P-tau231 specific antibodies and their use in an alzheimer's disease adjunct diagnostic kit

Abstract

The application discloses a p-Tau231 specific antibody and application thereof in an Alzheimer's disease auxiliary diagnosis kit, and belongs to the technical field of immune analysis. The amino acid sequences of CDRH1-CDRH3 of the antibody heavy chain are sequentially shown as SEQ ID NO:1-SEQ ID NO:3, the amino acid sequence of CDRL1 of the light chain is shown as SEQ ID NO:4, the amino acid sequence of CDRL2 of the light chain is QA, and the amino acid sequence of CDRL3 of the light chain is shown as SEQ ID NO:5. The application further provides an antibody targeting p-Tau231 and a chemiluminescence kit for detecting p-Tau231. The antibody has high affinity and high sensitivity, and the production cycle is obviously shortened, is more suitable for being applied to the field of in-vitro diagnosis reagents as a core raw material, and can be used for the auxiliary diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] This application belongs to the field of immunoassay technology and relates to a p-Tau231 specific antibody and its application in an auxiliary diagnostic kit for Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is an age-related, progressive, and irreversible neurodegenerative disease that typically occurs in old age or pre-old age. Clinically, it manifests as a progressive decline in memory, thinking, analytical judgment, spatial awareness, mood, and daily living abilities, accompanied by various neuropsychiatric symptoms and behavioral disturbances, all without impairment of consciousness.

[0003] Studies have found that the main pathological manifestations of Alzheimer's disease (AD) include extracellular senile plaques, intracellular neurofibrillary tangles (NFTs), and cholinergic dysfunction, ultimately leading to neuronal damage and necrosis and eventually spongiform encephalopathy. Among these, senile plaques composed of β-amyloid protein and intracellular neurofibrillary tangles composed of phosphorylated tau protein (p-Tau) are the main neuropathological features of AD. Currently, various pathogenesis theories attempt to explain these changes, including the Aβ-amyloid cascade hypothesis, the Tau protein hypothesis, the inflammation hypothesis, and other mechanisms such as neurovascular damage, oxidative stress, and mitochondrial dysfunction.

[0004] The Tau protein hypothesis posits that excessive phosphorylation of Tau protein leads to a decrease in the levels of normal Tau protein and other microtubule-associated proteins, resulting in microtubule degradation and impaired axonal transport. Simultaneously, excessively phosphorylated Tau protein readily aggregates into insoluble fibers and larger fibrillary tangles. Ultimately, the loss of microtubule stability and the formation of neurofibrillary tangles damage neuronal and synaptic function. To date, approximately 84 sites involved in aberrant phosphorylation of Tau protein have been identified, representing 20% ​​of all Tau amino acids. These include 4 phosphorylation sites on tyrosine, 45 on serine, and 35 on threonine. Sites prone to phosphorylation include pT181, pS200, pT217, pT231, pS396, pS400, and pS404. Studies have shown that pathological changes in Tau protein are more strongly associated with cognitive decline than β-amyloid (Aβ), especially phosphorylated Tau protein at threonine 231 (p-Tau231), which is considered a specific biomarker for Alzheimer's disease (AD). p-Tau231 shows a strong correlation with Aβ-PET, with its elevation typically preceding positive Aβ-PET results. Furthermore, the upward trend is continuous and linear throughout the entire disease course (from preclinical to dementia), without a significant plateau phase. Therefore, it can serve as a biomarker for the pathological state of Aβ in preclinical Alzheimer's disease, providing a new direction for early diagnosis and treatment, and highlighting the importance of studying the mechanisms of Tau protein. Summary of the Invention

[0005] This application provides a specific antibody against p-Tau 231, specifically a p-Tau 231-specific antibody, Anti-pTau231-rRmab-1. The monoclonal antibody prepared using single-B cell technology in this application exhibits higher sensitivity and a significantly shorter production cycle. The kit prepared using Anti-pTau231-rRmab-1 has high sensitivity and a low detection limit, providing a tool for the auxiliary diagnosis of p-Tau231-related diseases, and further offering the application of this antibody or kit in the auxiliary diagnosis of Alzheimer's disease.

