Preparation of anti-p-tau217 antibody and its application in alzheimer's disease detection kit

Abstract

The application provides a preparation of an anti-p-Tau217 antibody and application thereof in an Alzheimer's disease detection kit, and belongs to the technical field of antibodies and immunodetection, and comprises a heavy chain variable region and a light chain variable region, a coding gene of the heavy chain variable region comprises a nucleotide sequence shown in SEQ ID NO. 1, a coding gene of the light chain variable region comprises a nucleotide sequence shown in SEQ ID NO. 3, the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO. 2, the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 3, and a preparation method is provided. The application provides a preparation of an anti-p-Tau217 antibody and application thereof in an Alzheimer's disease detection kit, and realizes successful expression of a high-affinity monoclonal anti-p-Tau217 antibody and use of the high-affinity monoclonal anti-p-Tau217 antibody in detection of Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies and immunoassay technology, and in particular to the preparation of anti-p-Tau217 antibodies and their application in Alzheimer's disease detection kits. BACKGROUND

[0002] Tau is a microtubule-associated protein that can interact with tubulin under normal physiological conditions, promote microtubule assembly, and regulate microtubule stability and dynamics. However, under certain pathological conditions, Tau protein may be modified, mainly through phosphorylation, which can lead to the production of abnormal aggregates that are toxic to neurons. When the Tau protein is post-translationally hyperphosphorylated, it dissociates from the microtubule, leading to its self-aggregation and microtubule disassembly.

[0003] Alzheimer's disease (AD) is the main cause of senile dementia, and once it occurs, it cannot be reversed. Early diagnosis, early prevention, early detection, and early treatment are of great significance for preventing and delaying the development of Alzheimer's disease. Aβ42, total tau protein (t-tau), and phosphorylated tau protein (p-tau) in cerebrospinal fluid are the main biomarkers reflecting the pathophysiological changes of AD. Neurofibrillary tangles formed by Tau protein aggregation are one of the decisive features of Alzheimer's disease, which makes Tau an attractive therapeutic target. Current drug strategies targeting Tau protein can be summarized as inhibiting Tau aggregation, inhibiting Tau phosphorylation, reducing Tau levels, and Tau immunization. There are many Tau-specific monoclonal antibodies in clinical trials. With the progress of research, p-Tau217 has become one of the hottest AD early screening in recent years and is considered to be the most promising biomarker. Roche's Elecsys pTau-217 plasma biomarker detection has obtained FDA breakthrough device designation, marking a further breakthrough in blood testing for Alzheimer's disease. p-Tau217 has become a popular biomarker for Alzheimer's disease, and many detection companies have commercialized the use of this marker. Although Aβ42 / 40, etc. are also biomarkers, Aβ detection in blood is difficult, while p-Tau217 detection has more potential. SUMMARY

[0004] Therefore, the present application provides a preparation of anti-p-Tau217 antibodies and their application in Alzheimer's disease detection kits, which successfully expresses high-affinity monoclonal anti-p-Tau217 antibodies and is used for the detection of Alzheimer's disease.

[0005] In order to realize the above-mentioned purpose, the application provides a preparation of anti-p-Tau217 antibody, which comprises a heavy chain variable region and a light chain variable region, the coding gene of the heavy chain variable region comprises a nucleotide sequence shown in SEQ ID NO. 1, and the coding gene of the light chain variable region comprises a nucleotide sequence shown in SEQ ID NO. 3.

[0006] Optionally, the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO. 2, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 3.

[0007] Optionally, the method comprises the following steps: preparing an immunogen, immunizing mice by using the immunogen and an adjuvant, detecting the antibody titer of the serum of the mice, taking the cells of the mouse with the highest serum antibody titer to perform fusion and screening to obtain a 1-1F cell strain of anti-217 site phosphorylated polypeptide, injecting the cell strain into the abdominal cavity of a mouse after amplification culture to obtain ascites containing monoclonal antibodies, and processing and purifying the ascites containing the monoclonal antibodies to obtain the anti-p-Tau217 antibody.

