Monoclonal antibodies against emtbr-tau243 protein fragments and uses and products thereof

Abstract

The application provides a kind of anti-eMTBR-tau243 protein fragment monoclonal antibody and its application and product, it is related to the field of biotechnology.The anti-eMTBR-tau243 protein fragment monoclonal antibody provided in the application can specifically bind tau protein fragment (aa225-242), and tau protein fragment (aa1-256) and full-length tau protein (2N4R, aa1-441) comprising the fragment, and can be used for the preparation of the above-mentioned protein detection product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a monoclonal antibody against the eMTBR-tau243 protein fragment and its applications and products. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly. One of its typical pathological features is the hyperphosphorylation and accumulation of tau protein in the brain, forming neurofibrillary tangles (NFTs). Currently, early identification and dynamic monitoring of tau pathology have become crucial for AD diagnosis, staging, and drug development. However, while existing imaging techniques such as tau-PET can visually visualize tau deposition in the brain, they suffer from high costs, radiation exposure, and low equipment availability. Traditional cerebrospinal fluid (CSF) testing can reflect pathological changes in the central nervous system, but its clinical application is limited due to the need for lumbar puncture. Therefore, developing a simple, non-invasive, and reproducible blood-derived tau pathological biomarker has significant clinical translational value.

[0003] In recent years, a novel tau protein fragment—eMTBR-tau243 (endogenous microtubule-binding region tau-243)—has been discovered. This fragment originates from the microtubule-binding region (MTBR) released after endogenous enzymatic cleavage of tau protein. Its C-terminus undergoes specific cleavage at residue 256, specifically reflecting the level of neurofibrillary tangles in the brain. Unlike existing phosphorylated tau detected in blood (such as p-tau181 and p-tau217), which primarily reflect the early phosphorylation state of tau protein, eMTBR-tau243 shows a high degree of concordance with the time point of tau-PET positivity during the course of Alzheimer's disease (AD), particularly showing significant increases in the stages of mild cognitive impairment (MCI) and dementia, and is closely related to the rate of cognitive decline. More importantly, this biomarker can effectively distinguish AD from other 3R or 4R tau protein diseases, demonstrating good disease specificity. Based on these characteristics, eMTBR-tau243 has been considered one of the most promising blood-derived biomarkers for assessing the tau aggregate burden in late-stage AD.

[0004] Currently, the detection of eMTBR-tau243 mainly relies on immunoprecipitation combined with mass spectrometry (IP-MS). While this method offers high sensitivity, it is complex, expensive, and has low throughput, making it difficult to apply in routine clinical laboratories or large-scale screening. Developing high-affinity antibodies that specifically recognize eMTBR-tau243 is a prerequisite for the translational application of immunoassay platforms (such as CLIA, PEA, SIMOA, and NULISA). Specific enrichment of the target fragment using immunoprecipitation (IP) antibodies can significantly improve detection sensitivity in complex biological samples (such as plasma) and avoid cross-reactivity from N-terminal or C-terminal interfering fragments, laying the foundation for developing high-throughput, standardized, and low-cost diagnostic kits. Therefore, developing specific IP antibodies against eMTBR-tau243 has a clear practical need and significant application prospects for promoting this biomarker from a research tool to clinical diagnostics, companion diagnostics, and drug efficacy monitoring.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a monoclonal antibody against the eMTBR-tau243 protein fragment to address the aforementioned technical problems.

[0007] A second objective of this invention is to provide biological materials.

[0008] A third objective of this invention is to provide a method for preparing a monoclonal antibody against the above-mentioned eMTBR-tau243 protein fragment.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned monoclonal antibody against the eMTBR-tau243 protein fragment in the preparation of detection products for the eMTBR-tau243 protein fragment.

[0010] The fifth objective of this invention is to provide a marker for the eMTBR-tau243 protein fragment.

[0011] The sixth objective of this invention is to provide a kit for detecting the eMTBR-tau243 protein fragment.

[0012] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a monoclonal antibody against the eMTBR-tau243 protein fragment, wherein the variable region of the monoclonal antibody against the eMTBR-tau243 protein fragment comprises: a complementarity-determining region CDR1-VH as shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH as shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH as shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL as shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL as shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL as shown in SEQ ID NO.6.

[0013] As a further technical solution, the amino acid sequence of the heavy chain variable region VH of the monoclonal antibody against the eMTBR-tau243 protein fragment is shown in SEQ ID NO.7; As a further technical solution, the amino acid sequence of the light chain variable region VL of the monoclonal antibody against the eMTBR-tau243 protein fragment is shown in SEQ ID NO.8; Secondly, the present invention provides a biomaterial selected from any one of the following: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a monoclonal antibody encoding the anti-eMTBR-tau243 protein fragment; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid in a, or containing the vector in b, or expressing a monoclonal antibody against the eMTBR-tau243 protein fragment.

