Preparation method of p-tau231 monoclonal antibody, p-tau231 monoclonal antibody and antibody variable region

Abstract

The invention discloses a preparation method of a p-tau231 monoclonal antibody, the p-tau231 monoclonal antibody and an antibody variable region, and relates to the technical field of antibodies, the preparation method comprises the following steps: polypeptide design and synthesis: designing and synthesizing a phosphorylated polypeptide and a non-phosphorylated control polypeptide based on the 224th-238th amino acid sequence of tau protein, the phosphorylated polypeptide sequence is CKKVAVVRT (p) PPKSPSS, the phosphorylated polypeptide sequence is CKKVAVVRT (p) PPKSPSS, the phosphorylated polypeptide sequence is CKKVAVVRT (p) PPKSPSS, and the non-phosphorylated control polypeptide sequence is CKKVAVVRT (p) PPKSPSS; threonine (Thr231) at the 231st site is subjected to phosphorylation; the sequence of the non-phosphorylated polypeptide is CKKVAVVRTPPKSPSS, and the sequence of the non-phosphorylated polypeptide is Immunogen coupling: introducing cysteine residues to the N end of the phosphorylated polypeptide, and connecting the phosphorylated polypeptide with carrier protein KLH through a sulfydryl chemical coupling method to form an immunogen compound; and preparation of the conjugate for detection: introducing cysteine residues to the N ends of the phosphorylated polypeptide and the non-phosphorylated polypeptide, and respectively coupling with a carrier protein BSA to form the antigen complex for detection. The p-tau231 monoclonal antibody prepared by the technical scheme of the invention can be specifically combined with a p-tau231 antigen, has no specific reaction with non-phosphorylated polypeptide and tau polypeptide, and can be applied to early diagnosis of AD (Alzheimer's disease).

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to a method for preparing a p-tau231 monoclonal antibody, a p-tau231 monoclonal antibody, and an antibody variable region. Background Art

[0002] Alzheimer's disease (AD), as the main subtype of dementia, accounts for 60% to 80% of dementia cases and is a neurodegenerative disease characterized by cognitive decline and memory loss. It is estimated that by 2030, the number of global dementia patients will increase to 78 million, and the global expenditure on dementia will reach $2.8 trillion. The typical pathological features of AD mainly include Aβ plaques caused by β-amyloid protein deposition and neurofibrillary tangles generated by the hyperphosphorylation of microtubule-associated tau protein. The main diagnostic methods for AD include cognitive function assessment, imaging examination, cerebrospinal fluid examination, gene mutation detection, and hematological examination.

[0003] Recent studies have found that by using ultrasensitive detection techniques, AD-related pathological markers such as p-tau231 can be detected in the blood. p-tau231 (phosphorylated tau protein, threonine at position 231) can be used as an early biomarker to help identify patients in the mild cognitive impairment (MCI) stage; it can also be used to monitor the progression of AD and evaluate the treatment effect; it can also be used in combination with other biomarkers to improve the diagnostic accuracy of AD. The detection methods for p-tau231 mainly include enzyme linked immunosorbent assay (ELISA), Immunoprecipitation Mass Spectrometry (IP-MS), Liquid Chromatography-Mass Spectrometry (LC-MS) technology, Single Molecule Array (Simoa), and chemiluminescence-based detection methods. For methods that require antigen-antibody binding for detection, monoclonal antibodies that specifically recognize p-tau231 are needed. However, the number of such monoclonal antibodies on the market is small, the sensitivity is low, and the specificity is poor. Therefore, there is an urgent need to develop a p-tau231 antibody with high sensitivity and good specificity to solve the above problems. Summary of the Invention

[0004] The technical solution for the present invention to solve the above technical problems is to provide a method for preparing a p-tau231 monoclonal antibody, including the following steps: Step 1 Polypeptide Design and Synthesis: Based on the amino acid sequence at positions 224 - 238 of the tau protein, a phosphorylated polypeptide and a non - phosphorylated control polypeptide were designed and synthesized, where: The phosphorylated polypeptide sequence is CKKVAVVRT(p)PPKSPSS, and the threonine at position 231 (Thr231) is phosphorylated; The non - phosphorylated polypeptide sequence is CKKVAVVRTPPKSPSS; Step 2 Immunogen Conjugation: Using the N - terminal cysteine residue of the phosphorylated polypeptide, it was linked to the carrier protein KLH by thiol chemical conjugation to form an immunogen complex; Step 3 Preparation of Conjugates for Detection: Using the N - terminal cysteine residues of the phosphorylated polypeptide and the non - phosphorylated polypeptide, they were respectively conjugated to the carrier protein BSA to form antigen complexes for detection.

