P-Tau217 single molecule detection kit and detection antibody thereof

Abstract

The invention provides a single-molecule diagnostic kit based on p-Tau217. The detection kit comprises a magnetic bead coated p-Tau217 capture antibody, a biotin labeled Tau detection antibody and streptavidin-beta-galactosidase (SbetaG), ultra-sensitive detection is achieved through a high-specificity antibody and magnetic bead coupling technology in combination with a Simoa platform instrument, the detection sensitivity is high, and the detection sensitivity is high. The kit is suitable for early diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to a single-molecule detection kit for phosphorylated Tau217 (p-Tau217) and a detection antibody thereof, as well as application of the detection kit for diagnosing early-stage Alzheimer's disease. Background Art

[0002] Early diagnosis of Alzheimer's disease (AD) relies on highly sensitive detection of biomarkers in cerebrospinal fluid or blood, among which phosphorylated Tau217 (p-Tau217) protein is a key phosphorylation marker discovered in recent years.

[0003] Currently, the main clinical detection methods for P-Tau217 include radioimmunoassay, homogeneous enzyme immunoassay, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and gas chromatography. Radioimmunoassays require reagents with short half-lives, high costs, and are radioactive, posing significant risks to the human body. They also sometimes produce cross-reactions and false positives. Homogeneous enzyme immunoassays are complex to operate. ELISAs have disadvantages such as low automation, complex operation, and time-consuming procedures. High-performance liquid chromatography and gas chromatography require expensive instruments and high reagent quality requirements, making them unsuitable for clinical testing. Thus, existing detection methods have various drawbacks, and a lack of specific single-molecule detection methods for p-Tau217 is lacking. Summary of the Invention

[0004] The present invention achieves ultrasensitive detection of p-Tau217 by designing high-affinity antibodies, optimizing antigen epitopes, and combining a single-molecule immunoassay array (Simoa) instrument, and the detection kit has high stability.

[0005] The present invention provides a p-Tau217 capture antibody, the heavy chain amino acid sequence of the p-Tau217 capture antibody is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2.

[0006] On the other hand, the present invention provides a Tau detection antibody, wherein the heavy chain amino acid sequence of the Tau detection antibody is shown in SEQ ID NO: 3, and the light chain amino acid sequence is shown in SEQ ID NO: 4.

[0007] On the other hand, the present invention provides a p-Tau217 single molecule detection kit, which comprises magnetic beads coated with p-Tau217 capture antibody, biotin-labeled Tau detection antibody, and SβG; The magnetic beads coated with p-Tau217 capture antibody working solution consisted of the following: 1.5×10 7 ~3×10 70.2 μg / mL of magnetic bead coated p-Tau217 antibody, 20 mM Tris, 25 mM MES, 1% BSA, 1% sucrose, 0.5% trehalose, 0.1% glycerol and 0.05% Proclin 300; The biotin-labeled Tau detection antibody working solution is composed of: 0.2 μg / mL biotin-labeled Tau detection antibody, 20 mM phosphate buffer, 1% BSA, 1% sucrose, 0.1% glycerol and 0.05% Proclin 300; The SβG working solution is composed of: 100 pM SβG, 20 mM phosphate buffer, 1% BSA, 0.1% glycerol and 0.05% Proclin 300.

[0008] In some embodiments, the magnetic bead coated p-Tau217 capture antibody working solution is composed of: 0.2 μg / mL of magnetic bead coated p-Tau217 antibody, 20 mM Tris, 25 mM MES, 1% BSA, 1% sucrose, 0.5% trehalose, 0.1% glycerol and 0.05% Proclin 300; The biotin-labeled Tau detection antibody working solution is composed of: 0.2 μg / mL biotin-labeled Tau detection antibody, 20 mM phosphate buffer, 1% BSA, 1% sucrose, 0.1% glycerol and 0.05% Proclin 300; The SβG working solution is composed of: 100 pM SβG, 20 mM phosphate buffer, 1% BSA, 0.1% glycerol and 0.05% Proclin 300.

[0009] The present application has carried on the thorough research to the magnetic bead coated capture antibody working solution, the biotin-labeled detection antibody working solution, SβG working solution, in the above-mentioned working solution, including Tris buffer, phosphate buffer, BSA, sucrose, glycerol, Proclin 300 and the like component, provides the stable detection environment, can effectively avoid the interference, improves the sensitivity and accuracy of detection kit. More importantly, in the magnetic bead coated capture antibody working solution, add 20-50 mM MES, which is conducive to maintaining the stability of the magnetic bead coated antibody, and the detection kit has good long-term storage stability. And the inventor optimizes the number of magnetic bead coated p-Tau217 capture antibody, the concentration of Tau detection antibody, the use concentration of SβG, wherein, 1.5 x 10 7 ~ 3 x 10 7The magnetic beads were coated with p-Tau217 capture antibody at a concentration of 1.5×10 / mL, the concentration of biotin-labeled Tau detection antibody was 0.1-0.5 μg / mL, and the concentration of SBG was 100-200 pM; preferably, 1.5×10 7 ~2×10 7 The magnetic beads with a concentration of 0.1 μg / mL were coated with p-Tau217 capture antibody, the concentration of biotin-labeled Tau detection antibody was 0.2 μg / mL, and the concentration of SBG was 100 pM. It showed superior effects in parameters such as the minimum detection limit, lower limit of quantification, upper limit of quantification, and detection dynamic range, and the effect was better.