[0014] Thirdly, the present invention provides a method for preparing the above-mentioned monoclonal antibody against the eMTBR-tau243 protein fragment, which is obtained by the cell fermentation method described above.

[0015] Fourthly, the present invention provides the application of the above-mentioned monoclonal antibody against the eMTBR-tau243 protein fragment in the preparation of detection products for the eMTBR-tau243 protein fragment.

[0016] Fifthly, the present invention provides a marker for the eMTBR-tau243 protein fragment, comprising the monoclonal antibody against the eMTBR-tau243 protein fragment and the marker; The monoclonal antibody against the eMTBR-tau243 protein fragment was conjugated with a marker.

[0017] As a further technical solution, the labeling material includes enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0018] In a sixth aspect, the present invention provides a kit for detecting the eMTBR-tau243 protein fragment, the kit comprising a monoclonal antibody against the eMTBR-tau243 protein fragment or a marker of the eMTBR-tau243 protein fragment.

[0019] As a further technical solution, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic microparticle detection kit, an immunofluorescence detection kit, or an immunoblotting detection kit.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The monoclonal antibody against the eMTBR-tau243 protein fragment provided by this invention can specifically bind to the tau protein fragment (aa225-242), as well as the tau protein fragment containing this fragment (aa1-256) and the full-length tau protein (2N4R, aa1-441), and can be used in the preparation of the above-mentioned protein detection products. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 : Flow sorting results; Figure 2 Results of immunoblotting analysis; Figure 3 Immunohistochemical staining results of the brains of AD mice; Figure 4 Immunohistochemical staining results of the brain of normal mice; Figure 5 Results of IP testing of endogenous tau protein in mouse brain tissue; Figure 6 IP results for tau protein fragments (aa1-256) and full-length tau protein (2N4R, aa1-441). Detailed Implementation

[0023] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0025] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable region can be called "VH," and the light chain variable region can be called "VL." These domains are usually the most variable parts of the antibody and contain antigen-binding sites. The variable regions of both the heavy and light chains consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held tightly together by the FRs to form the variable region. Typically, the VL / VH variable regions of the heavy and light chains are obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0026] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0027] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0028] In a first aspect, the present invention provides a monoclonal antibody against the eMTBR-tau243 protein fragment, wherein the variable region of the monoclonal antibody against the eMTBR-tau243 protein fragment comprises: a complementarity-determining region CDR1-VH as shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH as shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH as shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL as shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL as shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL as shown in SEQ ID NO.6.

[0029] The amino acid sequences of SEQ ID NO.1-SEQ ID NO.6 are shown in Table 1.

[0030] Table 1

[0031] In some alternative embodiments, the amino acid sequence of the heavy chain variable region VH of the monoclonal antibody against the eMTBR-tau243 protein fragment is shown in SEQ ID NO.7: QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYDMVWVRQAPGEGLEYIGFMHKSGSAYYASWAKGRFTISRTSTTVDLKMTSLTMEDTATYFCARGPSYCSGFNLWGQGTLVTVSS (SEQ ID NO. 7).

[0032] In some alternative embodiments, the amino acid sequence of the light chain variable region VL of the monoclonal antibody against the eMTBR-tau243 protein fragment is shown in SEQ ID NO. 8: AAVMTQTPSSVSAAVGGTVRISCQSSKSVVHNNWLSWYQQKPGQPPKLLIYGASTLASGVPSRFKGSGCGTQFILTISDVQCDDAATYYCLGGYSSSSDDGFGGGTEVVVK (SEQ ID NO. 8).

[0033] Secondly, the present invention provides a biomaterial selected from any one of the following: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a monoclonal antibody encoding the anti-eMTBR-tau243 protein fragment; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid in a, or containing the vector in b, or expressing a monoclonal antibody against the eMTBR-tau243 protein fragment.

[0034] Thirdly, the present invention provides a method for preparing the above-mentioned monoclonal antibody against the eMTBR-tau243 protein fragment, which is obtained by the cell fermentation method described above.

[0035] This preparation method is simple and efficient, and can obtain a large number of monoclonal antibodies against the eMTBR-tau243 protein fragment through fermentation.

[0036] Fourthly, the present invention provides the application of the above-mentioned monoclonal antibody against the eMTBR-tau243 protein fragment in the preparation of detection products for the eMTBR-tau243 protein fragment.

[0037] The monoclonal antibody against the eMTBR-tau243 protein fragment provided by this invention can specifically recognize the tau protein fragment (aa225-242), as well as the tau protein fragment containing this fragment (aa1-256) and the full-length tau protein (2N4R, aa1-441), and therefore can be used for the detection of the above proteins.

