Monoclonal antibody binding to microtubule associated protein tau with 231thr phosphorylation and use thereof

Abstract

Disclosed in the present invention is a monoclonal antibody binding to microtubule associated protein tau with 231Thr phosphorylation (pTau231). The present invention provides an amino acid sequence of a variable domain of the monoclonal antibody and an amino acid sequence of a complementarity determining region (CDR) comprised therein. The monoclonal antibody of the present invention has the ability to specifically recognize and significantly bind to the pTau231, and can be used as a reagent raw material for the development and production of pTau231 clinical in vitro diagnostic (IVD) medical device products based on the principle of immunological test.

Description

Monoclonal antibody for binding to tubulin-associated protein tau phosphorylated at threonine 231 and application thereof TECHNICAL FIELD

[0001] The present application relates to a monoclonal antibody specifically binding to tubulin-associated protein tau phosphorylated at threonine 231 (pTau231) and the application of the antibody in immunodetection technology and product development and production. BACKGROUND

[0002] In Alzheimer's disease (AD) patients, tau protein is abnormally phosphorylated at threonine 181, 217, 231, etc. (pTau181, pTau217, pTau231, etc.), and aggregated to form neurofibrillary tangles, leading to the 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.

[0003] Studies have shown that plasma pTau231 reaches an abnormal level, and the Aβ load is minimal. Plasma pTau231 and pTau217 are most relevant to Aβ positron emission tomography (PET) retention in the early accumulation region, and are associated with longitudinal increases in Aβ PET uptake in individuals without significant Aβ pathology at baseline. Plasma pTau231 and pTau217 better capture the earliest brain Aβ changes before the appearance of significant Aβ plaque pathology, and are promising AD blood biomarkers.

[0004] The core of pTau231 immunodetection technology and products is a monoclonal antibody that specifically recognizes pTau231. The antigen binding sensitivity, specificity and pairing ability of the monoclonal antibody largely determine the quality of the detection kit. 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 determining the physical condition of the human body. It is widely used in infectious diseases, heart disease, cancer, pregnancy testing, etc.

[0005] pTau231 is extremely low 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 pTau231 rather than other proteins, is also essential. Since the identification of pTau231 relies on the recognition of a single phosphorylation site, this undoubtedly places extremely high demands on the specificity of the antibody. Currently on the market, the selection of pTau231 monoclonal antibodies, especially monoclonal antibodies that do not cross-react with other important tau protein phosphorylation sites such as pTau181 and pTau217, and the supporting detection technology are extremely limited, and 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 pTau231 as a reliable biomarker. Therefore, high-quality anti-pTau231 monoclonal antibodies are a key condition for the development and production of pTau231 immunodetection kits. SUMMARY

[0006] To solve the above problems, the present application prepares a monoclonal antibody that specifically recognizes and significantly binds to pTau231 antigen (and has no binding activity to tau antigen (Tau231) that is not phosphorylated at threonine 231). This ensures that the antibody recognizes naturally active phosphorylated tau protein antigen and accurately recognizes phosphorylation at threonine 231. Tests have shown that while the antibody binds to phosphorylated threonine at position 231 of the tau protein antigen, it also has dependent recognition of specific key amino acids near threonine 231, thus having the advantages of high sensitivity and strong specificity.

[0007] 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.

[0008] Specifically, the present application uses the following technical solutions:

[0009] The monoclonal antibody or antigen-binding portion thereof that binds to pTau231 antigen is named pTau231-1, and the protein sequence thereof comprises:

[0010] a heavy chain variable region complementarity determining region CDR1 with an amino acid sequence as set forth in SEQ ID NO 1,

[0011] a heavy chain variable region complementarity determining region CDR2 with an amino acid sequence as set forth in SEQ ID NO 2,

[0012] a heavy chain variable region complementarity determining region CDR3 with an amino acid sequence as set forth in SEQ ID NO 3,

[0013] a light chain variable region complementarity determining region CDR1 having an amino acid sequence as set forth in SEQ ID NO 4,

[0014] a light chain variable region complementarity determining region CDR2 having an amino acid sequence as set forth in SEQ ID NO 5,

[0015] a light chain variable region complementarity determining region CDR3 having an amino acid sequence as set forth in SEQ ID NO 6.