[0010] In some embodiments, the detection kit further comprises an enzyme reaction substrate RGP (resorufin-β-D-galactopyranoside) solution.

[0011] In some embodiments, the detection kit further comprises a p-Tau217 calibrator.

[0012] In some embodiments, the kit further comprises a sample diluent, wherein the sample diluent is composed of: 25-50 mM phosphate buffer, 100-200 mM NaCl, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, and 0.05-0.1% Proclin 300.

[0013] The kit provided by the present invention improves upon the immunofluorescence method, employing a dual-antibody detection approach. Both the capture and detection antibodies are capable of binding to sites on the phosphorylated Tau protein. The biotin-labeled NHS-PEG4-Biotin on the detection antibody binds to streptavidin-β-galactosidase (SβG), which in turn reacts with the enzyme substrate RGP to produce a fluorescent signal for quantitative analysis of p-Tau217. Studies have found that the volume ratio of the three components (p-Tau217 capture antibody working solution coated with magnetic beads, biotin-labeled Tau detection antibody working solution, and SβG working solution) is 1-2:1-2:4-6. A preferred volume ratio of 1.25:1:5 results in enhanced detection signals, higher sensitivity, and the highest signal-to-noise ratio.

[0014] On the other hand, the present invention also provides a detection method of a p-Tau217 detection kit, the method comprising the following steps: (1) Dilute the sample to be tested with sample diluent and mix well; (2) 100 μL of the diluted sample, 25 μL of the magnetic bead-coated p-Tau217 capture antibody working solution, and 20 μL of the biotin-labeled Tau detection antibody working solution were added to a 96-well microplate in sequence and incubated in a constant temperature shaker at 37°C and 800 rpm for 30 minutes; (3) Wash the plate, add 100 μL of streptavidin-β-galactosidase working solution, and incubate in a constant temperature shaker at 37°C and 800 rpm for 10 minutes; (4) Wash the plate, add 100 μL of enzyme reaction substrate RGP, and place the 96-well microplate after reaction into the Simoa SRX instrument for detection.

[0015] On the other hand, the present invention also provides a method for preparing magnetic beads coated with p-Tau217 capture antibodies, comprising the following steps: 1) Add the capture antibody to the ultrafiltration tube, add buffer, centrifuge, and add buffer to obtain the capture antibody solution; 2) Wash the magnetic beads, then add NHS and EDC solution, mix well, incubate for 30 minutes, and wash the magnetic beads; 3) Add the magnetic beads from step 2) to the solution from step 1), incubate at 25°C for 2-3 hours, and wash the beads. 4) Add magnetic bead blocking solution, incubate at 25°C for 2-3 hours, and wash the magnetic beads.

[0016] In some embodiments, in step 1), the buffer is 50 mM MES buffer with a pH of 5-6.

[0017] In some embodiments, in step 2), the concentration of the NHS solution is 0.1-0.5 mg / mL, and the concentration of the EDC solution is 0.1-0.5 mg / mL. Preferably, the concentrations of both the NHS and EDC solutions are 0.1 mg / mL.

[0018] In some embodiments, in step 1), the concentration of the p-Tau217 capture antibody is 0.2 mg / mL. The inventors of this application also experimented with different concentrations. At higher concentrations, steric hindrance reduced efficiency, leading to decreased coupling efficiency. The highest coupling efficiency was achieved at a p-Tau217 capture antibody concentration of 0.2 mg / mL.

[0019] In some embodiments, in step 3), the weight ratio of magnetic beads to antibodies is 100:1. The inventors of this application have also tried different ratios during the experiment. Larger ratios resulted in poor antibody labeling, while smaller ratios affected the substrate signal. Overall, a weight ratio of 100:1 was preferred for best results.

[0020] In some embodiments, the magnetic bead blocking solution in step 4) is 50 mM phosphate containing 1% BSA.

[0021] In some embodiments, the pH of the magnetic bead blocking solution in step 4) is 8.0.

[0022] In some embodiments, the magnetic beads are magnetic microparticles having carboxyl functional groups.

[0023] In some embodiments, the magnetic beads have a particle size of 2-3 μm.

[0024] On the other hand, the present invention also provides a method for preparing a biotin-labeled Tau detection antibody, comprising the following steps: (1) Add the detection antibody to the ultrafiltration tube, add buffer, centrifuge, and add buffer to obtain the detection antibody solution; (2) Prepare a biotin solution, mix the biotin solution with the solution in step (1), and react at 25°C with shaking for 0.5-1 hour; (3) Transfer the liquid after the reaction in step (2) to an ultrafiltration tube, add buffer, centrifuge, and collect the solution.

[0025] In some embodiments, in step (1) and step (3), the buffer is 0.1 M PBS buffer, and the pH of the buffer is 7.4.