[0006] On the one hand, this application provides an antibody or antigen-binding fragment thereof that specifically binds to p-Tau 231.

[0007] In some embodiments, the antibody or antigen-binding fragment comprises at least one, two, three, four, five, or six CDRs selected from the following: (a) a heavy chain variable region CDR-H1 comprising a heavy chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 1; (b) a heavy chain variable region CDR-H2 comprising a heavy chain variable region CDR-H2 comprising a heavy chain variable region CDR-H3 comprising a heavy chain variable region CDR-H3 comprising a heavy chain variable region CDR-H3 comprising a heavy chain variable region CDR-H4 comprising a heavy chain variable region CDR-H5 ... (e) a light chain variable region CDR-L1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4; and (f) a light chain variable region CDR-L2 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 5.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises at least one, at least two, or all three of the VH CDR sequences selected from the following: (a) a heavy chain variable region CDR-H1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 1; (b) a heavy chain variable region CDR-H2 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 2; and (c) a heavy chain variable region CDR-H3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or antigen-binding fragment comprises at least one, at least two, or all three of the VL CDR sequences selected from the following: (a) a light chain variable region CDR-L1 comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 4; (b) a light chain variable region CDR-L2 comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of QA; and (c) a light chain variable region CDR-L3 comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 5.

[0009] In some embodiments, the antibody or antigen-binding fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the following: (i) a heavy chain variable region CDR-H1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR-H2 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR-H3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 3; and (b) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the following: The VL domain of the CDR sequence includes: (i) a light chain variable region CDR-L1 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 4; (ii) a light chain variable region CDR-L2 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of QA; and (iii) a light chain variable region CDR-L3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment of this application comprises: CDR-H1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR-H2 with an amino acid sequence as shown in SEQ ID NO: 2, CDR-H3 with an amino acid sequence as shown in SEQ ID NO: 3, CDR-L1 with an amino acid sequence as shown in SEQ ID NO: 4, CDR-L2 with an amino acid sequence of QA, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO: 5.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises at least one or two heavy chain variable regions selected from: (a) a heavy chain variable region VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 6; and (b) a light chain variable region VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO: 7.

[0011] On the one hand, this application provides a polynucleotide that encodes the aforementioned antibody or antigen-binding fragment.

[0012] On the one hand, this application provides a vector containing the polynucleotide of this application.

[0013] In some embodiments, the vector includes a viral vector, an expression vector, or a recombinant expression vector. In some embodiments, the expression vector can be any suitable recombinant expression vector selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, ZapII (Stratagene), λEMBL4, and λNM1149 can also be used. In some embodiments, the expression vector is pcDNA3.1.

[0014] On the one hand, this application provides a host cell that contains the vector of this application or whose genome integrates the polynucleotides described in this application.

[0015] In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a 293 cell.

[0016] On the one hand, this application provides a kit for detecting p-Tau231.

[0017] In some embodiments, the kit comprises the antibody or antigen-binding fragment of this application. In some embodiments, the kit is used for non-diagnostic immunoassay of p-Tau231. In some embodiments, the kit is a chemiluminescence immunoassay, electrochemiluminescence immunoassay, or ELISA. In some embodiments, the kit is a chemiluminescence immunoassay kit based on a double-antibody sandwich principle, comprising: magnetic beads coated with a first antibody and a second antibody labeled with a chemiluminescent agent. In some embodiments, the chemiluminescent agent is selected from at least one of acridinium ester, alkaline phosphatase (ALP), and horseradish peroxidase (HRP).

[0018] In some embodiments, the second antibody is the antibody or antigen-binding fragment described in this application. In some embodiments, the amino acid sequences of the heavy chains CDRH1-CDRH3 of the first antibody are as shown in SEQ ID NO: 18-SEQ ID NO: 20, respectively; the amino acid sequence of the light chain CDRL1 is as shown in SEQ ID NO: 21; the amino acid sequence of the light chain CDRL2 is RAS; and the amino acid sequence of the light chain CDRL3 is as shown in SEQ ID NO: 22. In some embodiments, the first antibody comprises a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO: 16 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO: 17.