[0008] Optionally, the immunogen is a phosphorylated polypeptide.

[0009] Optionally, the antibody titer of the serum of the mice is P / N≥2.1 and the highest dilution multiple.

[0010] Optionally, the screening is performed by using p-Tau181, p-Tau217 and p-Tau231 polypeptides and t-Tau protein.

[0011] In order to realize the above-mentioned purpose, the application further provides an application of the preparation of anti-p-Tau217 antibody in an Alzheimer's disease detection kit, wherein the kit comprises reagent A and reagent B.

[0012] The preparation of the reagent A comprises the following steps: uniformly mixing p-Tau217 monoclonal antibodies and biotin at a certain ratio, reacting at room temperature, dialyzing the reactants in a PBS buffer system, and obtaining a solution of biotinylated p-Tau217 monoclonal antibodies; uniformly mixing the biotinylated p-Tau217 monoclonal antibodies solution and streptavidin magnetic microspheres after washing and resuspension in a Tris-HCl buffer, reacting at room temperature, washing the magnetic microspheres after reaction with the Tris-HCl buffer, removing the supernatant, resuspending the magnetic microspheres in a magnetic microsphere buffer system, and obtaining a p-Tau217 monoclonal antibody magnetic microsphere mother liquor, i.e., the reagent A.

[0013] Optionally, the preparation of the reagent B comprises the following steps: uniformly mixing t-Tau antibodies and acridinium ester at a certain ratio, reacting at room temperature, adding a lysine termination solution, dialyzing the reactants in a PBS buffer system, and obtaining the reagent B.

[0014] Optionally, the magnetic microsphere buffer system is prepared by adding 3% BSA, 2% trehalose, 0.05% magnetic microsphere dispersant, 0.25% Tween 20 and 0.05% Proclin 300 into a PBS buffer system, thoroughly mixing and dissolving, and then adding pure water to a certain amount; the concentration of the reagent A is 0.7-0.8 mg / mL when used.

[0015] Optionally, the PBS buffer system has a concentration of 0.15 M and a pH of 7.3.

[0016] The above technical solutions of the present application at least have the following beneficial effects:

[0017] The preparation method provided by the present application screens specific antibodies for p-Tau217 protein and successfully expresses monoclonal anti-p-Tau217 antibodies with high affinity. The anti-p-Tau217 antibodies can bind to p-Tau217 protein with high specificity, so the monoclonal antibodies can be used to identify or detect AD patients. Meanwhile, the present application combines magnetic particles and chemiluminescence technology to prepare a p-Tau217 detection kit, which has important application value and significance for large-scale early screening of AD. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 OD and P / N graphs of mouse serum in Example 1 of the present application;

[0019] Figure 2 VH and VL gene fragments (500 bp) of mice in Example 1 of the present application;

[0020] Figure 3 Activity graph of p-Tau217 monoclonal antibody in Example 2 of the present application;

[0021] Figure 4 Purity graph of p-Tau217 monoclonal antibody in Example 2 of the present application;

[0022] Figure 5 Correlation coefficient graph of p-Tau217 concentration detected by the kit and Simoa single molecule immunization method in Example 3 of the present application. DETAILED DESCRIPTION

[0023] To make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the following will combine the drawings of the embodiments of the present application to further describe the present application in detail. Figures 1-5 The technical solutions of the embodiments of the present application are described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, not all the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by those skilled in the art belong to the scope of protection of the present application.

[0024] Example 1. Preparation of monoclonal antibody specifically recognizing 217 phosphorylation site

[0025] Preparation of immunogen

[0026] The immunogen is CRTPSLP(pT)PPTR, which is a phosphorylated polypeptide. The P-Tau217 polypeptide used in this study is synthesized by Shenguo Biotechnology Co., Ltd.

[0027] 2. Mouse immunization

[0028] The P-Tau217 polypeptide was mixed with a new immunoadjuvant, squalene, and used to immunize 6-8 week old BALB / c mice by subcutaneous injection. The immunization dose was 50 ug per mouse. Two weeks later, the same method and dose were used for the second immunization. After two immunizations, the tail blood was collected to determine the serum antibody titer by ELISA gradient dilution. According to the results, it was determined whether to boost the immunization.