[0026] In some embodiments, in step (1) and step (3), the ultrafiltration tube has a molecular weight cutoff of 30KD, and the centrifugation conditions are 2-8°C, 4000g, and 15 minutes.

[0027] In some embodiments, in step (1), the concentration of the detection antibody is 0.2 μg / mL.

[0028] In some embodiments, in step (2), the biotin is NHS-PEG4-Biotin; In some embodiments, in step (2), the mass ratio of the detection antibody to biotin is 40:1. The inventors of this application also tried different ratios of biotin-labeled antibodies during the experiment. If the biotinylation ratio is too high, nonspecific binding increases due to excessive labeling. Among them, the mass ratio of the detection antibody to biotin is 40:1, which has the best effect and the highest signal-to-noise ratio (S / N) (>70).

[0029] On the other hand, the present invention also provides a method for preparing streptavidin-β-galactosidase, comprising the following steps: A) Streptavidin and β-galactosidase were dialyzed overnight with dialysate to remove interfering substances. B) The dialyzed streptavidin was mixed with succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and reacted at room temperature for 1-2 hours to generate maleimide-activated streptavidin and remove unbound SMCC. C) The dialyzed β-galactosidase was reacted with Traut's reagent at room temperature for 1-2 hours to remove unbound Traut's. D) Mix maleimide-activated streptavidin with the modified β-galactosidase, react overnight at 4°C, add β-mercaptoethanol, incubate at room temperature for 0.5-1 hour to terminate the reaction, and separate the coupled product.

[0030] In some embodiments, the dialysate in step A) is 0.1 M phosphate buffer, and the pH of the buffer is 7.2.

[0031] In some embodiments, in step B), the molar ratio of streptavidin to SMCC is 1:25.

[0032] In some embodiments, in step C), the molar ratio of β-galactosidase to Traut's reagent is 1:15.

[0033] In some embodiments, in step D), the molar ratio of maleimide-activated streptavidin to modified β-galactosidase is 1:3.

[0034] In another aspect, the present invention provides a kit for detecting the Alzheimer's disease biomarker p-Tau217, wherein the kit is as defined above.

[0035] On the other hand, the present invention provides the use of p-Tau217 as a biomarker in the preparation of a detection reagent or kit for diagnosing Alzheimer's disease.

[0036] The detection kit provided by the present invention has the advantages of small sample volume, accurate detection results, high detection sensitivity, good specificity, wide linear range, good reproducibility, short detection time, and is convenient for fully automatic detection instruments, thus overcoming many shortcomings of existing immunoassay technologies. The beneficial effects of the present invention also include: 1. The present invention uses a high-affinity p-Tau217 capture antibody. The p-Tau217 capture antibody and biotinylated Tau detection antibody can specifically bind to the p-Tau217 antigen and form a "sandwich" structure with the antigen to achieve ultrasensitive detection of p-Tau217.

[0037] 2. The present invention optimizes the number of magnetic beads coated with p-Tau217 capture antibodies, the concentration of Tau detection antibodies, and the concentration of SβG used, wherein 1.5×10 7 ~3×107 The magnetic beads were coated with p-Tau217 capture antibody at a concentration of 0.1-0.5 μg / mL, the concentration of biotin-labeled Tau detection antibody was 0.1-0.5 μg / mL, and the concentration of SBG was 100-200 pM; preferably, 1.5×10 7 ~2×10 7 The magnetic beads were coated with p-Tau217 capture antibody at a concentration of 0.1 μg / mL, the concentration of biotin-labeled Tau detection antibody was 0.2 μg / mL, and the concentration of SBG was 100 pM. The results showed superior effects in parameters such as the minimum detection limit, lower limit of quantification, upper limit of quantification, and dynamic range of detection, and the effect was better.

[0038] 3. This invention optimizes the working solutions of magnetic bead-coated p-Tau217 capture antibodies, Tau detection antibodies, and SβG. The present invention has conducted in-depth research on these working solutions. These solutions, including Tris buffer, phosphate buffer, BSA, sucrose, glycerol, and Proclin 300, provide a stable detection environment, effectively avoid interference, and improve the sensitivity and accuracy of the detection kit. This is of great significance for distinguishing Alzheimer's disease from non-Alzheimer's disease and for the early diagnosis of Alzheimer's disease.

[0039] 4. This study optimized the preparation process of the p-Tau217 capture antibody during kit preparation, including the activation time (30 minutes) of EDC and NHS and the blocking conditions (phosphate buffer containing 1% BSA). In the preparation of the biotinylated Tau detection protein, the effect of different ratios of biotinylated detection antibody on sensitivity was investigated. The optimized synergistic effect significantly improved the sensitivity of the kit (detection limit reached 0.003 pg / mL).

[0040] 5. The detection kit prepared by the present invention has good stability and is suitable for large-scale clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the ROC curve of the sample. DETAILED DESCRIPTION

[0042] The following examples are used to illustrate the present invention, but they are not intended to limit the scope of the present invention.

[0043] The methods used in the following embodiments are conventional methods unless otherwise specified, and the materials used are commercially available unless otherwise specified.

[0044] Unless otherwise specified herein, % refers to mass percentage.

