Monoclonal antibodies against phosphorylated Tau217 and their kits

Abstract

This application relates to a monoclonal antibody that specifically recognizes phosphorylated Tau217 protein, its preparation method, and a phosphorylated Tau217 protein detection kit containing the antibody. This antibody can be used for early auxiliary diagnosis, disease monitoring, and efficacy evaluation of Alzheimer's disease. The kit of this invention has a blank limit of 0.51 pg / mL, a detection limit of 1.04 pg / mL, a quantitation limit of 3.93 pg / mL, a linear range of 0.5–100 pg / mL (linear correlation coefficient r ≥ 0.99), intra-assay precision CV ≤ 8%, inter-assay precision CV ≤ 15%, good anti-interference performance, kit stability of 12 months, on-board stability after opening for 30 days, and good correlation with imported reference kits.

Description

Technical Field

[0001] This application belongs to the fields of clinical in vitro diagnostics, medical immunology, and neuromedical testing, and relates to a monoclonal antibody, specifically a monoclonal antibody that specifically recognizes phosphorylated Tau217 protein, its preparation method, and a phosphorylated Tau217 protein detection kit containing the antibody. This antibody can be used for early auxiliary diagnosis, disease monitoring, and efficacy evaluation of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease primarily characterized by impairments in memory, cognitive abilities, emotion, and behavior. It is the most common neurodegenerative disease globally, accounting for 60-70% of all cognitive impairment cases in the elderly. More than 50 million people worldwide suffer from AD, and this number is projected to reach 150 million by 2050. The most prominent pathological feature of AD is the deposition of β-amyloid (Aβ) protein in the brain, hyperphosphorylation of Tau protein, and decreased or even lost neuronal and synaptic function. AD often has a slow and insidious onset, and currently lacks effective treatments and prevention methods. Early diagnosis and treatment are crucial, but early diagnosis of AD remains highly challenging, primarily due to the lack of ideal diagnostic biomarkers, especially those with strong correlations to cognitive function changes and prognosis.

[0003] Tau protein is a microtubule-associated protein that, under normal circumstances, promotes microtubule assembly, stabilizes microtubule structure, and maintains neuronal function through its microtubule-binding domain. Under AD pathological conditions, Tau protein undergoes abnormal hyperphosphorylation, leading to a significant decrease in its affinity for microtubules. It dissociates from microtubules and aggregates into insoluble paired helical filaments, which in turn form neurofibrillary tangles, ultimately resulting in neuronal dysfunction and death.

[0004] Among them, p-Tau217, the phosphorylated product of threonine 217 in the Tau protein, is a highly specific biomarker for AD pathology: it is significantly elevated in the plasma of AD patients, while it is usually not elevated in other common neurodegenerative diseases, and can distinguish Aβ-PET positive and negative individuals with high precision. In 2024, the National Institute on Aging and the Alzheimer's Disease Association (NIA-AA) revised the AD diagnostic criteria and included plasma p-Tau217 in the core biomarker system, making it the only blood biomarker that can be used for the clinical diagnosis of AD, providing core support for non-invasive large-scale screening of AD.

[0005] Currently, the main methods for detecting p-Tau217 in clinical and laboratory settings include enzyme-linked immunosorbent assay (ELISA), single-molecule array technology (SimoA), and mass spectrometry. Among these, SimoA technology has extremely high sensitivity, but the equipment is expensive, the detection throughput is limited, and the operation is complex; ELISA is cumbersome to operate, has a long detection time, and its sensitivity is insufficient for trace detection needs; mass spectrometry has high requirements for sample pretreatment and is not suitable for routine clinical use.

[0006] Chemiluminescence immunoassay, with its advantages of high sensitivity, wide linear range, ease of operation, and high degree of automation, has become the mainstream technology for clinical immunoassay. Magnetic microparticle chemiluminescence assays utilize magnetic microparticles as a solid-phase carrier, combined with the biotin-streptavidin system or directly coated with antibodies, enabling rapid, efficient, and fully automated immunoassay. However, currently, there is a lack of high-sensitivity p-Tau217 quantitative detection kits based on magnetic microparticle chemiluminescence assays in China. The core bottleneck lies in the lack of highly specific and high-affinity anti-p-Tau217 monoclonal antibodies. Therefore, developing a monoclonal antibody that specifically recognizes p-Tau217 is of significant clinical and market value for developing highly sensitive p-Tau217 detection kits. Summary of the Invention

[0007] Based on the needs of existing research, this application provides a new monoclonal antibody that specifically recognizes p-Tau217, a sandwich detection kit containing the antibody based on a magnetic microparticle chemiluminescence platform, and successfully completed the quantitative detection of p-Tau217 in human plasma using this kit.