[0038] Fifthly, the present invention provides a marker for the eMTBR-tau243 protein fragment, comprising the monoclonal antibody against the eMTBR-tau243 protein fragment and the marker; The monoclonal antibody against the eMTBR-tau243 protein fragment was conjugated with a marker.

[0039] This marker can be used for specific labeling of the eMTBR-tau243 protein fragment.

[0040] In some alternative embodiments, the labeling material includes, but is not limited to, enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0041] In a sixth aspect, the present invention provides a kit for detecting the eMTBR-tau243 protein fragment, the kit comprising a monoclonal antibody against the eMTBR-tau243 protein fragment or a marker of the eMTBR-tau243 protein fragment.

[0042] This kit can be used for the detection of eMTBR-tau243 protein fragments.

[0043] In some alternative implementations, the kit includes, but is not limited to, immunochromatographic assay kits, ELISA kits, immunomagnetic microparticle assay kits, immunofluorescence assay kits, or immunoblotting assay kits.

[0044] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0045] Example 1: Preparation of rabbit monoclonal antibody against eMTBR-tau243 protein fragment 1. Immunogen preparation In this embodiment, the human tau protein (Uniprot protein number: P10636-8) polypeptide fragment aa225-256 was selected as the immunogen, and its amino acid sequence is: CKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNV (SEQ ID NO.9).

[0046] 2. Rabbit immunization Reagent: adjuvant, 75% alcohol.

[0047] Consumables: Syringes.

[0048] Sample: Immunogen peptide conjugated with KLH (keyhole hemocyanin).

[0049] Rabbit immunization: Step a): Animal selection. Use New Zealand White rabbits, around 2.5 kg in young adulthood. Choose healthy animals with glossy fur and free movement. After selecting the animal, raise it for about two weeks to allow it to acclimatize.

[0050] Step b): Prepare for the experiment and mark the rabbits.

[0051] Step c): Remove the antigen from the -20°C freezer and thaw it at room temperature, avoiding repeated freeze-thaw cycles. Label the syringe with the project number and animal number.

[0052] Step d): Extract the antigen (the antigen should be completely mixed). The antigen concentration for the first immunization is 1 mg / ml, and for rabbits it is 0.5 ml / rabbit. The antigen concentration for the second to fourth immunizations is halved.

[0053] Step e): Draw the adjuvant, with a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. Ensure the adjuvant is thoroughly mixed before drawing it into the syringe.

[0054] Step f): Connect the two syringes with the syringe connecting tube and emulsify them completely. The emulsification standard is: the emulsified immunogen should not disperse when dropped into 37 ℃ water to be considered qualified.

[0055] Step g): Rabbits were given multiple subcutaneous injections, 0.2 ml at each point. A second immunization was given 14 days after the first, with a 7-day interval between the second, third, and fourth immunizations. Blood samples were collected from the rabbits after all four immunizations for titer testing.

[0056] Step h): The procedure for collecting a small serum sample is as follows: The experimental rabbit is restrained in a frame, and the ear is gently tapped to dilate the central auricular artery. The area is disinfected with 75% alcohol. The rabbit's ear is held in place with the left hand, and the syringe is held in the right hand. The needle is inserted into the central auricular artery at a point one-third of the way from the distal end, parallel to the artery and directed towards the heart. 8 ml of blood is collected at a time. After blood collection, pressure is applied with a cotton ball to stop the bleeding.

[0057] 3. Rabbit serum titer detection The immunogenicity of rabbit serum was detected by indirect ELISA. Rabbits with an OD450nm value exceeding 0.6 under a serum dilution of 1:64000 were considered to have qualified titers and could proceed to the next step.

[0058] Reagents: Goat anti-rabbit-HRP (Huaan Biotechnology: HA1001), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Sangon Biotech, domestically produced analytical grade.

[0059] Consumables: Microplate (Hangzhou Shengyou).

[0060] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), ELISA reader (MD: Cmax plus).

[0061] Indirect ELISA procedure: Step a): Dilute the antigen to 2 μg / mL with coating buffer, add 50 μL / well to the microplate, cover and coat overnight at 4°C.

[0062] Step b): Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 150 μL / well, and incubate at 37℃ for 1 h.

[0063] Step c): Shake off the liquid in the wells and add 50 μL of serum at different dilution ratios to each well of the microplate. Cover the plate and incubate at 37°C for 30 min.

[0064] Step d): Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute goat anti-rabbit-HRP to the working concentration (1:30000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37°C for 45 min.