[0016] Further, the above 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.

[0017] The monoclonal antibody or antigen-binding portion thereof binding to the pTau231 antigen according to the present application can also be a protein sequence obtained by amino acid mutation or modification based on the aforementioned pTau231-1 heavy chain and light chain variable region amino acid sequences.

[0018] The present application also relates to an immunoglobulin or genetic engineering product thereof of the aforementioned antibody or antigen-binding portion thereof, an antibody conjugate comprising the aforementioned antibody or antigen-binding portion thereof, a hybridoma cell secreting the aforementioned antibody or antigen-binding portion thereof, a nucleic acid encoding the aforementioned antibody or antigen-binding portion thereof, an expression vector comprising the aforementioned nucleic acid, and a host cell comprising the aforementioned expression vector.

[0019] 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 for detecting pTau231 are disclosed. The half maximal effective concentration (EC 50 ) of the monoclonal antibody (pTau231-1) for recognizing the antigen can reach 0.27 μg / ml. (2) The invented monoclonal antibody (pTau231-1) can accurately recognize the 231 th phosphorylated threonine and some specific amino acids near it, and has no cross-reaction with tau antigens phosphorylated at the 181 th threonine (pTau181) and the 217 th threonine (pTau217), which can distinguish the two important tau antigen phosphorylation sites pTau181 and pTau217, and achieve specific and accurate detection of the pTau231 phosphorylation site. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : ELISA detection of the recognition activity of the antibodies against the antigens and the cross-reactivity, "tau Ag": phosphorylated tau antigen, "SP pT231 ": 231 th Threonine phosphorylated polypeptide (15 amino acids on each side of the 231 th Threonine of the tau antigen), "SP T231 ": 231 th Threonine non-phosphorylated polypeptide (15 amino acids on each side of the 231 th Threonine of the tau antigen), "SP pT181 ": 181 th Threonine phosphorylated polypeptide (15 amino acids on each side of the 181 th Threonine of the tau antigen), "SP pT217": 217th Threonine phosphorylated polypeptide (15 amino acids on each side of the 217th Threonine of the tau antigen).

[0021] Figure 2: ELISA detection of the half maximal effective concentration (EC50) of the recognition of the antigens by the pTau231 -1 antibody. 50 ), coated antigen: SP pT231, 231 th Threonine phosphorylated polypeptide (15 amino acids on each side of the 231 th Threonine of the tau antigen). DETAILED DESCRIPTION

[0022] The term "antibody" as used herein refers to any form of antibody or fragment thereof that is capable of displaying the desired biological activity. Accordingly, it is used in the broadest sense consistent with that terminology, 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.

[0023] 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 compared to binding to other epitopes.

[0024] The term "antigen binding portion" as used herein includes fragments or derivatives of an antibody that substantially retain its 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.

[0025] "CDR" as used herein is according to the IMGT numbering system "THE INTERNATIONAL IMMUNOGENETICS INFORMATION " (www.imgt.org). As common knowledge in the art, "CDRs" can also be annotated and defined by other ways well known in the art, including but not limited to Kabat numbering system, Chothia numbering system, and the tools websites including but not limited to AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0026] The term "EC 50 " as used herein refers to the concentration for 50% of maximal effect (EC 50 ), which refers to the concentration that elicits 50% maximal effect.

[0027] The following is the specific preparation and testing process of the monoclonal antibody. Unless otherwise specified, the reagents and materials used in the experimental process described in the present 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 in accordance with the provisions of "Immunology Terms" (Science Press 2008) of the National Committee for Technical Terminology, unless otherwise specified.

[0028] 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 Slake Experimental Animal Co., Ltd.

[0029] Example 1 Preparation of antigen

[0030] (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.) in pcDNA3.1 mammalian expression vector.