[0005] To solve the above - mentioned technical problems, the present invention also provides a monoclonal antibody against p - tau231, the amino acid sequence of its light - chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of its heavy - chain variable region is as shown in SEQ ID NO: 2 or the amino acid sequence of its heavy - chain variable region is as shown in SEQ ID NO: 3.

[0006] To solve the above - mentioned technical problems, the present invention also provides an antibody variable region for a monoclonal antibody against p - tau231. The CDR1 of the light - chain variable region with the amino acid sequence SEQ ID NO: 1 is QSVYNNNY, the CDR2 is SAS, and the CDR3 is LGDFDCTSADCGV; The CDR1 of the heavy - chain variable region with the amino acid sequence SEQ ID NO: 2 is QSVYNNNY, the CDR2 is IKGGGNT, and the CDR3 is TSSGSDY; Or the CDR1 of the heavy - chain variable region with the amino acid sequence SEQ ID NO: 3 is GIDLSRNG, the CDR2 is IKGGGSA, and the CDR3 is ASSGSDY.

[0007] The monoclonal antibody against p - tau231 prepared by the technical solution of the present invention can specifically bind to the p - tau231 antigen and has no specific reaction with the non - phosphorylated polypeptide and the tau polypeptide. Therefore, it can be applied to the early diagnosis of AD. The Kd values of the binding of the two monoclonal antibodies to the antigen both reach 10 -10 , and the antibody has extremely strong antigen - binding activity, affinity and specificity. Brief Description of the Drawings

[0008] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0009] Figure 1 Rabbit antigen affinity sorting B cells diagram of the present invention; Figure 2 SDS-PAGE diagram of antibody purification of the present invention; Figure 3 ELISA indirect evaluation of the Kd value of the antibody against p-tau231 antigen of the present invention; Figure 4 ELISA indirect evaluation of the Kd value of the antibody against tau antigen of the present invention. Detailed implementation manners

[0010] The present invention provides a method for preparing a p-tau231 monoclonal antibody, a p-tau231 monoclonal antibody and an antibody variable region, aiming to prepare a p-tau231 monoclonal antibody for specifically binding to the p-tau231 antigen.

[0011] The following will illustrate the method for preparing the p-tau231 monoclonal antibody and the p-tau231 monoclonal antibody proposed by the present invention in specific embodiments: Example 1: A method for preparing a p-tau231 monoclonal antibody, comprising the following steps: (1) Polypeptide design and synthesis: Based on the amino acid sequence at positions 224-238 of the tau protein, a phosphorylated polypeptide and a non-phosphorylated control polypeptide are designed and synthesized, where: The phosphorylated polypeptide sequence is CKKVAVVRT(p)PPKSPSS, and the 231st threonine (Thr231) is phosphorylated; The non-phosphorylated polypeptide sequence is CKKVAVVRTPPKSPSS; (2) Immunogen conjugation: Using the N-terminal cysteine residue of the phosphorylated polypeptide, it is connected to the carrier protein KLH by thiol chemical conjugation to form an immunogen complex; (3) Preparation of the conjugate for detection: Using the N-terminal cysteine residues of the phosphorylated polypeptide and the non-phosphorylated polypeptide, they are respectively conjugated to the carrier protein BSA to form the antigen complex for detection.

[0012] Tau full-length protein was purchased from Nanjing Lideng Biotechnology Co., Ltd.

[0013] The positive control antibody, namely phospho-Tau (Thr231) Antibody, was purchased from Suzhou Renduan Biomedical Technology Co., Ltd.

[0014] Example 2: A p-tau231 monoclonal antibody (clone number: 1M1F1) was prepared according to the preparation method of the p-tau231 monoclonal antibody described in Example 1. The amino acid sequence of its light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 2.