[0065] Step e): Develop color, terminate and read the value. Discard the liquid in the wells. Add washing buffer to the microplate at a rate of 180 μL / well and wash the microplate 3 times. Add 100 μL of freshly prepared TMB chromogenic substrate to each reaction well and incubate at 37℃ for 10 min. Then add 90 μL / well of stop solution to terminate the reaction and measure the OD value at 450 nm on the microplate reader.

[0066] The results are shown in Table 2.

[0067] Table 2

[0068] 4. Isolation of rabbit peripheral blood lymphocytes (PBMCs) Reagents: Rabbit peripheral blood lymphocyte separation kit (Solarbio: P8760), red blood cell lysis buffer (Solarbio: R1010), PBS (BasalMedia: B310KJ-500ml).

[0069] Consumables: 50mL centrifuge tubes (BD: 352070), 10mL pipettes (Hangzhou Lanjieke: 352070).

[0070] Equipment: Biosafety cabinet (Sujing: BSC-1300IIB2), centrifuge (Thermo: THM#75004530).

[0071] Isolation of rabbit peripheral blood lymphocytes (PBMCs): Step a): Take 20 ml of anticoagulated blood from the rabbit with the best titer after the fourth immunization, place it on ice, and dilute the whole blood with an equal volume of PBS.

[0072] Step b): Add an equal volume of rabbit peripheral lymphocyte separation solution to a centrifuge tube, and spread the diluted blood evenly on top of the separation solution.

[0073] Step c): Centrifuge at 1800 rpm with a horizontal rotor for 30 min at room temperature.

[0074] Step d): After centrifugation, distinct layers will appear: the top layer is diluted plasma, the middle layer is a clear separation medium, the white membrane layer between the plasma and the separation medium is the lymphocyte layer, and the bottom of the centrifuge tube contains red blood cells and granulocytes. Carefully aspirate the white membrane layer cells into a clean 50mL centrifuge tube, and wash the white membrane layer cells with 10mL PBS. Centrifuge at 1500rpm for 5min.

[0075] Step e): Discard the supernatant, resuspend the cells in 20 mL of PBS, centrifuge at 1500 rpm for 5 min. Repeat step e) and discard the supernatant for later use.

[0076] Step f): Add 5 ml of erythrocyte lysis buffer and gently vortex or invert to mix. Incubate at 37°C for 5 minutes. After erythrocyte lysis, the solution should be clear and transparent.

[0077] Step g): Centrifuge at 1500 rpm for 5 minutes to precipitate white blood cells, and carefully aspirate the supernatant.

[0078] Step h): Resuspend the cells in 20 mL of PBS and centrifuge at 1500 rpm for 5 min.

[0079] Step i): Repeat step h) and discard the supernatant. The peripheral blood lymphocytes obtained are then kept for later use.

[0080] 5. Sorting of B cells that specifically bind to antigens Antigen-positive B cells are selected negatively by T cell surface markers, and B cell surface immunoglobulin-positive cells are selected positively. Then, specific B cells are sorted from peripheral blood mononuclear cells by sorting antigens labeled with fluorescent dyes.

[0081] Reagents: PBS (Basal Media: B310KJ-500ml), fetal bovine serum (Sijiqing: 11011-8611), mouse anti-rabbit CD4 antibody-FITC, mouse anti-rabbit IgG-PE, mouse anti-rabbit IgM-biotin, streptavidin-PE / Cy7, BrilliantViolet 421™-streptavidin, Alexa Fluor®647-streptavidin, biotin-labeled antigen-BSA.

[0082] Consumables: 15mL centrifuge tubes (BD: 352097), EP tubes (AXYGEN: MCT-150-C).

[0083] Equipment: Biosafety cabinet (Sujing: BSC-1300IIB2), flow cytometer (Sony: MA900), centrifuge (Thermo: THM#75004530).

[0084] Immunogen-specific binding to B cell sorting: Step a): Set the flow cytometry sorting parameters (IgG, IgM, antigen-BSA) and experimental groups (blank cell control group, compensated single positive tube control group, reduced fluorescence control group, experimental group).

[0085] Step b): For IgM and antigen (antigen-BSA) positive indicators, IgM-Biotin and antigen (antigen-BSA)-Biotin should be pre-incubated with streptavidin-PE / Cy7 and Brilliant Violet 421™ / Alexa Fluor® 647-Streptavidin for 20 min, respectively, and then blocked with excess BSA-Biotin for 20 min before sorting and detection. In step e, these should be added to the corresponding experimental group.

[0086] Step c): Take peripheral blood lymphocytes, resuspend them in 10 mL PBS, count the cells, aliquot the cells into 1.5 mL EP tubes and centrifuge at 1500 rpm for 5 min.

[0087] Step d): Carefully discard the supernatant and resuspend in 100 μL PBS.

