Preparation of anti-p-Tau217 antibody and application of anti-p-Tau217 antibody in Alzheimer's disease detection kit

Abstract

The invention provides preparation of an anti-p-Tau217 antibody and application of the anti-p-Tau217 antibody in an Alzheimer's disease detection kit, and belongs to the technical field of antibodies and immunodetection, the anti-p-Tau217 antibody 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, and a coding gene of the light chain variable region comprises a nucleotide sequence shown in SEQ ID NO.2. The coding gene of the light chain variable region comprises a nucleotide sequence shown in SEQIDNO.3, the heavy chain variable region comprises an amino acid sequence shown in SEQIDNO.2, and the light chain variable region comprises an amino acid sequence shown in SEQIDNO.3. The invention also discloses a preparation method. The invention provides preparation of an anti-p-Tau217 antibody and application of the anti-p-Tau217 antibody in an Alzheimer's disease detection kit. The successful expression of the monoclonal anti-p-Tau217 antibody with high affinity is realized, and the monoclonal anti-p-Tau217 antibody is used for detecting the Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical fields of antibodies and immunoassay, and particularly to the preparation of anti-p-Tau217 antibody and its application in an Alzheimer's disease detection kit. Background Art

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

[0003] Alzheimer's disease (AD) is the main cause of senile dementia. Once the disease occurs, it is irreversible. 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 the aggregation of Tau protein 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 hotspots for early screening of AD in recent years and is considered the most promising biomarker. Roche's Elecsys pTau-217 plasma biomarker test has received 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 several diagnostic companies have commercially used this biomarker. Although Aβ42 / 40, etc. are also biomarkers, the detection of Aβ in blood is difficult, while the detection of p-Tau217 is more promising. Summary of the Invention

[0004] In view of this, the present invention provides the preparation of an anti-p-Tau217 antibody and its application in an Alzheimer's disease detection kit, achieving the successful expression of a monoclonal anti-p-Tau217 antibody with high affinity and its use in the detection of Alzheimer's disease.

[0005] To achieve the above object, the present invention provides a preparation of an anti-p-Tau217 antibody, which includes a heavy chain variable region and a light chain variable region. The coding gene of the heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO.1, and the coding gene of the light chain variable region includes the nucleotide sequence shown in SEQ ID NO.3.

[0006] Optionally, 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.3.

[0007] Optionally, it includes the following steps: preparing an immunogen, immunizing a mouse with the immunogen and an adjuvant, detecting the antibody titer of the mouse serum, fusing and screening the cells of the mouse with the highest antibody titer in the mouse serum to obtain the 1-1F cell line against the phosphorylated polypeptide at the 217 site, amplifying and culturing the cell line and then injecting it into the abdominal cavity of a mouse to obtain ascites containing monoclonal antibody, and treating and purifying the ascites containing monoclonal antibody to obtain the anti-p-Tau217 antibody.

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

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

[0010] Optionally, the screening uses p-Tau181, p-Tau217, p-Tau231 polypeptides and t-Tau protein for reverse screening.

[0011] To achieve the above object, the present invention also provides an application of the preparation of an anti-p-Tau217 antibody in an Alzheimer's disease detection kit, and the kit includes reagent A and reagent B; The preparation of reagent A includes the following steps: mixing the p-Tau217 monoclonal antibody and biotin in proportion and reacting at room temperature, dialyzing the reactant in a PBS buffer system, and the dialyzed solution is a biotinylated p-Tau217 monoclonal antibody solution; washing and redissolving the streptavidin magnetic microspheres with Tris-HC1 buffer and then mixing them with the biotinylated p-Tau217 monoclonal antibody solution in proportion and reacting at room temperature, washing the reacted magnetic microspheres with Tris-HCI buffer, removing the supernatant, and redissolving them with a magnetic microsphere buffer system to obtain the magnetic microsphere mother solution of the p-Tau217 monoclonal antibody, that is, reagent A.

[0012] Optionally, the preparation of reagent B includes the following steps: mixing the t-Tau antibody and acridinium ester in proportion and reacting at room temperature, adding a lysine termination solution to react, and dialyzing the reactant in a PBS buffer system to obtain reagent B.

[0013] 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 to a PBS buffer system, mixing well until completely dissolved, and quantifying with pure water; the concentration of reagent A during use is 0.7 - 0.8 mg / mL.