[0045] Example 1 Preparation of p-Tau217 Antigen Antigen design: A synthetic peptide containing the p-Tau217 epitope peptide (amino acid sequence: VYKpSPVVSGDTSPRHL, where pS represents phosphorylated serine) was synthesized by GenScript Biotech Co., Ltd. This is a p-Tau217 peptide and is used for immunization and p-Tau217 calibrator.

[0046] Example 2 Preparation of monoclonal antibodies (1) Preparation of p-Tau217 capture antibody The p-Tau217 peptide was emulsified with Freund's complete adjuvant at a 1:1 ratio for primary immunization in rabbits, with 50–200 µg administered subcutaneously in a total volume of 0.5–1.0 mL per rabbit. Booster immunizations were administered every 14–21 days using the same dose but incomplete Freund's adjuvant. Seven to ten days after each booster immunization, 0.5–1.0 mL of blood was collected from the marginal ear vein. Anti-p-Tau217 peptide antibody titers in serum were assessed using an ELISA plate coated with p-Tau217 peptide (1 µg / mL, 50 µL / well). Three to five days before planned spleen harvest, p-Tau217 peptide was injected again in PBS (without adjuvant) to increase circulating B cell counts.

[0047] After the immunization procedure was completed, the rabbits were euthanized in accordance with animal ethics standards, and the spleens were aseptically removed and placed in ice-cold RPMI-1640 medium containing 1% FBS. Single-cell suspensions were prepared and viable cells were counted. Splenocytes were then mixed with immortalized rabbit B cell lines at a ratio of 2:1 or 3:1. PEG 1500 or PEG 3350 was slowly added to promote fusion. The cells were then diluted and neutralized with culture medium, centrifuged and resuspended in RPMI-1640 containing HAT medium, and plated into 96-well plates at 200 μL per well (cell density approximately 1 × 10 3 –1×10 4 ).

[0048] When cells reach approximately halfway across the bottom of the wells, positive clones are screened by ELISA using the culture supernatant. Purify through two to three rounds of limiting dilution until a stable clone with 100% positive results is obtained, expand, and freeze. The ELISA assay steps include: coating the well with 50 µL of p-Tau217 peptide at 1 µg / mL overnight at 4°C; blocking the wells with culture supernatant, incubating and washing the plates, and then adding an HRP-conjugated secondary antibody (1:5000 dilution). After color development is complete, the OD value is measured at 450 nm. A high-affinity positive clone is identified if the OD value is >2.3 and significantly higher than the negative control (0.053).

[0049] Cell culture supernatants from cell lines identified as high-affinity positive clones were directly loaded onto a Protein A / G affinity chromatography column to allow selective binding of the antibody to the matrix. Following binding, the column was washed with 20 mM sodium phosphate buffer (pH 7.0) to remove nonspecific proteins, and the antibody was eluted with 0.1 M Glycine-HCl (pH 2.7). The eluted antibody was immediately neutralized with 1 M Tris-HCl buffer (pH 8.5) to prevent inactivation. The concentration was then determined spectrophotometrically, and the aliquots were aliquoted as needed and stored at -80°C.

[0050] To obtain the antibody gene sequence, total RNA is extracted from hybridoma cells in the logarithmic growth phase (Trizol reagent) and synthesized into cDNA via reverse transcription. The resulting cDNA is then sequenced by a third-party sequencing company to obtain the gene sequence information of the target monoclonal antibody.

[0051] By analyzing the monoclonal antibody sequence structure, the obtained monoclonal p-Tau217 capture antibody was further sequenced and identified, and its heavy chain amino acid sequence and light chain amino acid sequence were determined as follows: Heavy chain amino acid sequence: QVQLKESGPGLVQPSQSLSITCTVSGFSLTAYGVSWVRQSPGKGLEWLGVIWSGGNTDYNSAFMSRLSISRDNSKSQVFLKMNSLQTDDTAIYYCAREGGYYAMDYWGQGTSVTVSS (SEQ ID NO: 1) CDR1:VSGFSLTAYGV CDR2:VIWSGGNTDYNSAFMS CDR3:AREGGYYAMDY Light chain amino acid sequence: QSVLTQPPSVSGAPGQRVTISCTRSSGSIASNYVQWYQEKPGQSPKLVIYEGSKLPSGVPSRFSGSGSGTAFTLRISRVEAEDVGVYYCGQSLSRPYTFGGGTKLEIK (SEQ ID NO: 2) CDR1:RSSGSIASNYVQ CDR2:EGSKLPS CDR3:GQSLSRPYT (2) Preparation of Tau detection antibodies Preparation method: Recombinant full-length Tau protein (sequence: MAPT gene encoding, UniProt ID P10636. Amino acid sequence: (SEQ ID NO:5).

[0052] Recombinant full-length Tau protein was emulsified with Freund's complete adjuvant at a ratio of 1:1 for the first immunization of rabbits, with each rabbit injected subcutaneously with 50-200 pg in a total volume of 0.5-1.0 mL. Boost immunization was performed every 14-21 days with the same dose but with Freund's incomplete adjuvant. Seven to ten days after each boost, 0.5-1.0 mL of blood was collected from the marginal ear vein, and the serum was tested for anti-recombinant full-length Tau protein antibody titer using ELISA, with the ELISA plate coated with recombinant full-length Tau protein (1 pg / mL, 50 pL / well). Three to five days before the planned spleen collection, recombinant full-length Tau protein diluted in PBS (without adjuvant) was injected again to increase the number of circulating B cells.