[0088] Step e): Add the corresponding antibody to each experimental group set in step a, and gently vortex to mix.

[0089] Step f): After incubating at room temperature for 20 min, resuspend in 800 uL PBS, centrifuge at 1500 rpm for 5 min.

[0090] Step g): Carefully discard the supernatant, resuspend the cells in 1 mL of PBS, and centrifuge at 1500 rpm for 5 min.

[0091] Step h): After repeating step g, carefully discard the supernatant. Add 1 mL of PBS containing 2% FBS to the experimental group for resuspension, and add 200 μL of PBS containing 2% FBS to the other groups for resuspension.

[0092] Step i): Place on ice and load onto the machine.

[0093] Step j): Adjust fluorescence compensation on the flow cytometer using a blank cell control group and a compensated single-positive control group, and then gate the flow cytometer by subtracting one fluorescence control group. Finally, load the experimental group samples and sort out the antigen-double-positive cell population, such as... Figure 1 As shown.

[0094] The sorting strategy is as follows: All events → FSC / SSC cell population screening Figure 1 (Top left image in the image) (A) → Single-cell screening ( Figure 1 (A in the middle) (B) → FITC / PE-Cy7 fluorescence screening ( Figure 1 (B in the middle) (C) → Further positioning of PE / SSC ( Figure 1 (C in the middle) (D) → Dual fluorescence detection using Alexa Fluor 647 and BV421 ( Figure 1 (D in the middle).

[0095] The obtained analysis subjects were single-cell populations; FITC and PE-Cy7 signals were mostly negative; the target cells mainly expressed the Alexa Fluor 647 marker; BV421 + Alexa Fluor 647 + Double-positive cells were collected into 96-well plates. Sorting of antigen-specifically bound B cells: Refer to patent "m6A binding protein and its application" (Publication No.: CN115975038A).

[0096] 6. Cloning and production of rabbit monoclonal antibodies To obtain rabbit monoclonal antibodies that recognize antigens, the selected positive B cells were reverse transcribed to obtain cDNA, which was then sequenced. The selected rabbit monoclonal antibody heavy chain variable region (VH) and light chain variable region (VL) were then constructed into an expression vector. Finally, the antibody was produced through expression in mammalian cells.

[0097] Single B cell lysis: Reagents: dithiothreitol, digitalis saponins, ribonuclease inhibitors, 20-polythymidine primers, random hexamer primers, nuclease-free water.

[0098] Consumables: PCR plates, pipette tips.

[0099] Equipment: PCR instrument.

[0100] Step a): Prepare single-cell lysis buffer according to Table 3 and dispense it into the wells of a PCR plate.

[0101] Table 3 Single-cell lysate

[0102] Step b): Sort single cells and inject them into the lysis buffer of the PCR plate wells, then keep on ice for later use.

[0103] Step c): Quick-freeze the 96-well PCR plate at -80°C until all samples are frozen, then place it in a 37°C metal bath until thawed. Repeat this freeze-thaw cycle three times, then place it on ice.

[0104] Acquisition of cDNA from a single B cell: Reagents: PrimeScript™ II 1st Strand cDNA Synthesis Kit (TAKARA: 6210A), RNase inhibitor, dNTP, H2O.

[0105] Consumables: PCR plates, pipette tips.

[0106] Equipment: PCR instrument.

[0107] Step a): Place the sample from the previous step in the PCR instrument and incubate at 65°C for 5 minutes. Immediately after incubation, place the sample on ice for at least 2 minutes. Step b): Prepare the reaction system and cDNA synthesis system according to the table below. Table 4 cDNA Synthesis System

[0108] Step c): Place the sample in a PCR instrument, incubate at 42°C for 1 h, inactivate at 80°C for 5 s, and store in a -20°C freezer for a short period after the incubation period; for long-term storage, store in a -80°C freezer.

[0109] Preparation of TAP fragments for antibody expression: Reagents: Taq DNA Polymerase (TAKARA: R001A); 2×Phanta Flash Master Mix (Dyeplus / minus) (Novazia: P520 / P510); 6×Loading Buffer (TAKARA: 9156); DL2000 DNA Marker (TAKARA: 3427A); TAE buffer, agarose.

[0110] Consumables: PCR plates, pipette tips.

[0111] Equipment: PCR instrument, horizontal electrophoresis apparatus, gel imaging system.

[0112] The components required for antibody expression (such as promoter and terminator sequences) are integrated and amplified with the antibody expression sequence by PCR. The PCR product containing the splicing fragment (TAP fragment) can be directly used for transfection in the suspension 293F system.

[0113] TAP fragment cell transfection: Reagent: Expi293™ Expression System Kit (Thermo, A14635).

[0114] Consumables: 96-well deep well plate, pipette tips.