[0008] The present invention aims to develop a monoclonal antibody that specifically recognizes p-Tau217. This antibody has the characteristics of high affinity and high specificity, and can be used to develop a magnetic particle chemiluminescence detection kit for detecting phosphorylated Tau217 protein in human plasma. The kit is simple to operate, has a wide linear range, fast detection speed, high sensitivity, no pollution, good specificity and low cost.

[0009] Detection principle: The phosphorylated Tau 217 protein (p-Tau217) detection kit of the present invention is based on the principle of magnetic microparticle chemiluminescence double antibody sandwich method, using magnetic microparticles as solid phase carrier, and realizing the detection of samples through immunoassay.

[0010] Step 1: Incubation reaction. Magnetic microparticles coated with the monoclonal antibody of this invention and acridine ester-labeled antagonistic anti-p-Tau217 monoclonal antibody are mixed with p-Tau217 antigen in the sample to be tested, and incubated at 37°C. The p-Tau217 in the sample specifically binds to the solid-phase coated antibody and the labeled antibody, respectively, forming a double-antibody sandwich immune complex of solid-phase antibody-p-Tau217 antigen-acidine ester-labeled antibody.

[0011] The second step is cleaning and separation. Under the influence of a magnetic field, magnetic particles are adsorbed onto the reaction tube wall, while unbound free proteins, impurities, and labeled antibodies are washed away by the cleaning solution, achieving efficient separation of conjugates and free substances.

[0012] Step 3: Luminescence reaction. Pre-excitation solution and excitation solution are added to the immune complex in sequence. The acridinium ester label is excited in an alkaline hydrogen peroxide environment and emits photons instantaneously.

[0013] Step 4: Reading the values. Use instruments to measure these photons. The test results are expressed as relative luminescence intensity (RLU). The luminescence intensity is positively correlated with the content of p-Tau217 protein in the sample.

[0014] Specifically, this application relates to the following: 1. A monoclonal antibody against phosphorylated Tau217, characterized in that: the monoclonal antibody comprises: a) Heavy chain variable region, which includes heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: The sequence of CDR-H1 is shown in the amino acid sequence of GFTFSDYYMY. The sequence of CDR-H2 is shown in the amino acid sequence of SINHDGDNTYYPDTVKG. The sequence of CDR-H3 is shown in the amino acid sequence of GRGMDV; b) Light chain variable region, which includes light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: The sequence of CDR-L1 is shown in the amino acid sequence of RASQSISSYLN. The sequence of CDR-L2 is shown in the amino acid sequence of AASSLQS. The sequence of CDR-L3 is shown in the amino acid sequence of QQSNSAPLT.

[0015] 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody comprises the antibody heavy chain variable region HCVR. and antibody light chain variable region LCVR, wherein: HCVR sequence as follows The amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVSSINHDGDNTYYPDTVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGRGMDVWGQGTLVTVSS is shown below; LCVR sequence as follows The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSAPLTFGGGTKVEIK is shown.

[0016] 3. A monoclonal antibody against phosphorylated Tau217, characterized in that: the monoclonal antibody comprises: a) Heavy chain variable region, which includes heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: The sequence of CDR-H1 is shown in the amino acid sequence of GFTFSDYYMY. The sequence of CDR-H2 is shown in the amino acid sequence of VISHDGDNTYYPDTVKG. The sequence of CDR-H3 is shown as the amino acid sequence of GRGFDV; b) Light chain variable region, which includes light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: The sequence of CDR-L1 is shown in the amino acid sequence of RASQSISSYLN. The sequence of CDR-L2 is shown in the amino acid sequence of AASSLQS. The sequence of CDR-L3 is shown in the amino acid sequence of QQSNSAPLT.

[0017] 4. The monoclonal antibody according to claim 3, wherein the monoclonal antibody comprises the antibody heavy chain variable region HCVR. and antibody light chain variable region LCVR, wherein: HCVR sequence as follows The amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAVISHDGDNTYYPDTVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGRGFDVWGQGTLVTVSS is shown below; LCVR sequence as follows The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSWPLTFGGGTKVEIK is shown.

[0018] 5. The application of the monoclonal antibody described in items 1-4 in the detection of phosphorylated Tau217, characterized in that the detection is for non-disease diagnostic purposes.

[0019] 6. A kit for detecting phosphorylated Tau217, wherein the kit comprises a capture antibody and an acridinium ester-labeled second antibody, the capture antibody being selected from any one of claims 1 to 2 as an anti-phosphorylated Tau217 monoclonal antibody.

[0020] 7. The kit according to claim 6, characterized in that the kit further comprises a detection diluent and a calibrator.

[0021] 8. The kit according to item 6 or 7, characterized in that the capture antibody is coated with superparamagnetic microparticles.

[0022] 9. The kit according to any one of claims 6-8, characterized in that the second antibody comprises a) Heavy chain variable region, which includes heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: The sequence of CDR-H1 is shown in the amino acid sequence of GFTFSDYYMY. The sequence of CDR-H2 is shown in the amino acid sequence of VISHDGDNTYYPDTVKG. The sequence of CDR-H3 is shown as the amino acid sequence of GRGFDV; b) Light chain variable region, which includes light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: The sequence of CDR-L1 is shown in the amino acid sequence of RASQSISSYLN. The sequence of CDR-L2 is shown in the amino acid sequence of AASSLQS. The sequence of CDR-L3 is shown in the amino acid sequence of QQSNSAPLT.

[0023] 10. A kit for detecting phosphorylated Tau217, wherein the kit comprises a capture antibody and an acridinium ester-labeled second antibody, the second antibody being a monoclonal antibody against phosphorylated Tau217 as described in any one of claims 3 to 4.

[0024] 11. The kit according to claim 10, characterized in that the kit further comprises a detection diluent and a calibrator.

[0025] 12. The kit according to item 10 or 11, characterized in that the capture antibody is coated with superparamagnetic microparticles.

[0026] 13. A method for quantitatively detecting the content of phosphorylated Tau217, wherein a capture antibody and a biotin-labeled second antibody are used, and a double-antibody sandwich ELISA is performed using magnetic microparticle chemiluminescence detection, wherein the capture antibody is a monoclonal antibody against phosphorylated Tau217 as described in any one of items 1 to 2, and the method is for non-disease diagnostic purposes.

[0027] 14. The method according to claim 13, wherein the second antibody is a monoclonal antibody against phosphorylated Tau217 as described in any one of claims 3 to 4.

[0028] Reagent kit performance characteristics This invention utilizes a magnetic microparticle chemiluminescence double-antibody sandwich method to develop a detection kit for phosphorylated Tau 217 protein (p-Tau217). When used with a fully automated chemiluminescence analyzer, it requires virtually no human intervention. Compared to existing detection methods, it offers the following advantages: Detection methods Magnetic microparticle chemiluminescence Enzyme-linked immunosorbent assay (ELISA) Detection time Approximately 10 minutes Approximately 90-120 minutes Linear range 0.5-100 pg / mL Approximately 5-50 pg / mL Detection limit 1.04 pg / mL Approximately 5 pg / mL Automation level Fully automatic Semi-automatic / Manual Precision (CV) Repeatability ≤8%, intermediate precision ≤15% Approximately 10-20% The reagent kit of this invention has a blank limit of 0.51 pg / mL, a detection limit of 1.04 pg / mL, a quantitation limit of 3.93 pg / mL, a linear range of 0.5-100 pg / mL (linear correlation coefficient r≥0.99), an intra-assay precision CV≤8%, an inter-assay precision CV≤15%, good anti-interference performance, a kit stability of 12 months, an on-board stability period of 30 days after opening, and good correlation with imported reference kits. Beneficial effects

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses hybridoma technology to screen and obtain monoclonal antibodies that specifically recognize p-Tau217. These antibodies recognize the backbone epitope, do not block phosphorylation sites, have extremely high specificity, and have a very low cross-reactivity rate with non-phosphorylated Tau proteins, thus effectively avoiding cross-interference.

[0030] 2. The antibody of this invention exhibits excellent binding activity with the p-Tau217 antigen, with an IC50 of 0.82 nM and an affinity at the nM level, providing a core foundation for the high-sensitivity detection of the kit.

[0031] 3. The magnetic microparticle chemiluminescence reagent kit developed based on the antibody of this invention has high detection sensitivity, with a blank limit of up to 0.51 pg / mL and a detection limit of up to 1.04 pg / mL, which is far superior to traditional ELISA kits and can meet the detection needs of low-concentration samples in the early stage of AD.