[0031] (2) The tau antigen protein was prepared by using mammalian cell transient transfection expression technology. The specific operation method of the experiment was carried out according to the description of ExpiCHO TM Expression System Kit (A29133, Thermo Fisher Scientific).

[0032] (3) The tau protein was affinity purified by Ni-IMAC magnetic beads (A50588, Thermo Fisher Scientific), and the specific operation of the experiment was performed according to the description in the product manual.

[0033] (4) The purified tau protein was phosphorylated in vitro by an AMPK series of kinases (purchased from Thermo Fisher Scientific), and the specific operation of the experiment was performed according to the description in the product manual. The phosphorylated tau antigen protein (tau Ag) was prepared.

[0034] (5) The 231 threonine phosphorylated tau epitope polypeptide (SP pT231) and the 231 threonine non-phosphorylated tau epitope polypeptide (SP T231) were used for antibody cross-reactivity screening. The polypeptide range consisted of 15 amino acids before and after the 231 Thr amino acid (Shanghai Shengong Biotechnology Co., Ltd.), with a purity of > 90%. The 181 threonine phosphorylated tau epitope polypeptide (SP pT181) and the 217 threonine phosphorylated tau epitope polypeptide (SP pT217) were also used for antibody cross-reactivity screening. The two polypeptides were prepared using the same design strategy and preparation method as SP pT231.

[0035] Example 2 Antibody Preparation

[0036] 1. Animal Immunization

[0037] (1) The experimental animals were Balb / c strain female 6-week-old mice. They were raised in a clean environment.

[0038] (2) Immunization procedure: After mixing tau Ag with Freund's complete adjuvant (purchased from Sigma-Aldrich) and emulsifying, multiple subcutaneous injections were performed for immunization. The dose of tau Ag was 0.05-0.2 mg / time / mouse. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant (purchased from Sigma-Aldrich) was used for the booster immunization. The interval between each immunization was 3 weeks, and a total of 3 immunizations were performed.

[0039] (3) After the third immunization, the mouse was subjected to recall stimulation before cell fusion. 0.05-0.2 mg of antigen tau Ag was dissolved in 0.5 ml of phosphate buffer solution (PBS) with pH 7.2, and was injected intraperitoneally.

[0040] (4) Three days after the recall stimulation, cell fusion and hybridoma construction were performed.

[0041] 2. Construction of Hybridoma Cell Strains

[0042] (1) The day before cell fusion, prepare feeder cell suspension: inject 2 ml of incomplete culture solution (RPMI1640, purchased from Thermo Fisher Scientific) into the abdominal cavity of a mouse, then aspirate, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend with complete culture solution (RPMI1640 + 10% FBS) containing fetal bovine serum, obtain abdominal cavity cells, adjust the cell concentration to 5 x 10 5 / ml, add 50 μl / well to a 96-well cell culture plate, and culture at 37°C in a 5% CO2 incubator.

[0043] (2) Take logarithmically growing myeloma cells SP2 / 0, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells with incomplete culture solution (RPMI1640), count the cells, take the desired number of cells, and wash twice with incomplete culture solution.

[0044] (3) Take the spleen of the mouse after immunization and recall stimulation, crush, remove large pieces of tissue, prepare immune spleen cell suspension, and wash twice with incomplete culture solution.

[0045] (4) Mix the myeloma cells and spleen cells together at a ratio of 1:10 or 1:5, wash once with incomplete culture solution in a 50 ml plastic centrifuge tube at 1200 rpm for 8 minutes.

[0046] (5) Discard the supernatant and aspirate the remaining liquid with a dropper to avoid affecting the concentration of polyethylene glycol (PEG).

[0047] (6) Gently tap the bottom of the centrifuge tube to slightly loosen the cell sediment.

[0048] (7) Fusion: add 1 ml of 50% PEG (Hybri-Max TM , Sigma-Aldrich, molecular weight 1450) preheated at 37°C, and stir while adding. Act for 90 seconds.