[0115] Equipment: constant temperature shaker, electric pipette.

[0116] Step a): Incubate the Expi293F medium in a 37°C water bath beforehand. Observe the cell state and growth under a microscope. Determine the passage ratio for the cell expansion experiment based on the cell growth volume and subsequent transfection requirements. Generally, the transfer density is controlled at 2x10⁻⁶ cells / day the day before transfection. 6 Approximately 100 live cells / mL. Remove the cell slurry from the CO2 cell shaker, count the cells, and calculate the required number of cells to transfer.

[0117] Select the optimal cell shake flask size based on the amount of cells to be transferred. Generally, the culture volume should not exceed 1 / 3 of the shake flask volume and should not be less than 1 / 5 of the shake flask volume. Add preheated Expi293 medium according to the cell density to achieve a final cell density of approximately 2 x 10⁶ viable cells / mL, which will be used for transfection the following day.

[0118] Step b): On the day of transfection, preheat the Expi 293 medium to room temperature in the dark. Determine the amount of cells to be transfected and prepare the corresponding number of 96-well plates. Determine the cell density according to the cell counting procedure, and dilute the cells with the preheated medium to a final cell density of 3 x 10⁻⁶ cells / well. 6For live cells / mL, aliquot 1ml / well into deep-well plates using a pipette and carefully place them on a cell culture shaker: 37℃, 8% CO2, 900rpm, 80% humidity. Take the appropriate number of 96-well cell culture plates and aliquot 1ug DNA + 60ul Opti-MEM (cold reagent) into each plate, mixing gently with a pipette (recommended antibody ratio HC:LC = 1:2). Separately, take the optimal volume of centrifuge tube and dilute sufficient transfection reagent to 3.2ul Expifectamine 293 + 60ul Opti-MEM (cold reagent) per ml of cells. Gently invert to mix and incubate at room temperature for 5 minutes. After 5 minutes, transfer the diluted transfection reagent to a disposable reservoir and pipette into the corresponding wells of the 96-well cell culture plates, incubating for 10-20 minutes. Using a pipette, add the settled ExpiFectamine 293 / DNA complex to the cell suspension and carefully place the mixture on a shaker: incubate at 37°C, 8% CO2, 900 rpm, and 80% humidity. 18-22 hours post-transfection, add 6 μL of transfection enhancer 1 and 60 μL of transfection enhancer 2 to 1 ml of the solution. Harvest the supernatant after 4-7 days for purification.

[0119] Step c): The supernatant was used to determine the specificity of the recognition of the tau protein polypeptide fragment aa225-256 by ELISA, and multiple antibodies were obtained. Among them, 1C9 had a higher effect. Subsequently, 1C9 was used for antibody heavy and light chain variable region sequencing, plasmid construction and antibody production.

[0120] Heavy and light chain variable region sequencing: Reagents: TAE buffer, agarose, nucleic acid dye.

[0121] Consumables: pipette tips.

[0122] Equipment: Nucleic acid electrophoresis instrument, nucleic acid imaging instrument.

[0123] The antibody nucleic acid sequence was extracted and sent to a gene sequencing company to obtain the variable region nucleic acid and protein sequences of the 1C9 heavy and light chains.

[0124] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.8.

[0125] Construction and production of cloning 1G1 expression plasmid: Based on the sequencing results of the variable regions of the heavy and light chains, the samples were sent to a gene synthesis company to construct an expression vector containing a signal peptide and a constant region. The synthesized plasmid was then mass-produced according to the transfection method described in "TAP Fragment Cell Transfection," with the amount of DNA added adjusted according to the production volume. After culturing for 4-7 days, the supernatant was harvested and purified.

[0126] Example 2: Application Identification of Rabbit Monoclonal Antibody 1C9 1. Indirect ELISA analysis: To confirm antibody specificity, an indirect ELISA was used. The experimental procedure is as follows: Step a): Coating: The immunogenic peptide and other cross-detection proteins (aa225-242 peptide, tau protein fragment (aa1-256) and full-length tau protein (2N4R, aa1-441)) were diluted to 1 μg / mL with coating buffer and added to the microplate in 50 μL / well. The plate was then capped and coated overnight at 4°C.

[0127] Step b): Blocking: Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 150 μL / well, and place it in a 37℃ thermostatic incubator for 1 h for blocking.

[0128] Step c): Add sample: Shake off the liquid in the well, serially dilute the anti-monoclonal antibody with PBS, and let it stand at room temperature for 10 min; add 100 μL of the mixture to the microplate and react at 37℃ for 45 min.

[0129] Step d): Add secondary antibody: Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute goat anti-rabbit-HRP to the working concentration (1:30000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37℃ for 45 min.