[0053] After completion of the immunization program, the rabbits were euthanized according to the animal ethics code, and the spleen was removed aseptically and placed in ice-cold RPMI-1640 medium containing 1% FBS to prepare a single-cell suspension and count the viable cells. Subsequently, the spleen cells were mixed with the immortalized rabbit B cell line at a ratio of 2:1 or 3:1, PEG 1500 or PEG 3350 was slowly added to promote fusion, and then diluted with medium to neutralize, centrifuged, resuspended in HAT medium-containing RPMI-1640, and inoculated into a 96-well plate at 200 pL per well (cell density of about 1 x 10 3 –1 x 10 4 )。

[0054] When the cells were cultured to about half the bottom of the well, the culture supernatant was screened for positive clones using ELISA. Two to three rounds of limiting dilution were used for purification until a stable clone with 100% positivity was obtained, and then the clone was expanded and cryopreserved. The ELISA detection steps included coating with recombinant full-length Tau protein at a concentration of 1 pg / mL overnight at 4°C, 50 pL per well; after blocking, adding the culture supernatant, incubating, washing the plate, adding HRP-labeled secondary antibody (1:5000 dilution), and measuring the OD value at 450 nm after color development termination. If the OD value is >2.3 and significantly higher than the negative control (0.053), it is determined to be a high-affinity positive clone.

[0055] The cell culture supernatant containing the cell strain determined to be a high-affinity positive clone was directly loaded into a Protein A / G affinity chromatography column to selectively bind the antibody to the filler. After binding was complete, 20 mM sodium phosphate buffer (pH 7.0) was used for washing to remove non-specific proteins, and 0.1 M Glycine-HCl (pH 2.7) was used to elute the antibody. The eluted antibody was immediately neutralized with 1 M Tris-HCl buffer (pH 8.5) to prevent inactivation, and then the concentration was determined by spectrophotometer, and then stored at -80°C as needed.

[0056] To obtain the antibody gene sequence, total RNA is extracted from hybridoma cells in the logarithmic growth phase (Trizol reagent) and synthesized into cDNA via reverse transcription. The resulting cDNA is then sequenced by a third-party sequencing company to obtain the gene sequence information of the target monoclonal antibody.

[0057] By analyzing the monoclonal antibody sequence structure, the obtained monoclonal detection antibody Tau was further sequenced and identified, and its heavy chain amino acid sequence and light chain amino acid sequence were determined as follows: Heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGFDYWGQGTLVTVSS (SEQ ID NO: 3) CDR1:SYAMS CDR2:SISGSGGSTYYADSVKG CDR3:EGGFDY and light chain amino acid sequence: DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 4) CDR1:RASQDISNYLN CDR2:YTSNLHS CDR3:QQGNTLPWT Example 3 A p-Tau217 Single Molecule Diagnostic Kit A p-Tau217 single molecule detection kit, comprising magnetic beads coated with p-Tau217 capture antibodies, biotin-labeled Tau detection antibodies, and SβG; The magnetic beads coated with p-Tau217 capture antibody working solution consisted of the following: 1.5×10 7 ~2×10 7 Magnetic beads at 100 μg / mL were coated with p-Tau217 antibody, 20 mM Tris, 25 mM MES, 1% BSA, 1% sucrose, 0.5% trehalose, 0.1% glycerol, and 0.05% Proclin300; The biotin-labeled Tau detection antibody working solution is composed of the following: 0.2 μg / mL biotin-labeled Tau detection antibody, 20 mM phosphate buffer, 1% BSA, 1% sucrose, 0.1% glycerol and 0.05% Proclin 300; The SβG working solution consists of the following: 100 pM SβG, 20 mM phosphate buffer, 1% BSA, 0.1% glycerol and 0.05% Proclin300.

[0058] The heavy chain amino acid sequence of the p-Tau217 capture antibody is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2.

[0059] The heavy chain amino acid sequence of the Tau detection antibody is shown in SEQ ID NO: 3, and the light chain amino acid sequence is shown in SEQ ID NO: 4.

[0060] Example 4 Detection Method of a p-Tau217 Single Molecule Detection Kit A detection method for a single molecule detection kit for p-Tau217, comprising the following steps: (1) Dilute the sample to be tested 3 times with sample diluent and mix thoroughly; (2) 100 μL of the diluted sample, 25 μL of the magnetic bead-coated p-Tau217 capture antibody working solution, and 20 μL of the biotin-labeled detection antibody working solution were added to a 96-well microplate in sequence and incubated in a constant temperature shaker at 37°C and 800 rpm for 30 minutes; (3) Wash the 96-well microplate after the reaction in step (2) three times using a magnetic bead washer; (4) After washing the plate, add 100 μL of streptavidin-β-galactosidase working solution and incubate in a constant temperature shaker at 37°C and 800 rpm for 10 minutes; (5) Wash the 96-well microplate after the reaction in step (4) three times using a magnetic bead washer; (6) Add 100 μL of enzyme reaction substrate RGP and place the 96-well microplate after the reaction into the Simoa SRX instrument for detection.