[0032] 4. The kit of this invention has a wide linear range, from 0.5 to 100 pg / mL, with a linear correlation coefficient r = 0.999878, enabling accurate quantification of samples of different concentrations.

[0033] 5. The reagent kit of this invention has a fast detection speed, requiring only 15 minutes of incubation throughout the entire process. When used with a fully automated chemiluminescence analyzer, it can achieve high-throughput automated detection, is easy to operate, and is suitable for routine clinical use.

[0034] 6. The reagent kit of this invention has good stability, can be stored at 2-8℃ for 12 months, remains stable for 30 days after opening, has a calibration curve validity period of 28 days, has low cost, and is easy to promote and apply in clinical practice.

[0035] 7. The reagent kit of this invention has good clinical relevance, and the detection results are highly consistent with those of imported reference reagent kits. It has comparable diagnostic efficacy and can be effectively used for early auxiliary diagnosis, disease monitoring and efficacy evaluation of Alzheimer's disease. Attached Figure Description

[0036] Figure 1 Schematic diagram of the reaction principle of the reagent kit.

[0037] Figure 2 Linear regression plot of the reagent kit.

[0038] Figure 3 This application assesses the clinical relevance of the reagent kit to imported reagent kits. Detailed Implementation

[0039] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are intended to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0040] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims. Example

[0041] Example 1: Screening and preparation of monoclonal antibodies Phosphorylated Tau217 target protein sequence p-Tau217 is not an independent protein isoform, but rather a product of site-specific phosphorylation modification of the human microtubule-associated protein Tau (encoded by the MAPT gene) at threonine 217 (Thr217, abbreviated as T). In clinical and research settings, the sequence numbering of p-Tau217 defaults to the longest central nervous system splice isoform of human Tau protein, the 2N4R isoform (441 amino acids), as the standard reference sequence. Its UniProtKB accession number is P10636, and the NCBI reference sequence is NP_005901.2. The full-length amino acid sequence is as follows: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTR EPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHK PGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL.

[0042] The core phosphorylation site is threonine at position 217 (Thr / T). Phosphorylation of the hydroxyl group at this site results in p-Tau217. The surrounding core recognition epitope sequence is: SRTPSLP(pT) PPTREPKK (pT is phosphorylated Thr217), which is the core region for antibody-specific recognition.

[0043] The monoclonal antibody of this invention is obtained through hybridoma screening, and the specific steps are as follows: 1. Immunogen preparation: The polypeptide antigen SRTPSLP(pT)PPTREPKK containing phosphorylated Thr217 was synthesized, conjugated with keyhole hemocyanin (KLH), and used as an immunogen to immunize animals to induce a specific immune response.

[0044] 2. Animal Immunization: The immunogen and Freund's adjuvant were thoroughly mixed and emulsified, and 6-8 week old BALB / c mice were immunized with an immunization dose of 50 μg / mouse. Booster immunizations were given every 2 weeks for a total of 4 immunizations. Serum was collected from the tail vein of the mice after the final immunization, and serum antibody titers were detected using an indirect ELISA method. Mice with the highest titers were selected for cell fusion.

[0045] 3. Cell fusion: Spleens from mice with the highest titer were collected, and spleen cell suspensions were prepared under sterile conditions. The spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 5:1, and cell fusion was performed using the PEG-1500-mediated cell fusion method.

[0046] 4. Hybridoma screening: The fused cells were seeded into 96-well culture plates and cultured in HAT selective medium to screen for hybridoma cells. After 10 days of culture, the cell culture supernatant was collected, and positive hybridoma cells that specifically bound p-Tau217 antigen and did not bind non-phosphorylated Tau antigen were screened using an indirect ELISA method.

[0047] 5. Subcloning and line establishment: The positive hybridoma cells obtained from screening were subcloned using the limiting dilution method. After 2-3 rounds of subcloning screening, monoclonal cell lines that could stably secrete specific antibodies were obtained and named Hybridoma-C1 and Hybridoma-D1, which were used to secrete the capture antibody and detection antibody of the present invention.

[0048] Sequencing analysis revealed that the monoclonal antibody Mab-pTau217-C1 secreted by Hybridoma-C1 has the following sequence: Heavy chain variable region (VH): EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVSSINHDGDNTYYPDTVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGRGMDVWGQGTLVTVSS Light chain variable region (VL, κ type): DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSAPLTFGGGTKVEIK This antibody has the advantages of no steric hindrance and extremely high specificity. It recognizes only the p-Tau217 proline-rich backbone, does not block phosphorylation sites, and has a cross-reactivity rate of <0.000003% with non-phosphorylated Tau protein, which can effectively avoid cross-interference.