[0015] Specifically, the amino acid sequence of the light chain variable region is: AQVLTQTASPVSAAVGGTVTINCQSSQS VYNNNYLSWFQQKPGQPPKLLIHSASTLYSGVSSQFKGSGSGTQFTLTIS DVQCDDAATYYCLGDFDCTSADCGVFGGGTEVVVR; CDR1 is QSVYNNNY, CDR2 is SAS, and CDR3 is LGDFDCTSADCGV; The full chain length is AQVLTQTASPVSAAVGGTVTINCQSSQS VYNNNYLSWFQQKPGQPPKLLIHSASTLYSGVSSQFKGSGSGTQFTLTIS DVQCDDAATYYCLGDFDCTSADCGVFGGGTEVVVR GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0016] The amino acid sequence of the heavy chain variable region is: QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN GINWVRQAPGKGLEWIVYIKGGGNTYYASWAKGRFTISKSSTTVDLKITT PTTEDTATYFCTSSGSDYWGPGTLVTVSS; CDR1 is QSVYNNNY, CDR2 is IKGGGNT, and CDR3 is TSSGSDY.

[0017] The full chain length is QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN GINWVRQAPGKGLEWIVYIKGGGNTYYASWAKGRFTISKSSTTVDLKITT PTTEDTATYFCTSSGSDYWGPGTLVTVSS GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTV APSTCSKPTCP PPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFKCKVHNKALPAPIEKTISKAR GQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。

[0018] Example 3: A p-tau231 monoclonal antibody (clone number: 1M1F3) was prepared according to the preparation method of the p-tau231 monoclonal antibody described in Example 1. The amino acid sequence of its light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 3.

[0019] Specifically, the amino acid sequence of the light chain variable region is: AQVLTQTASPVSAAVGGTVTINCQSSQS VYNNNYLSWFQQKPGQPPKLLIHSASTLYSGVSSQFKGSGSGTQFTLTIS DVQCDDAATYYCLGDFDCTSADCGVFGGGTEVVVR; CDR1 is QSVYNNNY, CDR2 is SAS, and CDR3 is LGDFDCTSADCGV; The full chain length is AQVLTQTASPVSAAVGGTVTINCQSSQS VYNNNYLSWFQQKPGQPPKLLIHSASTLYSGVSSQFKGSGSGTQFTLTIS DVQCDDAATYYCLGDFDCTSADCGVFGGGTEVVVR GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。

[0020] The amino acid sequence of the heavy chain variable region is: QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN GMNWVRQAPGKGLEWIAYIKGGGSAYYASWAKGRFTISKSPTTVDLKIAT PTTEDTATYFCASSGSDYWGPGTLVTVSS; CDR1 is GIDLSRNG, CDR2 is IKGGGSA, and CDR3 is ASSGSDY.

[0021] The full chain length is: QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN GMNWVRQAPGKGLEWIAYIKGGGSAYYASWAKGRFTISKSPTTVDLKIAT PTTEDTATYFCASSGSDYWGPGTLVTVSS GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTV APSTCSKPTCP PPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFKCKVHNKALPAPIEKTISKAR GQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。

[0022] Example 4: An antibody variable region for the p-tau231 monoclonal antibody of Example 2. The CDR1 of the light chain variable region amino acid sequence SEQ ID NO: 1 is QSVYNNNY, the CDR2 is SAS, and the CDR3 is LGDFDCTSADCGV; The CDR1 of the heavy chain variable region amino acid sequence SEQ ID NO: 2 is QSVYNNNY, the CDR2 is IKGGGNT, and the CDR3 is TSSGSDY; Or the CDR1 of the heavy chain variable region amino acid sequence SEQ ID NO: 3 is GIDLSRNG, the CDR2 is IKGGGSA, and the CDR3 is ASSGSDY.

[0023] Experimental verification: 1. Animal immunization and serum titer evaluation: Two 3-4-month-old female New Zealand white rabbits were selected as immunization targets. The above p-tau231 polypeptide conjugated with KLH (p-tau peptide-KLH) was used as the antigen. Freund's complete adjuvant and the antigen (150 μg) were thoroughly mixed at a volume ratio of 1:1 and injected subcutaneously at four points on the back and two points in the groin. The specific immunization protocol is shown in Table 1 below; Table 1: Immunization protocol

[0024] The sera after the fourth immunization were detected according to the experimental information in Table 2 below. The specific operations are as follows: Collect a small amount of blood from the back of the ear, and determine the serum titer by indirect ELISA: (1) Antigen coating: Dilute the antigen to 2 μg / mL with PBS buffer, add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate twice with a plate washer and pat dry. Add 200 μL of blocking solution per well and incubate in a 37°C incubator for 2 h. Then wash the blocked ELISA plate three times with a plate washer and pat dry.