[0130] Step e): Color development, termination and reading: Discard the liquid in the wells, add washing buffer to the microplate at a rate of 180 μL / well, and wash the microplate 3 times; add 100 μL of freshly prepared TMB chromogenic substrate to each reaction well, and incubate at 37℃ for 10 min; then add 90 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the microplate reader.

[0131] The results are shown in Table 5. Rabbit monoclonal antibody 1C9 can bind to the aa225-242 peptide, as well as the tau protein fragment containing this fragment (aa1-256) and the full-length tau protein (2N4R, aa1-441).

[0132] Table 5

[0133] 2. Western Blot (WB) Analysis: To detect the recognition specificity of the rabbit monoclonal antibody of this invention for endogenous proteins, negative and positive samples of tau protein were selected for detection. The experimental steps are as follows: Mouse, rat, and pig tissues (brain, spleen, cerebellum, and testis) were lysed, and proteins were extracted and subjected to 10% polyacrylamide gel electrophoresis. Subsequently, the proteins in the gel were transferred to a PVDF membrane using conventional methods. The PVDF membrane was placed in TBST blocking buffer containing 5% skim milk powder and incubated at room temperature for 1 hour. Then, monoclonal antibody 1C9 diluted 1:5000 was added, and the membrane was incubated overnight at 4°C. The membrane was then washed with TBST, and goat anti-rabbit secondary antibody was added, followed by incubation at room temperature for 1 hour. The membrane was washed again with TBST, and then ECL ultrasensitive chromogenic solution was added for development. The results are as follows. Figure 2 As shown.

[0134] 3. Immunohistochemistry (IHC): Before staining paraffin sections of brain tissue from mouse WT and AD models, dewaxing is necessary to ensure successful staining. The procedure is as follows: Xylene I 10 min; Xylene II 10 min; Xylene III 10 min; Anhydrous ethanol I 3 min; Anhydrous ethanol II 3 min; 95% ethanol 3 min; Rinse with running water 3 min.

[0135] Antigen retrieval: Pour approximately 1L of 1×Tris-EDTA (pH 9.0) into a high-temperature container and heat it to a boil using an induction cooker for 3-5 minutes, ensuring no bubbles are present on low heat. Place the tissue sections into the container and maintain the temperature at 90-95℃ on the induction cooker at low power for 20 minutes, then cover the pot. After cooling to room temperature for 10 minutes, rinse with running water and cool to room temperature.

[0136] Immunohistochemical staining: 1. Rinse the slides three times with pure water, 5 minutes each time.

[0137] 2. Blocking: Place the slice in a 3% hydrogen peroxide aqueous solution and incubate for 10 min.

[0138] 3. Rinse the slides twice with pure water, 5 minutes each time.

[0139] 4. After slightly drying the sections, wipe the water around the tissue sections with filter paper. Draw a circle around the tissue with a histochemical pen, add 1% BSA-prepared 10% negative goat serum to the circle, cover the tissue, and block at room temperature for 1 hour.

[0140] 5. Remove the blocking solution, then add primary antibody diluted with the recommended antibody diluent (usually 100 μl / tissue) to each section and incubate in a humidified chamber at room temperature for 1 h.

[0141] 6. Remove the antibody solution by rinsing three times with 1×TBST for 5 minutes each time.

[0142] 7. After patting the sections dry, add goat anti-rabbit or mouse secondary antibody (Huaan Biotechnology, catalog number HA1119) to the circle to cover the tissue, and incubate in a humidified chamber at room temperature for 20 minutes.

[0143] 8. Immerse three times with 1×TBST, 5 minutes each time.

[0144] 9. DAB staining: After the slide is dried, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive result is brownish-yellow. Observe until a positive signal appears or staining occurs for 5 minutes. Stop staining by rinsing the slide with a wash bottle or DAB beaker filled with tap water. Rinse with running tap water for 5-10 minutes.

[0145] Counterstaining and dehydration: The procedure was as follows: hematoxylin 10 min; rinse with running water for 3 min; blue promote for 1 min; rinse with running water for 3 min; 95% ethanol I for 1 min; 95% ethanol II for 1 min; 95% ethanol III for 1 min; anhydrous ethanol I for 1 min; anhydrous ethanol II for 1 min; xylene I for 1 min; xylene II for 1 min; oven at 65℃ for 2 min. Afterwards, the slides were mounted using neutral resin. Finally, the sections were scanned and the results were determined.

[0146] Immunohistochemical staining results showed that the rabbit monoclonal antibody 1C9 was accurately localized in the cytoplasm with a clean background and exhibited a significant enhancing trend in the brain of an AD mouse model. Figure 3 and Figure 4 As shown.