[0029] One week after the last immunization, the mouse blood was collected to detect the serum antibody titer. The following method can be used to detect the serum antibody titer: first, 10 ug / ml p-Tau217 polypeptide was used to coat a 96-well enzyme-labeled plate at 4°C for 24 hours. Then, 1% BSA was used for blocking at 37°C for 1 hour. Next, the sample was added at a dilution ratio of 1:2 and incubated at 37°C for 1 hour. Then, HRP-labeled secondary antibody was added and incubated at 37°C for 1 hour. Then, TMB substrate solution was added and reacted at room temperature for 15 minutes. Finally, 2M sulfuric acid was used to terminate the reaction, and the OD value at 450nm was read. The highest dilution ratio with P / N≥2.1 was taken as the serum antibody titer. The results are shown in Figure 1 .

[0030] As can be seen from Figure 1 , Figure 1 Figure A shows that after immunization, four mice all produced corresponding immune titers. Among them, the immune titer P / N value (immune mouse OD value / unimmunized mouse OD value) of mouse 2 was the highest at the same dilution ( Figure 1 Figure B), which was selected as the experimental object for the next step.

[0031] 3. Cell fusion

[0032] The BALB / c mouse with the highest P / N value of serum antibody titer was sacrificed by cervical dislocation, and was immersed in 75% alcohol for 5 min for sterilization. The mouse was fixed on a foam board with a large needle, the abdominal skin was cut, the abdominal cavity was opened, the surface fat and connective tissue of the spleen were stripped, the surface of the spleen was washed with Dulbecco's Modified Eagle Medium (DMEM), a 200-mesh cell screen was placed in a dish and was moistened with 10 mL of DMEM medium, the spleen was pressed with a syringe plug and was gently crushed, and was ground clockwise. The cells were collected in a 50-mL centrifuge tube, centrifuged at 1500 r / min for 3 min at room temperature, and the supernatant was discarded. 5 mL of red blood cell lysis solution was added in advance at room temperature, the cell debris was removed, 5 mL of DMEM medium was added, and the mixture was uniformly mixed and centrifuged at 1500 r / min for 3 min at room temperature, and the supernatant was discarded.

[0033] The SP2 / 0 cells were centrifuged at 1500 r / min for 3 min at room temperature, and the supernatant was discarded. The SP2 / 0 cells and the spleen lymphocytes were uniformly mixed in a 50-mL centrifuge tube at a ratio of 5:1 to 10:1, 10 mL of DMEM medium was added, and the mixture was centrifuged at 1500 r / min for 3 min at room temperature. After the supernatant was discarded, the following operations were performed at 37°C in a water bath: 1 mL of 37°C preheated polyethylene glycol 1500 (PEG1500) was slowly added dropwise into the centrifuge tube, and was oscillated while adding. Then, 1 mL of DMEM medium was added, and was oscillated for 1 min. Subsequently, 8 mL of DMEM medium was added to terminate the fusion, and the mixture was centrifuged at 1500 r / min for 3 min, and the cell supernatant was discarded. The fused cells were uniformly mixed in a medium containing high-concentration nucleotide precursors, and were then added dropwise into a 96-well cell culture plate in which feeder cells had been added.

[0034] 4. Cell screening

[0035] After the above cells grew to a medium size, the cell supernatant was aspirated for ELISA detection. The polypeptide used for detection was a 217-site phosphorylated polypeptide. After two days, the detection was performed again, and the hybridoma cell strain with positive results in both detections was subjected to counter-screening with p-Tau181, p-Tau217, p-Tau231 polypeptides and t-Tau protein. The positive clones that reacted with the 217-site phosphorylated polypeptide and did not react with other polypeptides or proteins were subjected to 3 times of cloning (each time of cloning was subjected to differential screening), and a hybridoma cell strain that could stably secrete antibodies was obtained, that is, the 1-1F cell strain that could stably secrete antibodies against the 217-site phosphorylated polypeptide was finally obtained.