[0061] Example 5 Preparation of p-Tau217 Single Molecule Diagnostic Kit 1. Preparation of magnetic beads coated with p-Tau217 capture antibody The preparation method of the magnetic beads coated with p-Tau217 capture antibody is as follows: 1) Add the p-Tau217 capture antibody to a 30 kD ultrafiltration tube, add 50 mM MES buffer, pH 5-6, centrifuge at 4000 g for 15 minutes at 2-8°C, and add 50 mM MES buffer to obtain a capture antibody solution with a concentration of 0.2 mg / mL. 2) Rinse the magnetic beads with 50 mM MES buffer. Add NHS (N-hydroxysuccinimide, concentration: 0.1 mg / mL) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, concentration: 0.1 mg / mL) solution to the washed magnetic beads, mix well, and incubate for 30 minutes to wash the magnetic beads. 3) Add the magnetic beads from step 2) to the solution from step 1) at a mass ratio of 100:1 (bead:antibody). Incubate at 25°C with rotation for 2 hours. Wash the beads with magnetic bead wash buffer (25 mM Tris, 150 mM NaCl, 2% gelatin). 4) Add magnetic bead blocking buffer (50 mM phosphate containing 1% BSA, pH 8.0), rotate and incubate at 25°C for 2 hours. Wash the magnetic beads with magnetic bead washing buffer (25 mM phosphate, 150 mM NaCl) to obtain magnetic beads coated with p-Tau217 capture antibody.

[0062] 2. Preparation of Biotinylated Tau Detection Antibody The preparation method of the biotin-labeled Tau detection antibody is as follows: (1) Add Tau detection antibody to an ultrafiltration tube, add 0.1 M PBS buffer, pH 7.4, centrifuge at 2-8°C, 4000 g, 15 minutes, add 0.1 M PBS buffer to obtain a detection antibody solution with a concentration of 0.2 μg / mL; (2) Prepare NHS-PEG4-Biotin solution with 0.1M PBS buffer and use within 30 minutes after preparation. Mix the biotin solution with the antibody solution to be labeled at a mass ratio of 40:1, and shake at 25°C for 30 minutes. (3) The liquid after the reaction in step (2) was transferred to an ultrafiltration tube, and antibody labeling buffer was added. The centrifugation was repeated 5 times, and the solution in the ultrafiltration tube was collected to obtain the biotin-labeled Tau detection antibody.

[0063] 3. Streptavidin-β-galactosidase (SβG) It can be commercially available or prepared by the following method. The preparation method of the streptavidin-β-galactosidase is as follows: A) Streptavidin and β-galactosidase were dialyzed overnight with 0.1 M phosphate buffer (pH 7.2) to remove interfering substances; B) The dialyzed streptavidin was mixed with succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) at a molar ratio of 1:25 and reacted at room temperature for 1 h to generate maleimide-activated streptavidin. Unbound SMCC was removed using a desalting column. C) The dialyzed β-galactosidase was mixed with Traut's reagent at a molar ratio of 1:15 and reacted at room temperature for 1 hour. Unbound Traut's reagent was removed using a desalting column. D) Maleimide-activated streptavidin and modified β-galactosidase were mixed at a molar ratio of 1:3 and reacted at 4°C overnight. β-Mercaptoethanol was added and the reaction was terminated by incubation at room temperature for 0.5 hours. The coupled product was separated using a Sephacryl S-300HR column, and the high molecular weight fraction was collected to obtain streptavidin-β-galactosidase.

[0064] Comparative Example 1: The only difference from Example 3 is that the magnetic beads coated with p-Tau217 capture antibody working solution is composed of the following: The magnetic beads coated with p-Tau217 capture antibody working solution is composed of the following: 1.5×10 7 ~2×10 7 Magnetic beads at 1000 μg / mL were coated with p-Tau217 antibody, 20 mM phosphate buffer, 1% BSA, 0.1% glycerol, and 0.05% Proclin300; The biotin-labeled Tau detection antibody working solution is composed of the following: 0.2 μg / mL biotin-labeled Tau detection antibody, 20 mM phosphate buffer, 1% BSA, 0.1% glycerol and 0.05% Proclin 300; The SβG working solution consists of the following: 100 pM SβG, 20 mM phosphate buffer, 1% BSA, 0.1% glycerol and 0.05% Proclin300.

[0065] Test Example 1: Effect of different antibody concentrations on magnetic bead coupling efficiency Detection Method: Magnetic beads coated with p-Tau217 capture antibody were prepared using the method described in the Examples. The effects of different p-Tau217 capture antibody concentrations (0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL) (diluted with Bead Conjugation Buffer) on magnetic bead coupling efficiency were investigated.

[0066] After blocking, the amount of unbound antibody in the supernatant was determined by the BCA assay, and the coupling efficiency was calculated. The results are shown in Table 1.