[0147] 4. Immunoprecipitation (IHC): IP results showed that rabbit monoclonal antibody 1C9 could enrich endogenous tau protein, as well as tau protein fragments (aa1-256) and full-length tau protein (2N4R, aa1-441).

[0148] I. Sample Preparation Add 100 µL of denaturing lysis buffer to mouse brain tissue. Vortex vigorously for 2–3 seconds. Transfer the suspension to a microcentrifuge tube. Denature at 95°C for 5 minutes. Add 0.9 mL of non-denaturing lysis buffer and mix gently. Dissolve the DNA by pipetting 5–10 times with a syringe. Incubate on ice for 5 minutes. Tissue lysate: Rapidly dissect the tissue on ice. Place the tissue in an EP tube and flash freeze in liquid nitrogen. Add approximately 5 mg of tissue to 300 µL of lysis buffer and homogenize using an electric homogenizer. Wash twice with 300 µL of lysis buffer and agitate at 4°C for 2 hours (if denaturation is required, refer to denaturation steps 2–5). Centrifuge at 12,000 rpm for 20 minutes at 4°C, and transfer the supernatant to a new EP tube on ice.

[0149] 2. Pre-removal of lysates: Add 100 µL of beads to 1 mL of lysis buffer and incubate at 4°C with rotation for 30 minutes. Centrifuge at 4°C and 500×g for 5 minutes. Transfer the supernatant to a new EP tube on ice (for immunoprecipitation). III. Immunoprecipitation (IP) Procedure: Add cell lysis buffer to a low-adsorption EP tube, and add the recommended amount of antibody. Incubate at 4°C with rotation for several hours (time depends on protein content and antibody affinity). Equilibrate the beads. Add 20–50 µL of the equilibrated bead mixture to the sample. Incubate at 4°C with rotation for 1–4 hours. Centrifuge at 4°C, 500×g for 3 minutes, and wash three times with lysis buffer. Add 25–50 µL of 2× loading buffer and boil at 100°C for 5 minutes. Centrifuge and use the supernatant for Western blotting. Results are as follows: Figure 5 and Figure 6 As shown, 1C9 can enrich artificially synthesized protein fragments containing their epitopes, and can also enrich endogenous tau protein in mouse brain tissue material.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against an eMTBR-tau243 protein fragment, characterized in that, The variable region of the monoclonal antibody against the eMTBR-tau243 protein fragment includes: complementarity-determining regions CDR1-VH (amino acid sequence as shown in SEQ ID NO.1), CDR2-VH (amino acid sequence as shown in SEQ ID NO.2), CDR3-VH (amino acid sequence as shown in SEQ ID NO.3), CDR1-VL (amino acid sequence as shown in SEQ ID NO.4), CDR2-VL (amino acid sequence as shown in SEQ ID NO.5), and CDR3-VL (amino acid sequence as shown in SEQ ID NO.6).

2. The monoclonal antibody against the eMTBR-tau243 protein fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody against the eMTBR-tau243 protein fragment is shown in SEQ ID NO.

7.

3. The monoclonal antibody against the eMTBR-tau243 protein fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region VL of the monoclonal antibody against the eMTBR-tau243 protein fragment is shown in SEQ ID NO.

8.

4. A biomaterial, characterized in that, The biomaterial is selected from any one of ac: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a monoclonal antibody encoding the anti-eMTBR-tau243 protein fragment of any one of claims 1-3; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing a monoclonal antibody against the anti-eMTBR-tau243 protein fragment of any one of claims 1-3.

5. A method for preparing a monoclonal antibody against the eMTBR-tau243 protein fragment according to any one of claims 1-3, characterized in that, It is obtained by cell fermentation as described in claim 4.

6. The use of the monoclonal antibody against the eMTBR-tau243 protein fragment as described in any one of claims 1-3 in the preparation of a detection product for the eMTBR-tau243 protein fragment.

7. A marker for an eMTBR-tau243 protein fragment, characterized in that, Includes the monoclonal antibody and marker against the eMTBR-tau243 protein fragment as described in any one of claims 1-3; The monoclonal antibody against the eMTBR-tau243 protein fragment was conjugated with a marker.

8. The marker for the eMTBR-tau243 protein fragment according to claim 7, characterized in that, The labeling agents include enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

9. A kit for detecting eMTBR-tau243 protein fragments, characterized in that, The kit comprises a monoclonal antibody against the eMTBR-tau243 protein fragment as described in any one of claims 1-3 or a marker of the eMTBR-tau243 protein fragment as described in claim 7 or 8.

10. The reagent kit according to claim 9, characterized in that, The kits include immunochromatographic assay kits, ELISA kits, immunomagnetic microparticle assay kits, immunofluorescence assay kits, or immunoblotting assay kits.

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