[0036] 5. Cell culture

[0037] The hybridoma cell strain stably secreting the antibody is subcultured in a carbon dioxide incubator, and is sequentially transferred from a 96-well plate to a 24-well plate, a 6-well plate, and a 10 cm cell plate. Then, the cells in the cell plate are collected, and are injected into the abdominal cavity of a mouse. Seven to ten days later, the ascites containing the monoclonal antibody is extracted from the abdominal cavity of the mouse.

[0038] 6. Purification of Monoclonal Antibody

[0039] The mouse ascites containing the monoclonal antibody is treated with a 50% saturated ammonium sulfate solution. Then, the obtained precipitate is dissolved in PBS, and is purified using a Protein A column to obtain a purified monoclonal antibody. The obtained monoclonal antibody is subjected to PCR amplification, and the PCR product is sent to a company for sequencing to obtain a sequence. The p-Tau217 antibody VH (heavy chain variable region), VL (light chain variable region) gene is as follows: Figure 2 .

[0040] The p-Tau217 antibody VH, VL sequence is as follows:

[0041] VH

[0042] SEQ ID NO. 1:

[0043] GAGCATTGGGAATTCGGGGTGCAGCTGCAGGAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAGATCTCCTGCAAGACTTCTGGATACACATTCACTGAATTCCCCATGTACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGAACTAGCAATAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGGCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCAAGAGGCACGGCTATGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGTACTCCAGAT

[0044] SEQ ID NO. 2:

[0045] EHWEFGVQLQESGPELVKPGASVKISCKTSGYTFTEFPMYWVKQSHGKSLEWIGGINPNNGGTSNNQKFKGKATLTVDKASSTAYMELRSLTSEDSAVYYCARGTAMYFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSYSR

[0046] The CDR regions of the VH were analyzed by IMGT as follows:

[0047] CDR1: GYTFTEFP

[0048] CDR2: INPNNGGT

[0049] CDR3: ARGTAMYFDV

[0050] VL

[0051] SEQ ID NO. 3:

[0052] CCTGACATTGAGCTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATAGGAGCTACACGTCATG

[0053] SEQ ID NO. 4:

[0054] PDIELTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIGATRH

[0055] The CDR regions of the VL were analyzed by IMGT as follows:

[0056] CDR1: KSVSTSGYSY

[0057] CDR2: LVS

[0058] CDR3: QHI

[0059] The sequences were aligned to the murine antibody heavy chain variable region and light chain variable region, i.e. VH and VL region, by GENBANK.

[0060] Example Two Identification of p-Tau217 Monoclonal Antibody Protein

[0061] 1. Activity detection of p-Tau217 monoclonal antibody

[0062] Coat p-tau217 polypeptide at 1 ug / mL, 150 ul per well, incubate at 37°C for 1.5 h; wash the plate 5 times, add 1% BSA for blocking, 150 ul per well, incubate at 37°C for 2 h; wash the plate 5 times, add p-tau217 monoclonal antibody diluted according to a specific ratio, 100 ul per well, incubate at 37°C for 1.5 h; wash the plate 5 times, add HRP secondary antibody, 100 ul per well, incubate at 37°C for 1.5 h; add TMB developing solution, 100 ul per well, develop for about 15 min, then terminate the reaction with termination solution, 50 ul per well; place it at OD450 nm for reading, and the analysis results are shown in Figure 3 .

[0063] Figure 3 The X-axis represents the antibody reaction concentration, with a value of 0~1.28 ug / ml, and the Y-axis represents the absorbance value (450 nm) of 1 ug / ml p-Tau217, with a value of 0~2.48.