[0049] The monoclonal antibody Mab-pTau217-D1 secreted by Hybridoma-D1 has the following sequence: Its variable region sequence is as follows: Heavy chain variable region (VH): EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAVISHDGDNTYYPDTVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGRGFDVWGQGTLVTVSS Light chain variable region (VL, κ type): DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSWPLTFGGGTKVEIK This paired antibody has the advantages of no steric hindrance and extremely high specificity, recognizing only p-Tau217 and having a cross-reactivity rate of <0.000003% with unphosphorylated Tau protein, which can effectively avoid cross-interference.

[0050] Example 2: Verification of antibody-antigen binding activity To verify the binding activity of the monoclonal antibody of this invention to the p-Tau217 antigen, the binding activity was determined using an indirect ELISA method. Simultaneously, the half-maximal inhibitory concentration (IC50) and affinity constant (KD) of the antibody were measured. 1. Experimental Procedure (1) Dilute the recombinant p-Tau217 antigen to 2 μg / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), coat 96-well microplates, 100 μL / well, and incubate overnight at 4°C.

[0051] (2) Discard the coating solution, wash 3 times with PBST washing buffer (PBS buffer containing 0.05% Tween-20), then add 200 μL / well of 5% skim milk blocking buffer and block at 37°C for 2 hours to block non-specific binding sites.

[0052] (3) Discard the blocking solution, wash 3 times, add serially diluted test antibody (concentration range of 0.01 nM-100 nM), 100 μL / well, and incubate at 37°C for 1 hour to allow the antibody to bind to the coated antigen.

[0053] (4) Discard the antibody solution, wash 3 times, add HRP-labeled goat anti-mouse IgG secondary antibody (diluted at a ratio of 1:5000), 100 μL / well, and incubate at 37°C for 1 hour.

[0054] (5) Discard the secondary antibody solution, wash 5 times, add TMB substrate color development solution, 100 μL / well, and develop color at room temperature in the dark for 15 minutes.

[0055] (6) Add 50 μL of 2 M H2SO4 stop solution per well to terminate the colorimetric reaction, and measure the OD value at 450 nm using an ELISA reader.

[0056] 2. Experimental Results By fitting the dose-response curve, the binding activity parameters of the two antibodies were obtained: Mab-pTau217-C1 0.82 <![CDATA[1.2×10 -9 ]]> Mab-pTau217-D1 0.65 <![CDATA[8.5×10 -10 ]]> The results show that both monoclonal antibodies of the present invention can bind specifically to the p-Tau217 antigen with high affinity, exhibiting excellent binding activity and no obvious non-specific binding, providing a core molecular basis for the high-sensitivity detection of the kit.

[0057] Example 3: Reagent Kit Preparation 1.1 Preparation of magnetic microparticle reagents Carboxyl magnetic beads with a particle size of 1-3 μm were selected, and the carboxyl groups on the surface of the magnetic beads were activated using a two-step EDC / NHS method. Anti-p-Tau217 coated monoclonal antibody (Mab-pTau217-C1) was mixed with the activated magnetic beads at a mass ratio of 1:10 and incubated by rotation at room temperature for 4 hours to allow the antibody to covalently couple to the surface of the magnetic beads. Unreacted active sites were then blocked for 2 hours with Tris buffer containing 1% BSA. The supernatant was removed by magnetic separation, and the beads were washed three times with washing buffer. Finally, the magnetic beads were resuspended in Tris buffer (0.1 M, pH 7.4) containing 10 g / L BSA and 0.6 g / L Proclin 300, and the magnetic particle concentration was adjusted to 0.4 mg / mL. The beads were stored at 2-8℃.

[0058] 1.2 Preparation of acridinium ester-labeled antibody solution The concentration of anti-p-Tau217 labeled monoclonal antibody (Mab-pTau217-D1) was adjusted to 2 mg / mL with PBS buffer. Acridinium ester labeling reagent activated with NHS was added at a molar ratio of acridinium ester to antibody of 15:1, and the reaction was gently stirred at room temperature for 1 hour. After the reaction, the labeled product was purified using a G25 gel filtration chromatography column to remove unbound free acridinium ester, and the antibody-labeled peak was collected. After dialysis, the protein concentration was determined by UV OD280 nm. Then, 5 g / L BSA, 0.02% methylisothiazolinone, and 0.02% bromonitrosodioxane were added, and the labeled antibody concentration was adjusted to 0.1 mg / L with MES buffer (0.1 M, pH 6.5). The mixture was stored at 2–8 °C protected from light.