[0025] (2) Incubate the primary antibody: Dilute the serum of the immunized rabbit with PBS buffer (PBS is used as a negative control), and add 100 μL per well to the corresponding wells according to the dilution multiples in Table 3 below after dilution. Incubate at 37°C for 1.5 h. After incubation, place the ELISA plate in a plate washer and wash 3 to 4 times and pat dry.

[0026] (3) Incubate the secondary antibody: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody to each corresponding well, incubate at 37°C for 45 min, place the ELISA plate in a plate washer and wash 4 to 5 times and pat dry.

[0027] (4) Color development: Add 100 μL of TMB color development solution to each well of the 96-well plate and incubate at 37 °C for 15 min.

[0028] (5) Termination and reading: Add 50 μL of termination solution to each well of the 96-well plate, then place it in an enzyme-linked immunosorbent assay (ELISA) reader for reading. Set the detection wavelength to 450 nm, read the detection results, and consider an OD value greater than 2.1 times that of the negative control as positive.

[0029] Table 2: Information on the four-immunization experiment

[0030] The data on the titer of the four-immunization serum detected by ELISA are shown in Table 3; Table 3: Data on the titer of the four-immunization serum detected by ELISA

[0031] From the detection results of the four-immunization ELISA titer, it can be seen that after the rabbit R0149 was immunized four times with the p-tau231 polypeptide, the titer of the detected serum against the p-tau231 polypeptide was ≥729K, while the titer of the detected serum of the rabbit R0149 after four immunizations was 81K. Therefore, the rabbit R0149 meets the sorting requirements.

[0032] 2. Preparation of PBMC: Collect the peripheral blood of the rabbit R0149 and prepare PBMC.

[0033] Transfer the peripheral blood into a 50 mL centrifuge tube, add an equal volume of sterile PBS solution for dilution, and mix gently; Take an appropriate number of new sterile centrifuge tubes, first add Ficoll-Paque PLUS cell separation solution, and then gently layer the diluted blood on top of the Ficoll-Paque PLUS cell separation solution in the centrifuge tube. The volume ratio of the separation solution to the cell suspension is 1:2; Place the prepared sample in a centrifuge and centrifuge at 2000 revolutions per minute at room temperature for 20 minutes; After centrifugation, take out the test tube, aspirate the mononuclear cell layer into another 50 mL sterile centrifuge tube, add sterile PBS to 50 mL, and centrifuge at 1500 revolutions per minute at room temperature for 10 minutes; If red blood cells are visible at the bottom layer, discard the supernatant, add 3 mL of red blood cell lysis solution, and incubate on ice for 12 minutes; otherwise, skip directly to the following steps; Add sterile PBS to 50 mL, and centrifuge at 1500 revolutions per minute at room temperature for 10 minutes; Discard the supernatant and repeat the washing step Carefully remove the supernatant, directly perform cell cryopreservation, and proceed to the next experiment.

[0034] 3. Flow cytometry sorting of MBC: Perform preliminary screening with the help of B cell surface markers. Different fluorescent labels are used for the immunoprotein and the secondary antibody against IgG. If the MBC surface can display IgG antibodies that specifically bind to the antigen, the cell will carry two fluorescent labels. Since the project aims to prepare antibodies specifically recognizing the p-tau231 polypeptide, the p-tau231 polypeptide labeled with biotin is used for positive screening, and the non-phosphorylated tau polypeptide labeled with FITC is used for negative screening. The target MBC can be sorted out one by one from the cell suspension by flow cytometry and placed in a 96-well plate.

[0035] Sorting results: PBMC prepared from R0149 peripheral blood was sorted by flow cytometry, and the cell populations were clearly separated ( Figure 1 as shown), and B cells with antigen specificity were obtained.

[0036] 4. High-throughput expression: (I) Cell lysis and amplification of light and heavy chain genes: Adopt a single-cell antibody gene amplification system to obtain the full sequence of the light chain and the Fab fragment of the heavy chain respectively.