[0064] 2. Specificity identification of p-Tau217 monoclonal antibody

[0065] Coat the enzyme-labeled plate with p-Tau217 polypeptide, p-Tau181 polypeptide, p-Tau231 polypeptide and t-Tau protein (5 ng / μL) respectively, add 50 μL of the monoclonal antibody cell supernatant to be detected, incubate at 37°C for 1 h, wash the plate with PBST for 3~5 times, add 1:1000 diluted HRP labeled goat anti-mouse IgG secondary antibody (100 μL / well), incubate at 37°C for 45 min, then wash with PBST for 3~5 times, add 100 μL / well TMB developing solution, avoid light for 15 min, add 2 M H2SO450 μL / well to terminate the reaction, and read the OD450 nm value of each detection well, with P / N>2.1 as the determination standard. The results are shown in Table 1.

[0066] Table 1. Specificity identification of anti-p-Tau217 monoclonal antibody

[0067]

[0068] From Table 1, the monoclonal antibody is a p-Tau217 polypeptide specific antibody, and does not react with p-Tau181, p-Tau231 polypeptide and t-Tau protein.

[0069] 3. Purity identification of the p-Tau217 monoclonal antibody

[0070] Formulate 5% separation gel and 15% concentration gel, and then load the purified antibody for electrophoresis under stable pressure. When the bromophenol blue reaches 1 cm from the lower edge of the separation gel, stop electrophoresis. After removing the gel film, place it in the Coomassie brilliant blue staining solution for 30 min, and finally decolorize it 2-3 times until the background is colorless. Scan the film with a scanner and store the image, and analyze the antibody purity with Gelpro32 software. Figure 4 The purity of the p-Tau217 monoclonal antibody is shown in the figure.

[0071] From Figure 4 It can be known that the p-Tau217 monoclonal antibody of the present application has obvious specific bands at 25 kD and 60 kD, and no other bands, and the total content of light chain and heavy chain (antibody purity) is 95.01%, so the obtained antibody has high purity.

[0072] 4. Affinity constant determination of the p-Tau217 monoclonal antibody

[0073] In the present study, the biofilm interference technology (BLI) is used to detect the affinity and kinetic parameters of the antibody and antigen binding. The antibody is immobilized on the sensor surface using the SA sensor, and then reacts with the gradient-diluted antigen, so as to obtain the information of the mutual influence between molecules by analyzing the changes of surface light interference. The specific operation method is referred to the instrument instruction, and the affinity test results are shown in Table 2.

[0074] Table 2. Affinity test of anti-p-Tau217 monoclonal antibody

[0075]

[0076] From Table 2, the affinity of the p-Tau217 antibody and the p-Tau217 antigen is 0.726 nM, indicating that the antibody prepared in the present application has high affinity with the antigen.

[0077] 5. Identification of the recognition site of the p-Tau217 monoclonal antibody

[0078] Bio-panning of phage library One well of 96-well plate was coated with 100 g / L Avastin 150 mΐ, at 4 °C overnight, after blocking, 100 mΐ of TBST diluted 10 mΐ of original peptide library was added, and incubated at room temperature for 1 h, and washed with 0.1% TBST for 10 times. 100 mΐ of elution solution (0.2 M Glycine HCl, 1 g / L BSA) was added for 8 min, and the elution solution was aspirated, and the above elution solution was neutralized with 15 mΐ of 1 M Tris-HCl. 1 mΐ of elution solution was taken to determine the titer, and the rest of the liquid was added to 20 mL of lysogeny broth (Luria-Bertan, LB) medium (containing ER2738 200 mΐ, tetracycline stock solution 20 mΐ) for amplification and purification. The second and third rounds of screening were performed according to the above steps, but were washed with 0.5% TBST, respectively.

[0079] Identification of positive phage After the completion of the third round of screening, 30 phage clones with good segmentation were selected for amplification and purification. The purified phage was added to a 96-well plate coated with Avastin and anti-p-tau217 monoclonal antibody in advance, and incubated at room temperature for 1 h, and HRP-labeled anti-M13 (1:2000) antibody was added and incubated for 1 h. o-phenylenediamine (OPD) was developed, and the A490 nm value was determined, and the A490 nm value higher than 5 times of the negative control (anti-p-tau217 monoclonal antibody) was taken as a positive clone.