[0059] 1.3 Preparation of Diluent for Testing Prepare 0.1 M Tris buffer, adjust pH to 7.4, add 5 g / L bovine serum albumin (BSA) and 0.6 g / L Proclin 300, stir thoroughly to dissolve, and then filter through a 0.22 μm filter membrane for sterilization to obtain the detection dilution. Store at 2-8℃ for later use.

[0060] 1.4 Preparation of calibrators and quality control samples Recombinant human p-Tau217 protein (expressed in HEK293 cells and phosphorylated) was used as the raw material. Tris buffer (0.05 M, pH 7.4) containing 5 g / L BSA and 0.6 g / L Proclin 300 was used as the matrix to prepare calibrators and quality control samples at predetermined concentrations. Calibrator concentrations were 0 pg / mL, 0.5 pg / mL, 5 pg / mL, 20 pg / mL, 50 pg / mL, and 100 pg / mL; quality control concentrations were 5.0 pg / mL and 50.0 pg / mL. High-precision pipettes and balances were used during preparation to ensure the accuracy and reproducibility of the concentrations.

[0061] Example 4: Detection Method of Anti-p-Tau217 Kit The reagent kit of this invention is used in conjunction with a fully automated chemiluminescence immunoassay analyzer, and the detection steps are as follows: (1) Reagent loading: Before loading the reagent kit, gently invert the kit at least 15 times to avoid generating air bubbles and ensure that the precipitated magnetic particles are completely suspended. Place the reagent kit into the corresponding position in the reagent compartment of the chemiluminescence analyzer, and input the reagent kit information into the instrument system via a barcode scanner.

[0062] (2) Calibration: Each kit contains batch-specific calibration information. The instrument automatically generates the master curve by scanning the master curve code in the kit. The master curve is then corrected using the calibrators included in the kit. After successful correction, the instrument will automatically fit and generate a new working curve for sample testing. The calibration curve is valid for 28 days.

[0063] (3) Sample pretreatment: The sample to be tested is EDTA-K2 anticoagulated plasma. After the sample is collected, it is centrifuged at 3000g for 10 minutes to remove the precipitate and suspended matter. Severe hemolysis, lipemia, and turbidity samples cannot be used. Frozen samples must be completely thawed, warmed to room temperature, and vortexed before use. The number of times a sample is repeatedly frozen and thawed should not exceed 1.

[0064] (4) Sample addition: Place the calibrator, quality control sample or the sample to be tested into the instrument sample chamber, with a sample volume of 30 μL.

[0065] (5) Incubation: Add reagents R1 (magnetic microparticle reagent) and R2 (acridoid ester labeled antibody solution) and incubate at 37°C for 15 minutes to allow p-Tau217 in the sample to bind with the two antibodies and form a double antibody sandwich immune complex.

[0066] (6) Cleaning: Adsorb magnetic beads under the action of magnetic field, and clean them 3 times with cleaning solution to remove unbound free substances.

[0067] (7) Luminescence detection: Pre-excitation solution and excitation solution are added to the reaction tube in sequence to trigger the luminescence reaction of acridine ester. The instrument detects and records the relative luminescence intensity (RLU) in real time.

[0068] (8) Result Calculation: The instrument automatically calculates the concentration of phosphorylated Tau 217 protein (p-Tau217) in the sample based on the standard curve, in pg / mL. Reference range: ≤5.00 pg / mL is considered normal, >5.00 pg / mL is considered high-risk for Alzheimer's disease, and further examination based on clinical information is recommended.

[0069] Example 5 Performance Evaluation 1. Analytical sensitivity verification Take the sample dilution as a blank sample, repeat the measurement 20 times, calculate the mean (M) and standard deviation (SD) to obtain M+2SD. Perform two-point regression fitting based on the concentration and luminescence value results between the zero concentration calibrator and the adjacent concentration calibrator. Substitute the luminescence value of M+2SD into the equation to find the corresponding concentration value, which is the blank limit.