[0037] (II) Construction of expression plasmids and plasmid extraction: Connect the amplified heavy chain gene of the antibody to the pcDNA3.4 vector containing the rabbit IgGFc fragment, and connect the antibody light chain gene to the blank pcDNA3.4 vector to construct a dual plasmid.

[0038] Use a commercial plasmid large-scale extraction kit for plasmid extraction.

[0039] (III) Transient transfection expression: (1) Inoculate cells one day before transfection to ensure that the cells grow to 3.0 - 5.0×106 cells / mL on the day of transfection, with a viability of 98%. Dilute the cell suspension with the medium and plate the cells in a 96-well deep well plate, 400 μL per well.

[0040] (2) Plate the transfection buffer into a 96-well plate, 100 μL per well. Then add the plasmid to the transfection buffer and pipette to mix evenly. Then add PEI max to the buffer and pipette to mix evenly. Incubate at 37°C for 10 min, and then add it to the cells in the 96-well deep well plate for continued culture.

[0041] (3) Add 25 μL of feeding medium to each well 20 h after transfection. After 5 days of expression, centrifuge at 4000 rpm for 10 min and collect the supernatant.

[0042] 5. Detection of cell supernatant: Indirect ELISA was used to detect whether the cell supernatant was positive. The cell supernatant was detected according to the experimental information in Table 4 below. The clone numbers and plate layout are shown in Table 5 below. The specific experimental procedures are as follows: (1)Antigen coating: Dilute the antigen to 2 μg / ml with PBS buffer, add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate 3 times with a plate washer and drain. Add blocking solution at 100 μL per well, incubate in an incubator at 37°C for 2 h, and then wash the blocked ELISA plate 3 times with a plate washer and drain.

[0043] (2)Incubation with primary antibody: Add 50 μL of the cell supernatant to each well. Take an equal amount of positive antibody as the positive control and PBS buffer as the negative control, and incubate at 37°C for 1 h. After incubation, place the ELISA plate in a plate washer and wash 3 times and drain.

[0044] (3)Incubation with secondary antibody: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody to each corresponding well, incubate at 37°C for 30 min, place the ELISA plate in a plate washer and wash 3 times and drain.

[0045] (4)Color development: Add the mixed chromogenic solution to the 96-well plate, 50 μL per well, and incubate at 37°C for 15 min.

[0046] (5)Termination and reading: Add the termination solution to the 96-well plate, 100 μL per well, and then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm, read the detection results, and an OD value greater than 2.1 times that of the negative control well is considered positive.

[0047] Table 4: Clone numbers and plate layout

[0048] Table 5: Experimental information on antigen specificity detection of cell supernatant

[0049] Table 6: Experimental results of phosphorylation polypeptide-BSA coating specificity detection

[0050] Table 7: Experimental results of non-phosphorylation polypeptide-BSA coating specificity detection

[0051] Table 8: Experimental results of tau full-length protein coating specificity detection

[0052] Compare the signal intensities, and finally select 2 clones (clone numbers: 1M1F1; 1M1F3) that have a stronger reaction signal with phosphorylated polypeptide-BSA and do not bind to non-phosphorylated polypeptide-BSA and tau full-length protein for subsequent expression and purification.

[0053] 7. Antibody expression and purification: (1) Perform gene sequencing on the 2 positive clones (clone numbers: 1M1F1; 1M1F3) in Example 2 and Example 3, compare the variable region gene sequences obtained by antibody sequencing and the amino acid sequences obtained by translation, and then perform expression and purification.

[0054] (2) Construct expression plasmids and transient transfection for expression: The specific experimental operations are the same as (2) and (3) in 4. (3) Antibody purification Centrifuge the cell culture medium 6 days after transfection and culture, filter the centrifuged secreted supernatant with a filter membrane, and dialyze it in a buffer of 100 mM Tris (pH 7.4), 150 mM NaCl, 100 mM Glycine, pH 7.0 at 4°C. After dialysis, purify it with a Protein A column, and detect the purity of the antibody by SDS-PAGE. The results are as Figure 2 shown: Under reducing conditions, the antibody has 2 electrophoretic bands, and the corresponding molecular weights of these two electrophoretic bands are consistent with the sizes of their respective light and heavy chains; under non-reducing conditions, the antibody has a single band, and the corresponding molecular weight of this electrophoretic band is consistent with the size of its respective intact antibody; these results indicate that the monoclonal antibody is expressed accurately. In addition, all electrophoretic bands have clear edges, indicating that the antibody obtained by the above expression and purification steps has a high purity.