[0080] 500 mΐ of the above phage stock solution was taken, 200 mΐ of PEG8000 / NaCl was added, and it was placed for 10 min, centrifuged for 10 min, the supernatant was discarded, the precipitate was resuspended in 100 mΐ of iodide buffer, 250 mΐ of ethanol was added, and it was incubated at room temperature for 10 min, centrifuged for 10 min, the supernatant was discarded, the precipitate was washed with 70% ethanol, and briefly vacuum dried. The precipitate was resuspended in 30 mΐ of trishydroxymethylaminomethane (Tris) + ethylene diaminetetraacetic acid (EDTA) buffer (10 mM Tris-HCl, 1 mM EDTA), and 5 mΐ of the above solution was sent to Shanghai Biosci Biotech Co., Ltd. for sequencing.

[0081] After three rounds of bio-panning screening, the yield of positive phage gradually increased, indicating that the selected phage was selectively enriched. The DNA sequence of the positive phage clone was determined, and the deduced amino acid sequence of the fusion twelve-peptide was deduced according to the measured DNA sequence. The results showed that the antigenic epitope of the anti-p-tau217 monoclonal antibody was located at P10636-8 (215-p217-219).

[0082] Example Three p-Tau217 Application to Alzheimer's Disease Detection Test Kit

[0083] Buffer preparation

[0084] 1.1 Magnetic microsphere buffer system preparation

[0085] Add 3% BSA, 2% trehalose, 0.05% magnetic microsphere dispersant, 0.25% Tween 20, 0.05% Proclin 300 in PBS buffer system (0.15 M, PH 7.3), mix thoroughly until completely dissolved, and add pure water to the specified amount.

[0086] 1.2 Acridinium ester buffer preparation

[0087] Add 0.8% BSA, 2% sucrose, 0.25% Tween 20, 0.05% Proclin 300 in MES buffer system (0.05 M, PH 6.0), mix thoroughly until completely dissolved, and add pure water to the specified amount.

[0088] 1.3 Calibrator buffer preparation

[0089] Add 1.5% BSA, 5% trehalose, 10 mM EDTA, 1.5% mannitol, 0.1% Tween 20, 0.1% Proclin 300 in PBS buffer system (0.15 M, PH 8.2), mix thoroughly until completely dissolved, and add pure water to the specified amount.

[0090] 2. Reagent A preparation

[0091] 2.1 Coupling of p-Tau217 monoclonal antibody with biotin

[0092] Mix 2 mg / mL of p-Tau217 monoclonal antibody with 35 mg / mL of biotin at a molar ratio of 1:20, react at room temperature for 2 h, then dialyze the reaction product in PBS buffer, replace the dialysis solution every two hours, repeat for 3 times, and store the dialyzed solution at 4°C for later use. The resulting biotinylated p-Tau217 monoclonal antibody solution is obtained.

[0093] 2.2 Coupling of biotinylated p-Tau217 monoclonal antibody with streptavidin magnetic beads

[0094] After washing with Tris-HCI buffer and reconstituting, mix 1 mg of streptavidin magnetic microspheres with 20 ug of biotinylated p-Tau217 monoclonal antibody, react at room temperature for 1.5 h, wash the reacted magnetic microspheres with Tris-HCI buffer 3 times, remove the supernatant, and reconstitute with magnetic microsphere buffer to 3 mg / mL.

[0095] 2.3 Preparation of reagent A-magnetic bead working solution

[0096] The magnetic microsphere stock solution coated with p-Tau217 monoclonal antibody was diluted to a final concentration of 0.75 mg / mL and stored at 4°C for standby.

[0097] 3. Preparation of reagent B

[0098] The t-Tau antibody was coupled with acridinium ester: 2 mg / mL t-Tau antibody was mixed with 6 mg / mL acridinium ester at a molar ratio of 1:20, and after 3 h of reaction at room temperature, 20 times the amount of 300 mg / mL lysine termination solution was added and reacted for 1 h. The reaction was dialyzed in a dialysis bag in PBS buffer, and the dialysis solution was replaced every two hours for three times. The dialyzed solution was stored at 4°C for standby.