[0019] On the one hand, this application provides the use of the antibody or antigen-binding fragment as described above in the preparation of reagents or kits for detecting p-Tau231.

[0020] On the one hand, this application provides the application of the antibody or antigen-binding fragment as described above and the kit as described above in the auxiliary diagnosis of Alzheimer's disease.

[0021] On the one hand, this application provides a method for preparing an antibody or antigen-binding fragment as described above, including culturing host cells as described in this application; and recovering the antibody or antigen-binding fragment.

[0022] Beneficial effects: This application provides a new antibody targeting p-Tau231 and a kit containing the aforementioned antibody. The antibody produced by this application using single B cell technology has high affinity, high sensitivity, and a significantly shortened production cycle, making it more suitable as a core raw material for use in the field of in vitro diagnostic reagents. Attached Figure Description

[0023] Figure 1 Image showing antigen-specific single B cells sorted by flow cytometry. Figure 2 This is a correlation curve between the detection results and clinical values ​​of the anti-p-Tau231 monoclonal antibody used in this application for chemiluminescence detection of a gradient of values ​​in clinical samples. Detailed Implementation

[0024] The present disclosure will be described in detail below with reference to the embodiments. However, the implementation of the present disclosure is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present disclosure. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present disclosure.

[0025] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. If multiple definitions exist for any term herein, those defined in this section shall prevail.

[0026] The technical solutions provided in this disclosure will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of this disclosure.

[0027] Example 1: Preparation of monoclonal antibodies against specific phosphorylation sites (p-Tau231) of microtubule-binding protein 1. Antigen preparation In this embodiment, positions 224-240 of the microtubule-binding protein were selected as the region of the p-Tau231 polypeptide. During the synthesis of the p-Tau231 polypeptide, a cysteine ​​C residue was added to the N-terminus to label the carrier protein KLH, serving as an immunogen.

[0028] During the synthesis of the p-Tau231 polypeptide, the carrier protein BSA was linked to the N-terminus via a cysteine ​​residue for the detection of the titer of animal immune serum and subsequent detection of the recombinant antibody expression supernatant.

[0029] The p-Tau231 polypeptide has the sequence: CKKVAVVR{pTHR}PPKSPSSAK (SEQ ID NO:8). Threonine is phosphorylated at position 231 during polypeptide synthesis.

[0030] The carrier proteins KLH and BSA are both self-developed proteins and are available for purchase on the market.

[0031] KLH-labeled p-Tau231 peptide was used to immunize New Zealand white rabbits, with each rabbit receiving 500 μg. For the initial immunization, the immunogen was mixed with an equal volume of Freund's adjuvant to form an emulsion, which was then injected subcutaneously at multiple sites. Three weeks later, 250 μg of the immunogen was mixed with an equal volume of Freund's adjuvant to form another emulsion, which was then injected subcutaneously at multiple sites for two booster immunizations. After three immunizations, PBMC samples were collected, coated with BSA-labeled p-Tau231 peptide, and serum titers were determined using ELISA. Rabbits with high serum titers received a booster immunization with 250 μg of the immunogen injected subcutaneously at multiple sites, followed by spleen harvesting.

[0032] 2. Preparation of single B cell suspension The isolation and preparation of lymphocytes from rabbit spleen samples involved physically grinding the rabbit spleen and filtering it through a porous mesh to prepare a single-cell suspension. This single-cell suspension was then used for cell staining and flow cytometry sorting.

[0033] 3. Antigen-specific single B cell sorting Antigen-specific single B cell sorting is based on the specific recognition of characteristic markers on the surface of lymphocytes by flow cytometry antibodies, and the use of flow cytometry to sort specific single B cells from a lymphocyte suspension.

[0034] In this embodiment, the original coupled FITC dyes are sorted.