[0049] (8) Slowly add incomplete culture solution preheated at 37°C to terminate the action of PEG. Centrifuge at 800 rpm for 6 minutes.

[0050] (9) Discard the supernatant and gently suspend with about 6 ml of complete culture solution.

[0051] (10) According to the number of 96-well culture plates used, add complete culture solution. Adjust the cell density to 5 x 10 5 / ml according to the number of spleen cells.

[0052] (11) Add the cell suspension after fusion to the 96-well plate containing feeder cells, 50 μl / well, and culture at 37°C in a 5% CO2 incubator.

[0053] (12) After 24 hours, add 2 × Add 100 μl / well of complete medium containing HAT (purchased from Sigma-Aldrich). Incubate at 37°C in a 5% CO2 incubator for 3-7 days.

[0054] 3. Positive monoclonal screening

[0055] (1) Prepare 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, resuspend with complete medium (RPMI 1640 + 10% FBS) containing fetal bovine serum, obtain abdominal cavity cells, adjust the cell concentration to 5 x 10 5 / ml.

[0056] (2) Count the positive well cells, and adjust the cell number to 1-5 x 10 3 / ml

[0057] (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 A, B, C three rows as 2 cells per well. The remaining 2.9 ml of cell suspension is supplemented with 2.9 ml of complete medium containing feeder cells, the cell number is 10 / ml, 100 μl / well, add D, E, F three rows, as 1 cell per well. The remaining 2.2 ml of cell suspension is supplemented with 2.2 ml of complete medium containing feeder cells, the cell number is 5 / ml, 100 μl / well, add G, H two rows, as 0.5 cells per well.

[0058] (4) After 4-5 days of culture, small cell clones can be seen under an inverted microscope, and 200 μl / well of complete medium is added.

[0059] (5) On the 8th-9th day, cell clones can be seen with the naked eye, and antibody detection is carried out in a timely manner. Note: The primary cloned hybridoma cells need to be added with HT (purchased from Sigma-Aldrich) in the complete medium.

[0060] The antibody detection steps are as follows:

[0061] (a) Antigen coating. Take the antigen (tau Ag or the above-mentioned phosphorylated / non-phosphorylated polypeptides: SP pT231, SP T231, SP pT181, SP pT217) and prepare a coating solution of 10 μg / ml with 50 mM carbonate buffer at pH 9.6, mix well. Add 100 μl / well to a 96-well ELISA detection plate. Place in a 4°C refrigerator overnight.

[0062] (b) Blocking. Wash the plate. Add blocking solution (0.5% m / v bovine serum albumin BSA in PBS), 200 μl / well. Incubate at 37°C for 2 hours.

[0063] (c) Add primary antibody (test sample). Wash the plate. Add diluted primary antibody solution (cell culture supernatant from hybridoma screening process) to the wells, 100 μl / well. Incubate at 37°C for 2 hours.

[0064] (d) Add secondary antibody. Wash the plate. Add diluted secondary antibody (ab6789, abcam, 1:2000 dilution) to the wells, 100 μl / well. Incubate at 37°C for 1 hour.

[0065] (e) Color development. Wash the plate. Prepare TMB substrate and add to the wells, 100 μl / well. Color development for 5-10 minutes. Stop the reaction by adding 2M sulfuric acid, 100 μl / well.

[0066] (f) Detection. Detect the absorbance OD value of the solution at 450 nm using a microplate reader.

[0067] According to the absorbance value, select the cells that produce positive results only for pTau231 polypeptide, continue to perform limited dilution and re-disperse cloning until single-well single clone positive cells are selected.

[0068] (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 select a single clone cell that can produce high antigen binding force of anti-phosphorylated pTau231 antibody, labeled as C-pTau231-1.