[0099] 4. Preparation of calibrators

[0100] The calibrator was p-Tau217 antigen (synthetic polypeptide), which was freeze-dried after being diluted with calibrator buffer to different concentrations.

[0101] The application test of the combined kit was performed on the gradient clinical samples with a fixed value, and the correlation between the test results of the kit and the clinical fixed value was compared. The gradient clinical samples were determined by Simoa single molecule immunization method.

[0102] The kit detection method is as follows: the sample to be tested, 50 uL of magnetic bead working solution, and 50 uL of acridinium ester working solution were sequentially added to the reaction cup, and incubated at 37°C. After washing with 200 uL of washing solution for 3 times, 100 uL of acridinium ester pre-priming solution was added and mixed, and then reacted for 2 min. An equal volume of priming solution was added, and the luminescence value was detected by a fully automatic magnetic particle chemiluminescence instrument. According to the p-Tau217 calibrator curve, the concentration of p-Tau217 in the sample to be tested was calculated.

[0103] The p-Tau217 concentration detected by Simoa single molecule immunization method was taken as the horizontal axis, and the p-Tau217 luminescence value detected by magnetic particle chemiluminescence method was taken as the vertical axis. The scatter plot of the p-Tau217 concentration of each sample was drawn, and the correlation coefficient of the p-Tau217 concentration values between the two detection methods was calculated. The results are shown in Figure 5 .

[0104] As shown in Figure 5 , the correlation coefficient of the two detection methods is R 2 =0.9898, which indicates that the prepared p-Tau217 specific monoclonal antibody has a strong correlation with the Simoa single molecule immunization method when applied to the magnetic particle chemiluminescence detection experiment, and can be used for downstream kit preparation.

[0105] The above are preferred embodiments of the present application. It should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the scope of protection of the present application.

Claims

1. An anti-p-Tau217 antibody, characterized in that, The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO. 2, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.

4.

2. The anti-p-Tau217 antibody according to claim 1, characterized in that The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO. 2, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.

4.

3. Use of an anti-p-Tau217 antibody according to any one of claims 1-2 for the manufacture of a test kit for Alzheimer’s disease, characterized in that, The kit comprises reagent A and reagent B. The preparation of the reagent A comprises the following steps: mixing anti-p-Tau217 antibody and biotin in proportion, reacting at room temperature, dialyzing the reaction product in PBS buffer system, and obtaining a biotinylated anti-p-Tau217 antibody solution; washing streptavidin magnetic microspheres with Tris-HCl buffer, reconstituting, mixing with the biotinylated anti-p-Tau217 antibody solution in proportion, reacting at room temperature, washing the magnetic microspheres with Tris-HCl buffer, removing the supernatant, reconstituting with a magnetic microsphere buffer system, obtaining a magnetic microsphere mother liquor of anti-p-Tau217 antibody, and obtaining reagent A.

4. Use of the anti-p-Tau217 antibody according to claim 3 for the manufacture of a test kit for Alzheimer’s disease, characterized in that, The preparation of the reagent B comprises the following steps: t-Tau antibody and acridinium ester are mixed in proportion, reacted at room temperature, added with lysine termination solution, reacted, dialyzed in PBS buffer system, and obtained reagent B.

5. The anti-p-Tau217 antibody of claim 3 in the preparation of an Alzheimer's disease detection kit, wherein the magnetic microsphere buffer system is prepared by adding 3% BSA, 2% trehalose, 0.05% magnetic microsphere dispersant, 0.25% Tween 20 and 0.05% Proclin 300 in a PBS buffer system, thoroughly mixing until completely dissolved, and adding pure water to a certain amount; and the concentration of the reagent A when used is 0.7-0.8 mg / mL.

6. The anti-p-Tau217 antibody of claim 4 in the preparation of an Alzheimer's disease detection kit, wherein the PBS buffer system is 0.15M, pH 7.3.

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