[0035] The anti-rabbit IgG secondary antibody is a self-developed antibody. It is conjugated with PE dye.

[0036] During cell labeling, DAPI dye is added to distinguish between dead and live cells.

[0037] Cell labeling protocols, B-cell sorting protocols: Dead / Live- / IgG+ / Antigen+.

[0038] Cell labeling procedure: Rabbit lymphocyte suspension, centrifuged at 300g for 5 min, added 5 mL of buffer, mixed by inverting, centrifuged at 300g for 5 min. Discard the supernatant, repeat once, take 30 μL of cell suspension for cell counting, and take 40 μL of cell suspension for blank control tubes and single-staining tubes to be labeled. The remaining cell suspension is used as sample tubes, centrifuged at 300g for 5 min, and resuspended with a small amount of PBS. Blank tubes are left untreated. For single-staining tubes, add PBS to 100 μL, and add 2 μL of PE, 2 μL of FITC, and 2 μL of LDAPI dye respectively. Sample tubes are labeled with PE dye at a ratio of 1.5 μL / 10 μL. 6 Cells, FITC dye 2 μg / 10 6 Calculate the actual amount of cells added, add the corresponding amount of antibody in the dark, and incubate at 4°C for 30 min. After antibody incubation, add 2 mL of buffer, mix gently, centrifuge at 300g for 5 min, and repeat the washing 3 times. Resuspend the cells in 1 mL of buffer, filter the cells, and wait for sorting.

[0039] After completing fluorescence compensation regulation, based on the live cell population, the cell population with double positive signals of PE and FITC was delineated. Figure 1 Antigen-specific B cells were sorted into 96-well plates, with only one cell per well. The plates needed to be immediately stored at low temperature after sorting; dry ice boxes were provided in this example for short-term storage. The wells contained cell lysis buffer, and the sorted 96-well PCR plates were directly used for single-cell antibody gene amplification.

[0040] 4. Preparation of cDNA from rabbit single B cells The preparation of single-cell cDNA libraries was based on SMART 5'RACE technology, and all reagents used were the Vazyme N711 kit from Nanjing Novizan Biotechnology Co., Ltd., which is commercially available. The amplification systems involved in the experiments described in the examples can be found in the N711 kit instructions.

[0041] Single B cell RNA reverse transcription: After sorting, thaw the 96-well plates and place them in a PCR instrument to run the program. After the program is completed, let them stand on ice for 2 min.

[0042] Single-stranded cDNA synthesis in B cells: After the reverse transcription reaction is complete, the single-stranded synthesis system can be added. After adding the system, gently mix the wells and place them in a PCR instrument to run the program. After the program is complete, incubate the samples on ice for 2 minutes.

[0043] Single-cell B-cell DNA double-strand synthesis: After the synthesis reaction of the cDNA single-strand product is completed, the double-strand synthesis system can be added. After adding the system, gently mix the well plate, centrifuge, and then place it in a PCR instrument to run the program. After the program is completed, incubate the well plate samples on ice.

[0044] 5. Amplification of antibody-encoding genes using rabbit single-cell B-cell PCR technology A single B-cell cDNA library can be used to retrieve genes encoding naturally paired antibody heavy and light chains.

[0045] All reagents used for gene amplification were the Vazyme P515 kit from Nanjing Novizan Biotechnology Co., Ltd., which are commercially available. The amplification systems used in the experiments described in this example can be found in the P515 kit instructions.

[0046] The upstream primer contains a homologous arm that interlocks with the 3' end of the promoter CMV gene sequence, so the antibody-encoding gene can be directly used to construct a linear expression cassette after retrieval.

[0047] The downstream primer for the antibody heavy chain encoding gene is located in a constant region and contains a homologous arm that interacts with the BGH-polyA gene sequence.

[0048] The downstream primer for the light chain encoding gene is located in a constant region and contains a homologous arm complementary to the BGH-polyA gene sequence. Therefore, after the antibody encoding gene is retrieved, it can be directly used to construct a linear expression cassette.

[0049] Amplification of the antibody heavy chain coding region gene; forward primer sequence is: caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 9).