[0055] 8. Evaluate the antibody affinity by indirect ELISA method: Perform antibody titration by indirect ELISA method to evaluate its antibody titer. The specific experimental operations are as follows: (1) Antigen coating: Dilute the antigen to 0.5 μg / ml with PBS buffer, add 100 μL per well to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and incubate overnight at 4°C. Wash the coated ELISA plate 3 times with a plate washer and drain it. Add 200 μL of blocking solution per well, incubate in an incubator at 37°C for 2 h, and then wash the blocked ELISA plate 3 times with a plate washer and drain it.

[0056] (2) Incubate the primary antibody: Add 100 μL of the antibody to each well, take an equal amount of positive antibody as the positive control, and PBS buffer as the negative control, and incubate at 37°C for 1.5 h. After incubation, place the ELISA plate in a plate washer and wash it 3 times and drain it.

[0057] (3) Incubation with secondary antibody: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody to the corresponding wells, incubate at 37°C for 45 min, wash the plate four times in a plate washer and dry.

[0058] (4) Color development: Add TMB color development solution to a 96-well plate, 50 μL per well, and incubate at 37°C in the dark for 25 min.

[0059] (5) Stop and read: Add stop solution to each well of the 96-well plate, 50 μL, and then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm and read the test results. An OD value greater than 2.1 times that of the negative control well is considered positive.

[0060] Table 9-1: ELISA coated p-tau231 antigen

[0061] Table 9-2: ELISA coated tau antigen

[0062] According to Table 9, curve fitting was performed using the "Prism" software to deduce the Kd values of the antibody for p-tau231 antigen and tau antigen. Figure 3 and Figure 4 As shown. The p-tau231 monoclonal antibodies 1M1F1 and 1M1F3 obtained by the above technical solution can bind to the p-tau231 antigen with a Kd of 10-9M, but have extremely low affinity to the tau antigen; while the commercial p-tau231 antibody and tau antibody used as controls have high affinity to both the p-tau231 antigen and the tau antigen. The p-tau231 monoclonal antibody obtained by this technical solution not only has extremely strong antigen binding activity and affinity, but also has high specificity.

[0063] Table 10: ELISA to evaluate antibody affinity

[0064] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a monoclonal antibody against p-tau231, characterized in that, It includes the following steps: Step 1, polypeptide design and synthesis: Based on the amino acid sequence at positions 224 - 238 of the tau protein, a phosphorylated polypeptide and a non - phosphorylated control polypeptide are designed and synthesized, where: The phosphorylated polypeptide sequence is CKKVAVVRT(p)PPKSPSS, with threonine at position 231 phosphorylated; The non - phosphorylated polypeptide sequence is CKKVAVVRTPPKSPSS; Step 2, immunogen conjugation: Using the N - terminal cysteine residue of the phosphorylated polypeptide, it is linked to the carrier protein KLH by thiol chemical conjugation to form an immunogen complex; Step 3, preparation of conjugate for detection: Using the N - terminal cysteine residues of the phosphorylated polypeptide and the non - phosphorylated polypeptide, they are respectively conjugated to the carrier protein BSA to form antigen complexes for detection.

2. A p-tau231 monoclonal antibody, prepared by the method for preparing a p-tau231 monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of its light - chain variable region is as shown in SEQ ID NO: 1, the amino acid sequence of its heavy - chain variable region is as shown in SEQ ID NO: 2, or the amino acid sequence of its heavy - chain variable region is as shown in SEQ ID NO:

3.

3. An antibody variable region for use in the p-tau231 monoclonal antibody according to claim 2, characterized in that, The CDR1 of the amino acid sequence SEQ ID NO: 1 of the light - chain variable region is QSVYNNNY, CDR2 is SAS, and CDR3 is LGDFDCTSADCGV; The CDR1 of the amino acid sequence SEQ ID NO: 2 of the heavy - chain variable region is QSVYNNNY, CDR2 is IKGGGNT, and CDR3 is TSSGSDY; Or the CDR1 of the amino acid sequence SEQ ID NO: 3 of the heavy - chain variable region is GIDLSRNG, CDR2 is IKGGGSA, and CDR3 is ASSGSDY.

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