[0014] Optionally, the PBS buffer system is 0.15 M and pH 7.3.

[0015] The above technical solutions of the present invention at least include the following beneficial effects: The preparation method provided by the present invention screens out specific antibodies against the p-Tau217 protein and successfully expresses monoclonal anti-p-Tau217 antibodies with high affinity. The anti-p-Tau217 antibodies can bind to the p-Tau217 protein with high specificity. Therefore, the monoclonal antibodies can be used to identify or detect AD patients. At the same time, this application combines magnetic microparticles and chemiluminescence technology to prepare a p-Tau217 detection kit, which has important application value and significance for the large-scale early screening of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the OD and P / N graph of mouse serum in Example 1 of the present invention; Figure 2 It is the VH and VL gene fragments (500 bp) of mice in Example 1 of the present invention; Figure 3 It is the activity graph of the p-Tau217 monoclonal antibody in Example 2 of the present invention; Figure 4 It is the purity graph of the p-Tau217 monoclonal antibody in Example 2 of the present invention; Figure 5 It is the correlation coefficient graph of the p-Tau217 concentration detected by the kit and the Simoa single molecule immunoassay in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention fall within the scope of protection of the present invention. Figures 1 to 5

[0018] Example 1. Preparation of Monoclonal Antibodies Specifically Recognizing the 217 Phosphorylation Site Preparation of Immunogen The immunogen is CRTPSLP(pT)PPTR, which is a phosphorylated polypeptide. The P-Tau217 polypeptide used in this study was synthesized by Sangon Biotech Co., Ltd.

[0019] 2. Mouse immunization The P-Tau217 polypeptide was mixed with the novel adjuvant squalene and used to immunize 6-8-week-old BALB / c mice by subcutaneous injection at a dose of 50 μg / mouse. Two weeks later, the second immunization was carried out in the same method and dose. After two immunizations, tail blood was taken and the serum antibody titer was measured by gradient dilution using the ELISA method. Whether to boost the immunization was determined according to the results.

[0020] One week after the last immunization, mouse blood was collected to detect the serum antibody titer. The following method can be used to detect the serum antibody titer: First, coat a 96-well ELISA plate with 10 μg / ml p-Tau217 polypeptide at 4 °C for 24 h. Secondly, block it with 1% BSA at 37 °C for 1 h. Then, add the serially diluted samples and incubate at 37 °C for 1 h. Subsequently, add the HRP-labeled secondary antibody and incubate at 37 °C for 1 h. Then, add the TMB substrate solution and react in the dark at room temperature for 15 min. Finally, terminate the reaction with 2 M sulfuric acid and read the OD value at 450 nm. The highest dilution factor with P / N≥2.1 was taken as the serum antibody titer. The results are shown in Figure 1 .

[0021] As Figure 1 can be seen, Figure 1 A shows that after immunization, all 4 mice produced corresponding immune titers. Among them, the P / N value (OD value of immunized mice / OD value of non-immunized mice) of mouse 2 was the highest at the same dilution ( Figure 1 B), and it was selected as the subject for the next experiment.

[0022] 3. Cell fusion The BALB / c mouse with the highest P / N value of serum antibody titer was sacrificed by cervical dislocation and soaked in 75% alcohol for 5 min for disinfection. The mouse was fixed on a foam board with a thumb tack, the abdominal skin was cut open, the abdominal cavity was opened, the fat and connective tissue on the surface of the spleen were dissected, and the surface of the spleen was rinsed with Dulbecco's Modified Eagle Medium (DMEM). A 200-mesh cell sieve was placed in a petri dish and moistened with 10 mL DMEM medium. The spleen was pressed with the piston of a syringe and gently crushed, ground clockwise, and the cells were collected into a 50 mL centrifuge tube. Centrifuge at 1500 r / min for 3 min at room temperature and discard the supernatant. Add 5 mL of erythrocyte lysate pre-incubated at room temperature to remove cell debris, then add 5 mL of DMEM medium, mix well, centrifuge at 1500 r / min for 3 min at room temperature, and discard the supernatant.