[0067] Table 1 Effect of different antibody concentrations on magnetic bead coupling efficiency The results showed that the coupling efficiency was the highest (>90%) when the p-Tau217 capture antibody concentration was 0.2 mg / mL, and the efficiency decreased at 0.3 mg / mL due to steric hindrance.

[0068] Test Example 2: Effect of EDC and NHS activation time on coupling effect Detection Method: Magnetic beads coated with p-Tau217 capture antibodies were prepared using the method described in the Examples. 0.2 mg / mL p-Tau217 antibody was then coupled to the beads for capture. The beads were activated with EDC and NHS for 15, 30, and 45 minutes (2-8°C). Antibody density on the bead surface was detected using a fluorescently labeled secondary antibody. The results are shown in Table 2.

[0069] Table 2 Effect of EDC and NHS activation time on coupling effect The results showed that when the magnetic beads were activated with EDC and NHS for 30 min, the antibody density was the highest (~5000 molecules / magnetic bead), and extending the activation time to 45 min resulted in magnetic bead aggregation.

[0070] Test Example 3: Inhibition of nonspecific binding by blocking buffer Magnetic beads coated with p-Tau217 capture antibodies were prepared using the method described in the Examples. The beads were blocked with phosphate buffer containing 1% BSA, 1% casein, and 1% skim milk powder. Sample diluent (without p-Tau217) was added, and the background signal (AEB) was measured. The results are shown in Table 3.

[0071] Table 3 Results of the inhibition of nonspecific binding by blocking buffer The results showed that the background signal in the 1% BSA group was the lowest (AEB was 0.0037), followed by the casein group (AEB was 0.015), and the background in the skimmed milk powder group was elevated due to lipid residue (AEB was 0.052).

[0072] Test Example 4: Effect of different ratios of biotin-labeled detection antibodies on detection sensitivity Biotin-labeled Tau detection antibodies were prepared using the method described in the Examples. Reactions were performed at antibody:biotin mass ratios of 10:1, 20:1, 40:1, and 60:1. The SA-β-Gal detection system was used to measure the average enzyme bound per bead (AEB) value and the fluorescence signal intensity at 1 pg / mL p-Tau217 at different ratios. The results are shown in Table 4.

[0073] Table 4 Effects of different ratios of biotin-labeled detection antibodies on detection sensitivity The results showed that when the mass ratio of Tau detection antibody:biotin was 40:1, the signal-to-noise ratio (S / N) was the highest (>70), while 60:1 resulted in increased nonspecific binding due to excessive labeling.

[0074] Test Example 5: Effects of different working fluids on detection sensitivity The detection kits described in Example 3 and Comparative Example 1 were used. The SA-β-Gal detection system was used to detect the background signal (AEB) and measure the fluorescence signal intensity at 1 pg / mL p-Tau217 at different test ratios. The signal-to-noise ratio (S / N) for Example 3 was >70, while that for Comparative Example 1 was 61.

[0075] Test Example 6: Thermal Accelerated Stability Test of Kit The working solution in the kit prepared in this example was placed in a 37°C accelerated heat chamber for one month, and the AEB values ​​were measured before and after. The results showed that compared to the kit prepared in Comparative Example 1, the change in the value measured by the kit described in Example 3 of the present invention was only 2% lower, while the change in the value in Comparative Example 1 was 10% lower. This demonstrates that the kit of the present invention has excellent stability.

[0076] Test Example 7: p-Tau217 Detection Kit Performance Index Test (1) Linear detection The p-Tau217 antigen was diluted with sample diluent to prepare calibrator solutions of varying concentrations. The p-Tau217 calibrators were then tested using the kit described in the Examples, and the AEB values ​​corresponding to each calibrator were measured. A standard curve was constructed using concentration as the abscissa and AEB as the ordinate. The results showed that the linear correlation coefficients for p-Tau217 were all >0.99.

[0077] (2) Minimum detection limit detection The zero-concentration calibrator was used as a sample for testing, and the measurement was repeated 20 times. The AEB values ​​of the 20 measurements were obtained, and the mean (M) and standard deviation (SD) were calculated to obtain M+2SD. A linear equation was obtained by performing a two-point regression fit based on the concentration-AEB ratio between the zero-concentration calibrator and the adjacent calibrator. The AEB value of M+2SD was substituted into the above equation to determine the corresponding concentration value, which is the minimum detection limit. The test results are shown in Table 5.

[0078] Table 5 Results of determination of the lowest detection line (3) Repeatability test Use two samples at different concentration levels (high and low), and test each concentration sample 10 times. Calculate the mean (M) and standard deviation (SD) of the 10 measurements. Then, calculate the coefficient of variation (CV) using the formula CV = SD / M × 100%. Where CV is the coefficient of variation, SD is the standard deviation of the 10 measurements, and M is the mean of the 10 measurements.

[0079] Table 6 p-Tau217 repeatability measurement results The results showed that the CV was within ≤8%.

[0080] (4) Accuracy testing Three replicate measurements were performed using p-Tau217 standard samples at both high and low concentrations. The relative deviation between the mean (denoted as M) and the labeled value was calculated. The relative deviation (B) of the measured concentration was calculated using the formula B = (MT) / T × 100%, where B is the relative deviation, M is the mean measured concentration, and T is the labeled value.