[0070] Low-concentration plasma samples were serially diluted, and the test results of samples with different concentrations were detected. The coefficient of variation (CV) for each concentration was calculated. The limit of detection was the concentration with CV ≤ 20%, and the limit of quantitation was the concentration with CV ≤ 10%.

[0071] The final validation results showed that the blank limit of this kit was 0.51 pg / mL, the detection limit was 1.04 pg / mL, and the quantitation limit was 3.93 pg / mL. This sensitivity can effectively meet the detection requirements of low-concentration samples in the early stage of AD, and is far superior to the detection limit of traditional ELISA kits.

[0072] 2. Linearity Range Verification A calibrator containing p-Tau217 protein was used as a high-concentration sample, and a calibrator diluent as a low-concentration sample. High-value samples approaching the upper limit of the linear range were diluted to several concentrations, with low-concentration samples approaching the lower limit of the linear range. Each concentration was tested three times, and the average value was calculated. The analyte concentrations (x-axis) and the average test results (y-axis) were then plotted using the least squares method to fit a linear relationship.

[0073] The results show that ( Figure 2 The linear regression equation is: Y = 0.992x + 0.3157. This kit has a linear correlation coefficient r = 0.999878 in the range of 0.5-100 pg / mL, indicating a good linear relationship and enabling accurate quantification of samples within this range.

[0074] 3. Accuracy Verification Two concentration levels of p-Tau217 protein, one high and one low, were prepared. After three repeated measurements, the relative deviation of the average value (denoted as M) from the labeled value was calculated.

[0075] 5 5.16 3.27 50 49.85 -0.31

[0076] The results showed that the mean detection value of the 5 pg / mL sample was 5.16 pg / mL, with a relative deviation of 3.27%; the mean detection value of the 50 pg / mL sample was 49.85 pg / mL, with a relative deviation of -0.31%, both within ±10%, indicating good accuracy.

[0077] 4. Precision Verification Quality control samples at concentrations of 5.0 pg / mL and 50.0 pg / mL were tested 10 times each, and the intra-batch coefficient of variation was calculated. Simultaneously, samples at these two concentrations were tested 10 times each using three different batches of kits, and the inter-batch coefficient of variation was calculated.

[0078] 1 5.02 49.15 2 4.93 48.96 3 5.16 50.26 4 5.12 49.66 5 5.18 51.31 6 5.19 49.73 7 5.07 49.22 8 4.81 48.11 9 4.83 48.58 10 5.05 50.50 M 5.0358 49.548 SD 0.1407 0.9534 CV 2.79% 1.92%

[0079] The results showed that the intra-assay precision CVs of this kit were 2.46% and 2.10%, and the inter-assay precision CVs were 2.79% and 1.92%, respectively, all less than 5%. The repeatability and stability of the test results were good, meeting the precision requirements for clinical testing.

[0080] 5. Specificity verification Interference experiments were conducted to detect potential interfering substances. Samples containing hemoglobin (500 mg / dL), triglycerides (520 mg / dL), and bilirubin (20 mg / dL) were prepared and detected using this kit. The recovery rate was calculated. Simultaneously, non-phosphorylated Tau protein at 1000 pg / mL was detected, and the cross-reactivity rate was calculated.

[0081] Interfering substances: When the hemoglobin in the sample is ≤500 mg / dL, triglycerides are ≤520 mg / dL, and bilirubin is ≤20 mg / dL, there is no interference with the detection results, and the recovery rate is between 90% and 110%.

[0082] Cross-reactivity: The cross-reactivity rate of 1000 pg / mL of non-phosphorylated Tau protein analogs was <0.000003%, indicating no significant cross-reactivity and extremely high specificity.

[0083] The results showed that the recovery rates of interfering substances were all between 90% and 110%, with no significant interference; the cross-reactivity rate of non-phosphorylated Tau protein was <0.000003%, with no cross-reactivity, indicating that the kit had good analytical specificity.

[0084] 6. Stability Verification (1) Long-term stability: The kit was stored at 2-8℃ and its performance was tested at 0, 3, 6, 9 and 12 months. All indicators met the requirements, indicating that the kit can be stably stored at 2-8℃ for 12 months.

[0085] (2) Stability after opening: After opening, the reagent was stored on a luminometer (2-8℃) and its performance was tested on days 0, 7, 14, 21 and 30. All indicators met the requirements, indicating that the reagent can be stably stored for 30 days after opening.