[0023] The SP2 / 0 cells were centrifuged at 1500 r / min for 3 min at room temperature. The supernatant was discarded. The SP2 / 0 cells and spleen lymphocytes were mixed evenly at a ratio of 5:1 to 10:1 in a 50 mL centrifuge tube. 10 mL of DMEM medium was added, and the mixture was centrifuged at 1500 r / min at room temperature for 3 min. After discarding the supernatant, the following operations were carried out under the condition of a 37 °C water bath: 1 mL of polyethylene glycol 1500 (PEG1500) preheated to 37 °C was slowly dropped into the centrifuge tube, while adding and oscillating. Then, 1 mL of DMEM medium was added and 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. The cell supernatant was discarded. The fused cells were evenly mixed in a medium containing a high concentration of nucleotide precursor substances, and then dropped into a 96-well cell culture plate with feeder cells added for culture.

[0024] 4. Cell screening When the above cells grew to medium size, the cell supernatant was aspirated for ELISA detection. The polypeptide used for detection was the phosphorylated polypeptide at position 217. After two days, the detection was carried out again. The hybridoma cell lines with both detection results being positive were re-screened with p-Tau181, p-Tau217, p-Tau231 polypeptides and t-Tau protein; the positive clones that were reactive to the phosphorylated polypeptide at position 217 and non-reactive to other polypeptides or proteins were cloned three times (differential screening was carried out for each cloning detection), and hybridoma cell lines that could stably secrete antibodies were obtained, that is, the 1-1F cell line against the phosphorylated polypeptide at position 217 was finally obtained.

[0025] 5. Cell culture The hybridoma cell lines that stably secreted antibodies were amplified and cultured in a carbon dioxide incubator, transferred from a 96-well plate to a 24-well plate, a 6-well plate, and a 10 cm cell plate in sequence. Then the cells in the cell plate were collected and injected into the abdominal cavity of mice. After 7 to 10 days, the ascites containing monoclonal antibodies was aspirated from the abdominal cavity of mice.

[0026] 6. Monoclonal antibody purification The mouse ascites containing monoclonal antibodies was treated with a 50% saturated ammonium sulfate solution. Then the obtained precipitate was dissolved in PBS and purified using a Protein A column to obtain the purified monoclonal antibody. The obtained monoclonal antibody was subjected to PCR amplification, and the PCR product was sent to the company for sequencing to obtain the sequence. The VH (heavy chain variable region) and VL (light chain variable region) genes of the p-Tau217 antibody are shown in Figure 2 .

[0027] The VH and VL sequences of the p-Tau217 antibody are as follows: VH SEQ ID NO.1: GAGCATTGGGAATTCGGGGTGCAGCTGCAGGAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAGATCTCCTGCAAGACTTCTGGATACACATTCACTGAATTCCCCATGTACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGAACTAGCAATAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGGCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCAAGAGGCACGGCTATGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGTACTCCAGAT SEQ ID NO.2: EHWEFGVQLQESGPELVKPGASVKISCKTSGYTFTEFPMYWVKQSHGKSLEWIGGINPNNGGTSNNQKFKGKATLTVDKASSTAYMELRSLTSEDSAVYYCARGTAMYFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSYSR The CDR regions of VH analyzed by IMGT are as follows: CDR1: GYTFTEFP CDR2: INPNNGGT CDR3: ARGTAMYFDV VL SEQ ID NO.3: CCTGACATTGAGCTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATAGGAGCTACACGTCATG SEQ ID NO.4: PDIELTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIGATRH The CDR regions of VL were analyzed by IMGT as follows: CDR1: KSVSTSGYSY CDR2: LVS CDR3: QHI It belongs to the variable regions of murine antibody heavy chain and light chain, namely VH and VL regions, by comparison with GENBANK.

[0028] Example 2 Identification of p-Tau217 monoclonal antibody protein 1. Activity detection of p-Tau217 monoclonal antibody Coat p-tau217 polypeptide at 1 μg / mL, 150 μL per well, and incubate at 37 °C for 1.5 h; wash the plate 5 times, add 1% BSA for blocking, 150 μL per well, and incubate at 37 °C for 2 h; wash the plate 5 times, add the p-Tau217 monoclonal antibody diluted at a specific ratio, 100 μL per well, and incubate at 37 °C for 1.5 h; wash the plate 5 times, add HRP secondary antibody, 100 μL per well, and incubate at 37 °C for 1.5 h; add TMB chromogenic solution, 100 μL per well, terminate the reaction with the termination solution after about 15 min of color development, 50 μL per well; place it at OD450 nm for reading, and the analysis results are shown in Figure 3 .