[0069] 4. Sequencing of the variable region gene of the C-pTau231-1 antibody of the positive clone

[0070] (1) RNA extraction: trypsin digestion to hybridoma cell suspension, centrifugation at 1000 rpm for 5 minutes (min), PBS washing twice, and determination of cells to 10 7; 1 ml TRI REAGENT (purchased from Sigma-Aldrich) was added to lyse, mixed with micropipette, after 5 min at room temperature, 200 μl chloroform was added (shaken vigorously, 5 min at room temperature, 5 min on ice); high-speed centrifugation 12000 g, 15 min, 4°C, the water phase (600 μl) was taken, and an equal volume of isopropanol was added (5 min at room temperature, 5 min on ice), immediately after adding isopropanol, mix well by inverting 5-10 times; high-speed centrifugation 12000 g, 15 min, 4°C, pour off the supernatant and dry with a pipette; 1 ml 75% ethanol was washed once, mixed evenly by blowing, centrifuged 7500 g, 5 min, 4°C to remove the supernatant; dry for 20 min at room temperature in the air, add 50 μl ddH2O (DEPC treated); detect OD value, 1% agarose electrophoresis to determine the quality and concentration of RNA.

[0071] (2) RT-PCR: After mixing the reverse transcription primer Random Primer and RNA in proportion, 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.

[0072] (3) 5' race PCR: using the terminal transferase activity of TdT enzyme, automatically add 3-5 dG residues to the 5' end of the first strand when reverse transcription is completed; then use the universal primer Poly C containing part of the adapter sequence as the upstream primer, and the gene-specific primer as the downstream primer, to perform PCR with G-cDNA as the template to amplify the cDNA fragment of the 5' end of the target gene. After obtaining the cDNA, 1 μl of cDNA was added to 3 μl of dGTP and 2 μl of TdT enzyme (purchased from Promega), and after 15 min at 37°C, it was quickly placed in a 70°C water bath for 10 min to obtain the G-tailed cDNA. In a 50 μl PCR reaction system, 1 μl of G-tailed cDNA template, 1 μl of Poly C and specific primers (Table 1) were added, and amplification and extension were performed under the action of 0.5 μl of Taq Platium (purchased from Qiagen);

[0073] Table 1 Primer sequences for amplifying antibody variable region genes

[0074] The reaction conditions are as follows:

[0075] (4) Enzymatic digestion, ligation, transformation: the PCR product was recovered, an A tail was added to the 3' end of the terminal smooth DNA segment, and then ligated with a pGEM-T Vector (purchased from Promega) at 16°C for more than 3 hours for transformation, or placed at 4°C overnight for ligation. After ligation, the competent bacteria DH5a were taken out and placed at room temperature until semi-melted. 5 μl of recombinant plasmid was 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 were selected by blue-white spot screening system, and whether they were positive bacteria was verified again by PCR and enzyme digestion system.

[0076] (5) Sequencing and sequence analysis: after obtaining the positive bacteria, plasmid sequencing was performed (Shanghai Sunway Biotech). After obtaining the sequencing results, the sequencing results were 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:

[0077] The full-length amino acid sequence of the H chain (heavy chain) variable region of pTau231-1 is SEQ ID NO 7, and the full-length amino acid sequence of the L chain (light chain) variable region is SEQ ID NO 8:

[0078] Among them,

[0079] The heavy chain variable region complementary determining region CDR1 is SEQ ID NO 1-GFTFSAYA;

[0080] The heavy chain variable region complementary determining region CDR2 is SEQ ID NO 2-ISNRGTYT;

[0081] The heavy chain variable region complementary determining region CDR3 is SEQ ID NO 3-ARRGYGSGRGYFDV;

[0082] The light chain variable region complementary determining region CDR1 is SEQ ID NO 4-ESVDSYDNSF;

[0083] The light chain variable region complementary determining region CDR2 is SEQ ID NO 5-FAS;

[0084] The light chain variable region complementary determining region CDR3 is SEQ ID NO 6-QQNNEDPWT;

[0085] The sequences were identified using the tools available on the database "THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM" (www.imgt.org).

[0086] 5. Antibody production and purification

[0087] (1) Resuscitation of positive clone C-pTau231-1: Incubate at 37°C, 5% CO2 incubator for 2-3 days. Then replace the culture medium with SFM Hybridoma Serum-free Medium (12045084, Thermo Fisher Scientific) and incubate for 3 days. Collect the culture supernatant, centrifuge and store at -20°C.