[0081] Table 7 p-Tau217 accuracy test results The results showed that the relative deviation was ≤10%.

[0082] Test Example 8: Clinical Performance Test of the p-Tau217 Detection Kit The p-Tau217 single molecule detection kit prepared in Example 3 of the present application was used to test plasma samples of 74 patients (35 of whom were without AD and 39 were AD patients) who were diagnosed with AD by the gold standard. The measurement results were analyzed by ROC curve. The results are as follows: Figure 1 As shown in the results, the area under the receiver operating characteristic curve was 0.946. When the critical value was 0.38 pg / mL, the sensitivity was 87.2% and the specificity was 94.3%.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. A p-Tau217 capture antibody, characterized in that: The heavy chain amino acid sequence of the p-Tau217 capture antibody is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO:

2.

2. A Tau detection antibody, characterized in that: The heavy chain amino acid sequence of the Tau detection antibody is shown in SEQ ID NO: 3, and the light chain amino acid sequence is shown in SEQ ID NO:

4.

3. A p-Tau217 single molecule detection kit, characterized by: The detection kit includes magnetic beads coated with p-Tau217 capture antibody, biotin-labeled Tau detection antibody, and SβG; The magnetic beads coated with p-Tau217 capture antibody working solution consisted of the following: 1.5×10 7 ~3×10 7 100 μg / mL magnetic beads coated with p-Tau217 capture antibody, 20-50 mM Tris, 25-30 mM MES, 1-2% BSA, 1-2% sucrose, 0.5-1% trehalose, 0.05-0.1% glycerol, and 0.05-0.1% Proclin300; The biotin-labeled Tau detection antibody working solution is composed of the following: 0.1-0.5 μg / mL biotin-labeled Tau detection antibody, 20-50 mM phosphate buffer, 1-2% BSA, 1-2% sucrose, 0.05-0.1% glycerol and 0.05-0.1% Proclin300; The SβG working solution consists of: 100-200 pM SβG, 20-50 mM phosphate buffer, 1-2% BSA, 0.05-0.1% glycerol and 0.05-0.1% Proclin300.

4. A p-Tau217 single molecule detection kit according to claim 3, characterized in that: The magnetic beads coated with p-Tau217 capture antibody working solution consisted of the following: 1.5×10 7 ~2×10 7 Magnetic beads at 100 μg / mL were coated with p-Tau217 antibody, 20 mM Tris, 25 mM MES, 1% BSA, 1% sucrose, 0.5% trehalose, 0.1% glycerol, and 0.05% Proclin300; The biotin-labeled Tau detection antibody working solution is composed of the following: 0.2 μg / mL biotin-labeled Tau detection antibody, 20 mM phosphate buffer, 1% BSA, 1% sucrose, 0.1% glycerol and 0.05% Proclin 300; The SβG working solution consists of the following: 100 pM SβG, 20 mM phosphate buffer, 1% BSA, 0.1% glycerol and 0.05% Proclin300.

5. The p-Tau217 single molecule detection kit according to claim 3, characterized in that: The detection kit also includes an enzyme reaction substrate RGP solution.

6. The p-Tau217 single molecule detection kit according to claim 3, characterized in that: The detection kit further comprises a sample diluent, which is composed of 25-50 mM phosphate buffer, 100-200 mM NaCl, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20 and 0.05-0.1% Proclin300.

7. The p-Tau217 single molecule detection kit according to claim 3, characterized in that: The method for preparing the magnetic beads coated with p-Tau217 capture antibodies comprises the following steps: 1) Add the capture antibody to the ultrafiltration tube, add buffer, centrifuge, and add buffer to obtain the capture antibody solution; 2) Wash the magnetic beads, then add NHS and EDC solution, mix well, incubate for 30 minutes, and wash the magnetic beads; 3) Add the magnetic beads from step 2) to the solution from step 1), incubate at 25°C for 2-3 hours, and wash the beads. 4) Add magnetic bead blocking solution, incubate at 25°C for 2-3 hours, and wash the magnetic beads.

8. The p-Tau217 single molecule detection kit according to claim 7, characterized in that: In step 3), the mass ratio of magnetic beads to antibodies is 100:

1.

9. The p-Tau217 single molecule detection kit according to claim 7, characterized in that: In step 4), the magnetic bead blocking solution is 50 mM phosphate containing 1% BSA.

10. The p-Tau217 single molecule detection kit according to claim 3, characterized in that: The method for preparing the biotin-labeled Tau detection antibody comprises the following steps: (1) Add the detection antibody to the ultrafiltration tube, add buffer, centrifuge, and add buffer to obtain the detection antibody solution; (2) Prepare a biotin solution, mix the biotin solution with the solution in step (1), and react at 25°C with shaking for 0.5-1 hour; (3) Transfer the liquid after the reaction in step (2) to an ultrafiltration tube, add buffer, centrifuge, and collect the solution.

11. A p-Tau217 single molecule detection kit according to any one of claims 3 to 10, characterized in that: The biotin is NHS-PEG4-Biotin.

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