[0029] Figure 3The X-axis represents the antibody reaction concentration, with values ranging from 0 to 1.28 ug / ml, and the Y-axis represents the absorbance value (at 450 nm) of 1 ug / ml p-Tau217, with values ranging from 0 to 2.48.

[0030] 2. Specificity identification of the anti-p-Tau217 monoclonal antibody Coat an ELISA plate with p-Tau217 polypeptide, p-Tau181 polypeptide, p-Tau231 polypeptide, and t-Tau protein (5 ng / μL) respectively. Add 50 μL of the supernatant of the monoclonal antibody cells to be tested, incubate at 37 °C for 1 h, wash the plate 3 - 5 times with PBST, add the HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:1000 (100 μL / well), incubate at 37 °C for 45 min, then wash 3 - 5 times with PBST. Add 100 μL / well of TMB chromogenic solution, react in the dark for 15 min, add 2 M H 2 SO 4 50 μL / well to terminate the reaction, read the OD450nm values of each detection well. The OD450nm value of each well is judged by P / N > 2.1. The results are shown in Table 1.

[0031] Table 1. Specificity identification of the anti-p-Tau217 monoclonal antibody

[0032] As can be seen from Table 1, the monoclonal antibody is a specific antibody for p-Tau217 polypeptide and does not react with p-Tau181, p-Tau231 polypeptides, and t-Tau protein.

[0033] 3. Purity identification of the anti-p-Tau217 monoclonal antibody Prepare a 5% separating gel and a 15% stacking gel. After loading the purified antibody, perform electrophoresis under constant voltage conditions. Stop electrophoresis when bromophenol blue reaches 1 cm from the bottom edge of the separating gel. Remove the gel film and place it in Coomassie Brilliant Blue staining solution for 30 min, and finally decolorize it 2 - 3 times with decolorizing solution until the background is colorless. Scan the film with a scanner and save the image, and analyze the antibody purity with Gelpro32 software. Figure 4 It is a diagram showing the purity of the anti-p-Tau217 monoclonal antibody.

[0034] From Figure 4 it can be seen that the anti-p-Tau217 monoclonal antibody of the present invention has obvious specific bands at 25 kD and 60 kD, without other impurity bands. The total content of the light chain and heavy chain (antibody purity) is 95.01%, and the obtained antibody has a high purity.

[0035] 4. Determination of the affinity constant of the anti-p-Tau217 monoclonal antibody In this study, biolayer interferometry (BLI) was used to detect the affinity and kinetic parameters of the binding between antibodies and antigens. An SA sensor was used to immobilize the antibody on the sensor surface, and then it was reacted with serially diluted antigens. Information on intermolecular interactions was obtained by analyzing the changes in surface optical interference. The specific operation method was referred to the instrument instruction manual, and the results of the affinity test are shown in Table 2.

[0036] Table 2. Affinity test of anti-p-Tau217 monoclonal antibody

[0037] As can be seen from Table 2, the affinity of the p-Tau217 antibody for the p-Tau217 antigen was 0.726 nM, indicating that the antibody prepared in this invention had a relatively high affinity for the antigen.

[0038] 5. Identification of the recognition site of the anti-p-Tau217 monoclonal antibody For the biopanning of the phage library, one well of a 96-well plate was coated with 150 μL of 100 g / L Avastin at 4 °C overnight. After blocking, 10 μL of the original peptide library diluted with 100 μL of TBST was added and incubated at room temperature for 1 h, followed by washing 10 times with 0.1% TBST. 100 μL of the eluent (0.2 M Glycine HCl, 1 g / L BSA) was added and incubated for 8 min. The eluent was aspirated, and then 15 μL of 1 M Tris-HCl was used to neutralize the above eluent. 1 μL of the eluent was taken to measure the titer, and the remaining liquid was added to 20 mL of lysogeny broth (Luria-Bertan, LB) medium (containing 200 μL of ER2738 and 20 μL of the tetracycline stock solution) for amplification and purification. The second and third rounds of screening were carried out according to the above steps, but washed with 0.5% TBST respectively.