[0086] (3) Transportation stability: The reagent kit was tested after 3 days of cold chain transportation at 2-8℃ and 3 days of transportation at room temperature. All indicators met the requirements, indicating that routine transportation conditions do not affect the performance of the reagent kit.

[0087] (4) Calibration frequency: After the kit was calibrated, performance tests were conducted on days 0, 7, 14, 21 and 28 respectively. All indicators met the requirements, indicating that the calibration curve was valid for 28 days.

[0088] Example 6 Clinical Relevance Assessment To verify the detection efficacy of the kit developed in this study, clinical performance evaluation was conducted. 104 clinical plasma samples were collected, including 30 patients with Alzheimer's disease (AD), 20 patients with mild neurodegenerative disease (MCI), 15 patients with other neurodegenerative diseases, and 39 healthy controls. An imported p-Tau217 reference kit was used for testing, and the data were exported. The concentration of p-Tau217 in plasma was determined using a self-developed magnetic microparticle chemiluminescence kit, and the results were compared with those obtained using the imported kit.

[0089] Statistical results show ( Figure 3 The correlation coefficient (R) between the two methods was 0.9935, with a positive concordance rate of 95.00%, a negative concordance rate of 92.50%, and an overall concordance rate of 93.48%. The Kappa value was 0.8672. The detection results showed a good linear correlation with the imported kit, indicating that the developed kit can meet the needs of early screening and auxiliary diagnosis of Alzheimer's disease in clinical practice.

Claims

1. A monoclonal antibody against phosphorylated Tau217, characterized in that: The monoclonal antibody comprises: a) Heavy chain variable region, which includes heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: The sequence of CDR-H1 is shown in the amino acid sequence of GFTFSDYYMY. The sequence of CDR-H2 is shown in the amino acid sequence of VISHDGDNTYYPDTVKG. The sequence of CDR-H3 is shown as the amino acid sequence of GRGFDV; b) Light chain variable region, which includes light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: The sequence of CDR-L1 is shown in the amino acid sequence of RASQSISSYLN. The sequence of CDR-L2 is shown in the amino acid sequence of AASSLQS. The sequence of CDR-L3 is shown in the amino acid sequence of QQSNSAPLT.

2. The monoclonal antibody according to claim 1, wherein, The monoclonal antibody contains the antibody heavy chain variable region HCVR. and antibody light chain variable region LCVR, wherein: HCVR sequence as follows The amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAVISHDGDNTYYPDTVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGRGFDVWGQGTLVTVSS is shown below; LCVR sequence as follows The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSWPLTFGGGTKVEIK is shown.

3. The application of the monoclonal antibody according to claims 1-2 in the detection of antiphosphorylated Tau217, characterized in that, The test is not for disease diagnosis purposes.

4. A kit for detecting phosphorylated Tau217, wherein, The kit includes a capture antibody and an acridinium ester-labeled second antibody, wherein the second antibody is a monoclonal antibody against phosphorylated Tau217 as described in any one of claims 1 to 2.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a detection diluent and calibrators.

6. The kit according to claim 4 or 5, characterized in that, The capture antibody is coated with superparamagnetic microparticles.

7. A method for quantitatively determining the content of phosphorylated Tau217, wherein, A double-antibody sandwich ELISA is performed using a capture antibody and a biotin-labeled second antibody, employing magnetic microparticle chemiluminescence detection, wherein the second antibody is a monoclonal antibody as described in any one of claims 1 to 2, and the method is for non-disease diagnostic purposes.

8. The method according to claim 7, wherein, The capture antibody comprises: a) Heavy chain variable region, which includes heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: The sequence of CDR-H1 is shown in the amino acid sequence of GFTFSDYYMY. The sequence of CDR-H2 is shown in the amino acid sequence of SINHDGDNTYYPDTVKG. The sequence of CDR-H3 is shown in the amino acid sequence of GRGMDV; b) Light chain variable region, which includes light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: The sequence of CDR-L1 is shown in the amino acid sequence of RASQSISSYLN. The sequence of CDR-L2 is shown in the amino acid sequence of AASSLQS. The sequence of CDR-L3 is shown in the amino acid sequence of QQSNSAPLT.

9. The method according to claim 8, wherein, The capture antibody contains the antibody heavy chain variable region HCVR. and antibody light chain variable region LCVR, wherein: HCVR sequence as follows The amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVSSINHDGDNTYYPDTVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGRGMDVWGQGTLVTVSS is shown below; LCVR sequence as follows The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSAPLTFGGGTKVEIK is shown.

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