[0050] Antibody heavy chain coding region gene amplification, reverse primer sequence is: tagtggatccgagctcggtacctcatttacccggagagcg (SEQ ID NO: 10).

[0051] The forward primer sequence for amplifying the antibody light chain coding region gene is as follows: caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 9).

[0052] Antibody light chain coding region gene amplification, reverse primer sequence is: tagtggatccgagctcggtacctcaacagtcacccctattg (SEQ ID NO: 11).

[0053] Extraction of antibody light and heavy chain encoding genes: Add to the PCR amplification system according to the kit instructions, mix gently in the well plate, place in the PCR instrument and run the program. After the program is completed, place the well plate samples on ice.

[0054] In this embodiment, the pairing positivity rate of the amplification products encoding the antibody light and heavy chains in the same 96-well plate was over 80%, and the bands were clear as detected by agarose gel electrophoresis, indicating that both the single-cell flow cytometry sorting and the encoding gene amplification experiment were effective. The amplification products were used for the construction of recombinant expression plasmids.

[0055] 6. Construction and expression of antibody heavy and light chain recombinant expression plasmids The recombinant expression vector, pcDNA3.1 (Invitrogen), was purchased from the ThermoFisher SCIENTIFIC website. Before recombinant construction, the expression vector was linearized by single digestion with HindIII restriction enzyme, which was purchased from the New England Biolabs website.

[0056] For efficient recombination of vectors and coding genes, choose the seamless cloning kit, and purchase the C115# kit from the Vazyme website.

[0057] Construction of recombinant expression plasmid: The amplified products encoding the antibody heavy and light chains were circularized with the pcDNA3.1 linearized vector using seamless cloning technology, and then transformed into E. coli DH5α competent cells. The plasmids were then plated on LB fixation medium plates and incubated overnight at 37°C with the plates inverted.

[0058] Selection of recombinant positive clones: Eight single colonies were selected from the heavy and light chains of the initial screening antibody, and the colony positivity rate was determined by PCR bacterial testing.

[0059] The bacterial detection PCR of the recombinant plasmid uses the upstream primer sequence of caagctggctagcgtttaaactt (SEQ ID NO:12).

[0060] The downstream primer sequence for antibody heavy chain bacterial detection PCR is: ctcatttacccggagagcg (SEQ ID NO:13).

[0061] The downstream primer sequence for antibody light chain bacterial detection PCR is: acctcaacagtcacccctattg (SEQ ID NO:14).

[0062] Recombinant positive clones were sent for testing: Five PCR-positive clones from both the antibody heavy chain and light chain were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0063] Rabbit antibody gene sequence analysis: The V region of the antibody sequence was determined using the IMGT database, and the antibody sequence was analyzed for the CDR1 / CDR2 / CDR3 regions of the heavy chain and light chain. The correct sequence number of the PCR-positive clones was then derived and determined.

[0064] Small-scale expression of recombinant expression plasmids: Cloning of the correct sequence and small-scale extraction of antibody light and heavy chain plasmids from bacterial culture. The plasmids were then co-transfected into HEK293 mammalian cells. Ten days after transfection, the cell supernatant was collected by centrifugation. The supernatant was used for antigen specificity assessment, and cell supernatant purification was performed after the initial ELISA screening results were available.

[0065] In this embodiment, 100 plasmids were transfected in each round, meaning that 100 monoclonal antibodies could be obtained in each round of transfection. A total of 12 rounds of transfection experiments were conducted.

[0066] 7. Evaluation of antigen specificity in recombinant expression supernatant Antigen-coated plates were prepared, and p-Tau231 peptide and Tau231 peptide (microtubule-binding protein with non-phosphorylated 231st site) were initially selected for screening. Due to their relatively short sequences, p-Tau231 peptide and Tau231 peptide had a cysteine ​​residue added to their N-terminus, and the antigen-coating process was completed using the SA-Biotin conjugation method.