[0039] Identification of positive phages After the third round of screening was completed, 30 well-separated phage clones were selected for amplification and purification respectively. The purified phages were added to 96-well plates pre-coated with Avastin and the anti-p-tau217 monoclonal antibody respectively, and incubated at room temperature for 1 h. Then, HRP-labeled anti-M13 (1:2000) antibody was added and incubated for 1 h. o-Phenylenediamine (OPD) was used for color development, and the A490 nm value was measured. A phage clone with an A490 nm value 5 times higher than that of the negative control (anti-p-tau217 monoclonal antibody) was regarded as a positive clone.

[0040] Take 500 μL of the above phage stock solution, add 200 μL of PEG8000 / NaCl, place for 10 min, centrifuge for 10 min, discard the supernatant, resuspend the precipitate in 100 μL of iodide buffer, add 250 μL of ethanol, react at room temperature for 10 min, centrifuge for 10 min, discard the supernatant, wash the precipitate with 70% ethanol, and dry briefly under vacuum. Resuspend the precipitate in 30 μL of tris(hydroxymethyl)aminomethane (Tris) + ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris-HCl, 1 mM EDTA), and take 5 μL of the above solution and send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0041] After three rounds of biopanning screening, the yield of positive phages gradually increased, indicating that the selected phages were selectively enriched. DNA sequence determination was performed on the positive phage clones, and the amino acid sequence of the fused dodecapeptide encoded by them was deduced based on the measured DNA sequences. The results showed that the antigen epitope targeted by the p-tau217 monoclonal antibody was located at P10636-8 (215-p217-219).

[0042] Example 3 Application of p-Tau217 in Alzheimer's disease detection kit Buffer preparation 1.1 Preparation of magnetic microsphere buffer system Add 3% BSA, 2% trehalose, 0.05% magnetic microsphere dispersant, 0.25% Tween 20, and 0.05% Proclin 300 to the PBS buffer system (0.15 M, pH 7.3), mix well until completely dissolved, and make up the volume with pure water.

[0043] 1.2 Preparation of acridinium ester buffer Add 0.8% BSA, 2% sucrose, 0.25% Tween 20, and 0.05% Proclin 300 to the MES buffer system (0.05 M, pH 6.0), mix well until completely dissolved, and make up the volume with pure water.

[0044] 1.3 Preparation of calibrator buffer Add 1.5% BSA, 5% trehalose, 10 mM EDTA, 1.5% mannitol, 0.1% Tween 20, and 0.1% Proclin 300 to the PBS buffer system (0.15 M, pH 8.2), mix well until completely dissolved, and make up the volume with pure water.

[0045] 2. Preparation of Reagent A 2.1 p-Tau217 monoclonal antibody conjugated to biotin 2 mg / mL p-Tau217 monoclonal antibody and 35 mg / mL biotin were mixed at a molar ratio of 1:20, and reacted at room temperature for 2 hours. The reactant was dialyzed in PBS buffer using a dialysis bag, and the dialysate was changed every two hours, and repeated 3 times. The dialyzed solution was stored at 4°C for use to obtain a biotinylated p-Tau217 monoclonal antibody solution.

[0046] 2.2 Biotinylated p-Tau217 monoclonal antibody coupled to streptavidin magnetic beads After washing and re-dissolving with Tris-HCl buffer, streptavidin magnetic microspheres were mixed with biotinylated p-Tau217 monoclonal antibody at a ratio of 1 mg: 20 ug and reacted at room temperature for 1.5 h. The magnetic microspheres were washed 3 times with Tris-HCl buffer, the supernatant was removed, and the magnetic microsphere buffer system was used to re-dissolve to 3 mg / mL. 2.3 Preparation of Reagent A-Magnetic Bead Working Solution 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 later use.

[0047] 3. Preparation of Reagent B The t-Tau antibody was coupled with acridinium ester: 2 mg / mL of t-Tau antibody and 6 mg / mL of acridinium ester were mixed at a molar ratio of 1:20, and reacted at room temperature for 3 h. Then, 20 times the amount of antibody and 300 mg / mL of lysine stop solution were added to react for 1 h. The reactants were dialyzed in PBS buffer using a dialysis bag, and the dialysate was changed every two hours, repeated 3 times, and the dialyzed solution was stored at 4°C for use.

[0048] 4. Preparation of Calibrators The calibrator is p-Tau217 antigen (synthetic peptide), which is diluted to different concentrations with calibrator buffer and then lyophilized.

[0049] The combined test kit was used to test clinical samples with a fixed value gradient, and the correlation between the test results of the kit and the clinical fixed value was compared. Clinical gradient samples were fixed using Simoa single molecule immunoassay.

