID NO 4 - QSIVHRNGNTY;

[0091] Complementarity determining region CDR2 of the light chain variable region: SEQ ID NO 5 - KVS;

[0092] Complementarity determining region CDR3 of the light chain variable region: SEQ ID NO 6 - FQGSHVPYT;

[0093] The above sequences are identified by analysis using the tools associated with the database “THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM” (www.imgt.org).

[0094] 5. Antibody production and purification

[0095] (1) Resuscitation of positive clones of C-pTau217-1: Incubate in a 37°C, 5% CO2 incubator for 2-3 days. Then replace the culture medium with SFM serum-free medium (12045084, Thermo Fisher Scientific) specially for hybridoma cell culture, and incubate for 3 days. Collect the culture supernatant, centrifuge, and store at -20°C. ​

[0096] (2) The culture supernatant was diluted 4-fold with pre-chilled phosphate buffered saline (PBS) and centrifuged at 2000 rpm for 5 minutes to remove the precipitate. Saturated ammonium sulphate solution was slowly added to the supernatant while stirring at 4°C until the final concentration of ammonium sulphate was 50%.

[0097] (3) The solution was left on ice for 30-60 minutes and then centrifuged at 5000 rpm for 10 minutes to remove the supernatant.

[0098] (4) The precipitate was dissolved in Tris-HCl buffer (40 mM NaCl).

[0099] (5) The dialysis bag was filled with the solution and dialysed against Tris-HCl buffer (20 mM NaCl) to remove the salt.

[0100] (6) The precipitate was removed by centrifugation.

[0101] (7) The protein content was measured at 280 nm after dilution of the solution (1:100 or higher).

[0102] (8) DEAE-cellulose column: equilibrated with 20 mM NaCl Tris buffer. The dialysed sample was diluted with an equal volume of Tris buffer. The sample was applied to the column bed at a 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.

[0103] Example 3 Detection of the recognition of the antibody to the antigen and cross-reactivity (ELISA)

[0104] (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, mixed well. 100 μl / well was added to a 96-well ELISA assay plate. It was placed in a refrigerator at 4°C overnight.

[0105] (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 in a 37°C incubator for 2 hours.

[0106] (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 well, 100 μl / well. 37°C, 2 hours.

[0107] (4) Addition of secondary antibody. The plate was washed. The diluted secondary antibody (ab6789, abcam, 1:2000 dilution for use) was added to the well. 100 μl / well. 37°C, 1 hour.

[0108] (5) Color development. Wash plate. Prepare TMB substrate and add to the wells, 100 μl / well. Color development for 5-10 minutes. Stop reaction by adding 2M sulfuric acid, 100 μl / well.

[0109] (6) Detection. Detect the absorbance OD value of the solution at 450 nm with 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. For comparison, some hybridoma clones secreted antibodies and some existing commercial antibodies obtained simultaneously during the antibody screening process in Example 2, appeared different degrees of cross-recognition reactivity with Tau217 and / or pTau181 and / or pTau231 in the cross-reactivity experiment.

[0110] (7) Draw the antibody concentration-OD value curve with the antibody dilution as the abscissa and the corresponding OD value as the ordinate. Use curve fitting tools (such as four-parameter logistic curve fitting) to determine the EC 50 value. As shown in Figure 2 , the half maximal effective concentration (EC 50 ) of the monoclonal antibody (pTau217-1) for antigen recognition can reach 0.21 μg / ml.

[0111] Example 4 Verification of genetically engineered antibody expression

[0112] The antibody heavy chain and light chain variable regions were constructed on a mouse IgG1 / κ expression plasmid (InvivoGen), and the ExpiCHO TM expression system kit (A29133, Thermo Fisher Scientific) was used to transfect cells, and the culture supernatant was harvested, and then the antibody was purified using the method listed in Example 2. The obtained antibody has significant recognition (ELISA) for tauAg as the hybridoma cell expressed antibody, proving that the obtained antibody sequence is the correct variable region sequence.

Claims

1. A monoclonal antibody or an antigen-binding portion thereof that binds 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, and a light chain variable region complementarity determining region CDR3 having an amino acid sequence as set forth in SEQ ID NO 6.

2. 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.

3. A monoclonal antibody or an antigen-binding portion thereof that binds to the pTau217 antigen, characterized in that the protein sequence comprises a heavy chain and a light chain variable region amino acid sequence as set forth in claim 2, obtained by amino acid mutation or modification.

4. An immunoglobulin or a genetically engineered product thereof comprising the antibody or antigen-binding portion thereof according to claims 1 to 3.

5. An antibody conjugate comprising the antibody or antigen-binding portion thereof according to claims 1 to 3.

6. A nucleic acid encoding the antibody or antigen-binding portion thereof according to claims 1 to 3.

7. An expression vector comprising the nucleic acid according to claim 6.

8. A host cell comprising the expression vector according to claim 7.

9. A hybridoma cell that secretes the antibody according to claims 1 to 3.

10. Use of the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3 in the preparation of a kit for detecting pTau217. ​ ​ ​ ​ ​ ​ ​

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