[0067] The Tau231 polypeptide has the sequence: CKKVAVVRTPPKSPSSAK (SEQ ID NO:15). During polypeptide synthesis, the threonine residue at position 231 is non-phosphorylated.

[0068] The screening protocol for p-Tau231 polypeptide monoclonal antibodies involves selecting antibodies that react with p-Tau231 polypeptide but do not react with Tau231 polypeptide; these are preliminarily identified as p-Tau231-specific monoclonal antibodies.

[0069] Indirect ELISA was used to detect cell supernatant. The cells were coated with self-produced rabbit secondary antibody Anti-Rabbit IgG mAb and HRP-labeled with goat anti-rabbit polyclonal antibody. The reactivity OD>1 indicated that the recombinant plasmid was normally expressed on 293 cells.

[0070] Indirect ELISA was used to detect cell supernatants, and the reactivity of the two antigen-coated plates was evaluated to obtain the initial screening results of the supernatants of the well plates (Tables 1-1 and 1-2 only show the detection data of supernatants of 50 cell lines).

[0071] Table 1-1: Antigen-antibody affinity data for some antibodies (top)

[0072] Table 1-2: Antigen-antibody affinity data for some antibodies (below)

[0073] Indirect ELISA results: The proportion of p-Tau231 peptide-specific antibodies was low. A total of 1200 monoclonal antibodies were initially screened and transfected. Antibodies that reacted with SA+Biotin-pTau231-Ag and did not react with SA+Biotin-Tau231-Ag were preliminarily screened and 100 specific monoclonal antibodies were selected.

[0074] The cell supernatant identified in the initial screening was purified by protein A to obtain a small amount of monoclonal antibody, with an average of 1-3 mg per strain.

[0075] 8. Recombinant antibody ELISA paired screening.

[0076] The recognition of the specific phosphorylation site (p-Tau231) of microtubule-binding protein was achieved through a pairing screening of universal antibodies against the non-phosphorylation site of Tau protein and p-Tau231 peptide-specific antibodies. Previously, the screening of highly sensitive and specific antibodies against the non-phosphorylation site of Tau protein had been completed. Therefore, in this embodiment, a sandwich ELISA experiment was conducted on 100 monoclonal antibodies initially screened by indirect ELISA to identify the superior monoclonal antibody that can specifically recognize the p-Tau231 peptide.

[0077] Rabbit-Anti-Tau-mAb-A, a universal antibody against the nonphosphorylation site of Tau protein, has the following heavy chain variable region sequence: QSVEESGGRLVKPDETLTLTCTVSGIDLSTYAMGWVRQAPGEGLEWIATIGISGGTYYASWAKGRFTISKTSTTVDLKMTSLTAADTATYFCASSRATTYPIWGPGTLVTVSS (SEQ ID NO:16); (CDR-H1: GIDLSTYA (SEQ ID NO:18); CDR-H2: IGISGGT (SEQ ID NO:19); CDR-H3: ASSRATTYPI (SEQ ID NO:20)) The light chain variable region sequence is as follows: DIVMTQTASPVSAAVGGTVTINCQASQSISTALAWYQQKPGQPPKLLIYRASTLASGVPSRFSGSGSGTQFTLTISDLECADAATYYCQGDYYTKSTSYLNGFGGGTEVVVK (SEQ ID NO: 17); (CDR-L1: QSISTA (SEQ ID NO:21); CDR-L2: RAS; CDR-L3: QGDYYTKSTSYLNG (SEQ ID NO:22)) Simultaneous detection data from the ELISA platform of clinical positive samples showed that the paired ELISA experiment selected the phosphorylated full-length microtubule-binding protein p-Tau as the screening agent.

[0078] One hundred monoclonal antibodies were initially screened out. The full-length p-Tau protein was detected by sandwich ELISA. Finally, the top 20 antibodies with the highest paired detection signal values ​​were selected and verified on a chemiluminescence platform to detect the antibodies in clinical samples (the detection results of 20 pairs of paired antibodies are shown as an example only).