[0050] The detection method of the kit is as follows: Add the sample to be tested, 50 μl of magnetic bead working solution, and 50 μl of acridinium ester working solution into the reaction cup in sequence. Incubate and react at 37 °C. After adding 200 μl of cleaning solution and repeating the cleaning 3 times, add 100 μl of acridinium ester pre-excitation solution, mix well and react for 2 min, then add an equal volume of excitation solution. Detect the luminescence value with a fully automatic magnetic particle chemiluminescence analyzer, and calculate the concentration of p-Tau217 in the sample to be tested according to the p-Tau217 calibrator curve.

[0051] Taking the p-Tau217 concentration detected by Simoa single molecule immunoassay as the horizontal axis and the luminescence value of p-Tau217 detected by magnetic particle chemiluminescence assay as the vertical axis, draw a scatter plot of the p-Tau217 concentration of each sample, and calculate the correlation coefficient of the p-Tau217 concentration values between the two detection methods. The results are shown in Figure 5 .

[0052] It can be seen from Figure 5 that 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 immunoassay when applied to the magnetic particle chemiluminescence detection experiment, and can be used for the preparation of downstream kits.

[0053] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation of an anti-p-Tau217 antibody, characterized in that: It comprises a heavy chain variable region and a light chain variable region, wherein the coding gene of the heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO.1, and the coding gene of the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO.

3.

2. The preparation of 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.

3.

3. The preparation of the anti-p-Tau217 antibody according to claim 1, characterized in that: The following steps are involved: An immunogen is prepared, mice are immunized with the immunogen and an adjuvant, the mouse serum antibody titer is detected, cells of mice with the highest mouse serum antibody titer are fused and screened to obtain a 1-1F cell line against the 217-site phosphorylated polypeptide, the cell line is amplified and cultured, and then injected into the mouse peritoneal cavity to obtain ascites containing the monoclonal antibody, and the ascites containing the monoclonal antibody is treated and purified to obtain an anti-p-Tau217 antibody.

4. The preparation of the anti-p-Tau217 antibody according to claim 3, characterized in that: The immunogen is a phosphorylated polypeptide.

5. The preparation of the anti-p-Tau217 antibody according to claim 3, characterized in that: The mouse serum antibody titer is P / N≥2.1 at the highest dilution multiple.

6. The preparation of the anti-p-Tau217 antibody according to claim 3, characterized in that: The screening was performed using p-Tau181, p-Tau217, p-Tau231 polypeptides and t-Tau protein for counter-screening.

7. A method for preparing an anti-p-Tau217 antibody according to any one of claims 1 to 6 and using it in an Alzheimer's disease detection kit, characterized in that: The kit comprises reagent A and reagent B; The preparation of the reagent A comprises the following steps: the p-Tau217 monoclonal antibody and biotin are mixed in proportion, reacted at room temperature, and the reactant is dialyzed in a PBS buffer system, and the dialyzed solution is a biotinylated p-Tau217 monoclonal antibody solution; the streptavidin magnetic microspheres are washed with a Tris-HCl buffer, redissolved, and mixed with the biotinylated p-Tau217 monoclonal antibody solution in proportion, reacted at room temperature, and the magnetic microspheres after the reaction are washed with a Tris-HCl buffer, the supernatant is removed, and redissolved with a magnetic microsphere buffer system to obtain a magnetic microsphere mother solution of the p-Tau217 monoclonal antibody, i.e., the reagent A.

8. The use of the preparation of the anti-p-Tau217 antibody according to claim 7 in an Alzheimer's disease detection kit, characterized in that: The preparation of the reagent B comprises the following steps: The t-Tau antibody and acridinium ester were mixed in proportion, reacted at room temperature, lysine stop solution was added to react, and the reactants were dialyzed in a PBS buffer system to obtain reagent B.

9. The use of the preparation of the anti-p-Tau217 antibody according to claim 7 in 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 to a PBS buffer system, mixing thoroughly until completely dissolved, and adding pure water for quantification; the concentration of the reagent A when used is 0.7-0.8 mg / mL.

10. The use of the preparation of the anti-p-Tau217 antibody according to claim 8 in an Alzheimer's disease detection kit, wherein the PBS buffer system is 0.15M, pH 7.3.

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