[0088] (2) Dilute the culture supernatant 4-fold with pre-cooled phosphate buffered saline (PBS), centrifuge at 2000 rpm for 5 minutes to remove the precipitate. Slowly add saturated ammonium sulfate solution into the supernatant while stirring at 4°C, so that the final concentration of ammonium sulfate is 50%.

[0089] (3) Place the solution in ice for 30-60 minutes, then centrifuge at 5000 rpm for 10 minutes to remove the supernatant.

[0090] (4) Dissolve the precipitate in Tris-HCl buffer (40 mM NaCl).

[0091] (5) Load the dialysis bag into Tris-HCl buffer (20 mM NaCl) for desalination.

[0092] (6) Centrifuge to remove the precipitate.

[0093] (7) After dilution (1:100 or higher), measure the protein content at 280 nm.

[0094] (8) DEAE-cellulose column: Equilibrate with 20 mM NaCl Tris buffer. Dilute the dialysis sample with Tris buffer. The sample enters the column bed at a speed of 1-2 ml / min, and is eluted with a linear gradient of NaCl. The monoclonal antibody is eluted at 40 mM and 80 mM NaCl. Measure OD280nm to collect the protein peak, and store the monoclonal antibody at -20°C.

[0095] Example 3 Detection of the recognition of the antibody to the antigen and cross-reactivity (ELISA)

[0096] ​(1) Antigen coating. Take the antigen (tau Ag of Example 1, the above-mentioned phosphorylated / non-phosphorylated polypeptide) and prepare a coating solution of 10 μg / ml with 50 mM carbonate buffer at pH 9.6, mix well. Add 100 μl / well to a 96-well ELISA detection plate. Place in a 4°C refrigerator overnight.

[0097] (2) Blocking. Wash the plate. Add blocking solution (0.5% m / v bovine serum albumin BSA dissolved in PBS), 200 μl / well. Incubate at 37°C for 2 hours.

[0098] (3) Add primary antibody (test sample). Wash the plate. Add the diluted primary antibody solution (monoclonal antibody purified in Example 2) to the wells, 100 μl / well. 37°C, 2 hours.

[0099] (4) 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.

[0100] (5) 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.

[0101] (6) Detection. Detect the absorbance OD value of the solution at 450 nm with a microplate reader. As shown in Figure 1, pTau231-1 has significant antigen recognition specificity for pTau231, and has no cross-reactivity with Tau231, pTau181 and pTau217. As a control, some hybridoma clones secreted antibodies obtained during the antibody screening process in Example 2 and some existing commercial antibodies appeared in the cross-reactivity experiment with different degrees of cross-recognition reactivity with Tau231 and / or pTau181 and / or pTau217.

[0102] (7) Take the antibody dilution as the abscissa and the corresponding OD value as the ordinate to draw the antibody concentration-OD value curve. 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 (pTau231-1) for antigen recognition can reach 0.27 μg / ml.

[0103] Example 4 Verification of genetically engineered antibody expression

[0104] The antibody heavy chain and light chain variable regions were constructed on a mouse IgG1 / κ expression plasmid (InvivoGen), and ExpiCHO TMThe expression system kit (A29133, Thermo Fisher Scientific) was used to transfect cells, and the culture supernatant was harvested and then subjected to antibody purification using the methods listed in Example 2. The obtained antibodies and hybridoma cells expressed the same antibodies with significant recognition of tau Ag (ELISA), proving that the obtained antibody sequences were correct variable region sequences.

Claims

1. A monoclonal antibody or an antigen-binding portion thereof that binds to the pTau231 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.

2. The monoclonal antibody or an 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 pTau231 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 genetic engineering product thereof comprising the antibody or an antigen-binding portion thereof according to claims 1 to 3.

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

6. A nucleic acid encoding the antibody or an 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 secreting the antibody according to claims 1 to 3.

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