[0079] Table 2: Detection data of p-Tau full-length protein paired antibodies (partial)

[0080] 9. Screening of recombinant antibodies using a chemiluminescence platform The antibodies selected from the sandwich ELISA assay were coated onto magnetic beads. The specific procedure was as follows: 2 mg of magnetic beads were washed twice with activation buffer, then a certain amount of EDC was added and the mixture was vortexed and the supernatant was discarded by magnetic aspiration. The precipitate was added to 1000 μL of coupling buffer, followed by 40 μg of antibody, and vortexed for 2 h. Then, 100 μL of blocking buffer was added, and the mixture was vortexed for 3 h. Finally, 1000 μL of TTBST was added to wash the magnetic beads, followed by 1000 μL of preservation buffer.

[0081] A series of biotin-labeled Tau protein peptides were artificially synthesized, and amino acids at specific positions were phosphorylated to serve as screening agents. Specific information about the screening agents is shown in Table 3. Table 3: Original Information Table for Chemiluminescence Platform Screening of Recombinant Antibodies

[0082] The streptavidin (SA) was labeled with acridinium ester, and the specific procedure was as follows: 100-(100 / C) SA Add 100 μL of coupling buffer to a 0.5 mL brown EP tube, and add (100 / C) SA Add 1 μL of SA to a 0.5 mL brown EP tube to achieve a final SA labeling concentration of 1 mg / mL. Add 5 mM acridine ester to the 0.5 mL brown EP tube, mix thoroughly using a vortex mixer, and then react vertically for 2 h at room temperature (20-25℃) to purify and remove free acridine ester.

[0083] The coated antibody, labeled SA, and screening antigen were prepared according to the above method. Each screening antigen was then detected using a fully automated chemiluminescence analyzer according to the set program. Antibodies that react with synthetic peptides 7# and 8# but do not react with other synthetic peptides (1#, 2#, 3#, 4#, 5#, 6#) are the preferred monoclonal antibodies that specifically recognize p-Tau231.

[0084] Of the 20 monoclonal antibodies screened by sandwich ELISA, one antibody specifically recognizing p-Tau231 was selected by chemiluminescence platform detection. Table 4 shows the detection results of three monoclonal antibodies as examples.

[0085] Table 4: Partial Detection Data of p-Tau231 Specific Antibody Chemiluminescence Platform

[0086] The selected specific antibody, Anti-pTau231-rRmab-1, has the following heavy chain variable region sequence: QCQEQLEESGGRLVTPGTPLTLTCRVSGIDLSSSGMSWVRQAPGKGLEYIGYISRSGSTYYASWAKGRFTISKTSTAVDLKVNSPTTEDTATYFCTRAGIWGPGTLVTVSS(SEQ ID NO:6); The light chain variable region sequence is as follows: AQVLTQTPSSVSAAVGGTVTINCQASQSVYNNNYLYWYQQKPGQPPKLLIYQASKLASGVPSRFKGSGSGTQFILNISDVQCDDAATYYCLGDYDCNVADCGAFGGGTEVVVK (SEQ ID NO: 7).

[0087] Table 5: Amino acid sequence of p-Tau231 antibody

[0088] Example 2: Application of anti-phosphorylated Tau 231 protein (p-Tau231) monoclonal antibody in chemiluminescence detection of clinical samples The selected p-Tau231-specific antibody Anti-p-Tau231-rRmab-1 was applied to chemiluminescence detection experiments to detect clinical samples with a set value gradient, and the correlation between the detection results and the clinical set values ​​was compared.

[0089] The concentration of p-Tau231 in the graded clinical samples was determined using the Simoa single-molecule immunoassay. The specific concentrations are shown in Table 6. Table 6: p-Tau231 concentration in clinical samples

[0090] Rabbit-Anti-Tau-mAb-A was fixed as the coating antibody. The specific coating method was as follows: 2 mg of magnetic beads were washed twice with activation buffer, then a certain amount of EDC was added and vortexed to mix. The supernatant was discarded by magnetic aspiration. 1000 μL of coupling buffer was added to the precipitate, followed by 40 μg of antibody. The mixture was vortexed to mix for 2 h, then 100 μL of blocking buffer was added, and the mixture was vortexed to block for 3 h. Finally, 1000 μL of LTBST was added to wash the magnetic beads, followed by 1000 μL of preservation buffer.

[0091] The selected p-Tau231-specific antibody Anti-p-Tau231-rRmab-1 was used as the labeling antibody for acridine ester labeling, and 100-(100 / C) was used. Ab Add 100 μL of coupling buffer to a 0.5 mL brown EP tube, and add (100 / C) Ab Add 1 μL of antibody solution to a 0.5 mL brown EP tube to achieve a final antibody labeling concentration of 1 mg / mL. Add 5 mM acridine ester to the 0.5 mL brown EP tube, mix thoroughly using a vortex mixer, and then react vertically for 2 h at room temperature (20-25℃) to purify and remove free acridine ester.

[0092] The coated antibody and labeled antibody were prepared according to the above method. The concentration of p-Tau231 in samples P01-P23 was detected using a fully automated chemiluminescence analyzer. The results are shown in Table 7. Table 7: Chemiluminescence Detection Results of Clinical Samples

[0093] A scatter plot of p-Tau231 concentration for each sample was created, with the p-Tau231 concentration detected by Simoa single-molecule immunoassay on the x-axis and the p-Tau231 signal value detected by chemiluminescence on the y-axis. Figure 2 The correlation coefficient of p-Tau231 between the two detection methods was calculated. The correlation coefficient between the two detection methods was R = 0.9909. 2 =0.9818, meaning that when the preferred p-Tau231-specific antibody Anti-p-Tau231-rRmab-1 is used in combination with a matching antibody for chemiluminescence detection experiments, the detection results show a strong correlation with the simoa single-molecule immunoassay. This preferred antibody can be used for downstream kit preparation.

[0094] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to p-Tau231, characterized in that, The amino acid sequences of the heavy chains CDRH1-CDRH3 are shown in SEQ ID NO:1-SEQ ID NO:3, respectively; the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:4; the amino acid sequence of the light chain CDRL2 is QA; and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO:

5.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, It includes: the heavy chain variable region VH as shown in SEQ ID NO: 6 and the light chain variable region VL as shown in SEQ ID NO:

7.

3. A kit for detecting p-Tau231, characterized in that, The kit includes magnetic beads coated with a first antibody and a second antibody labeled with a chemiluminescent agent; Wherein, one of the antibodies in the antibody pair consisting of the first antibody and the second antibody is the antibody or its antigen-binding fragment as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The second antibody in the antibody pair consisting of the first antibody and the second antibody is the antibody or its antigen-binding fragment as described in claim 1. The amino acid sequences of the heavy chains CDRH1-CDRH3 of the first antibody are shown in SEQ ID NO: 18-SEQ ID NO: 20, respectively. The amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:

21. The amino acid sequence of the light chain CDRL2 is RAS. The amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO:

22.

5. The reagent kit according to claim 4, characterized in that, The first antibody comprises: a heavy chain variable region VH as shown in SEQ ID NO: 16 and a light chain variable region VL as shown in SEQ ID NO:

17.

6. The reagent kit according to claim 3, characterized in that, The chemiluminescent agent is selected from at least one of acridine ester, alkaline phosphatase, and horseradish peroxidase.

7. The use of the antibody or its antigen-binding fragment as described in claim 1 in the preparation of a kit for detecting p-Tau231 protein.

8. The use of the antibody or antigen-binding fragment thereof as described in claim 1 in the preparation of a kit for the auxiliary diagnosis of Alzheimer's disease.

9. A biomaterial, characterized in that, Choose from any of the following: a. A polynucleotide encoding the antibody or antigen-binding fragment thereof as described in claim 1; b. A carrier containing the polynucleotide described in a; c. A host cell containing the vector described in b or whose genome integrates the polynucleotide described in a.

10. A kit for detecting p-Tau231 protein, characterized in that, It comprises the antibody or its antigen-binding fragment as described in claim 1.

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