A chemiluminescent detection kit for phosphorylated Tau-231 protein, an antibody variable region, and a preparation method thereof

Abstract

The present invention discloses a chemiluminescent detection kit for phosphorylated Tau-231 protein, an antibody variable region, and a preparation method thereof, relating to the field of in vitro detection technology. The kit includes an AE-labeled tau antibody, a dual-magnetic particle reagent, and a p-tau231 calibrator. The dual-magnetic particle reagent includes a p-tau231 antibody, the light chain variable region amino acid sequence of which is shown in SEQ ID NO: 1, and the heavy chain variable region amino acid sequence of which is shown in SEQ ID NO: 2. The technical solution of the present invention establishes a chemiluminescent detection kit for phosphorylated Tau-231 protein suitable for fully automatic chemiluminescence analyzers. The kit has high sensitivity, a wide linear range, and broad prospects for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of in vitro detection technology, and in particular to a chemiluminescence detection kit for phosphorylated Tau-231 protein, an antibody variable region, and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common form of dementia, accounting for 60% to 80% of all dementia cases. It is a neurodegenerative disease characterized by cognitive decline and memory loss. Typical pathological features of AD include amyloid-β plaques and neurofilament tangles caused by hyperphosphorylation of microtubule-associated tau proteins. The main diagnostic methods for AD include cognitive assessment, imaging, biomarker testing, and genetic testing.

[0003] Recent studies have demonstrated that ultrasensitive detection techniques can identify AD-related pathological markers in the blood, such as p-tau231 (tau protein phosphorylated at position 231). As an early-stage biomarker for AD, p-tau231 helps identify patients in the mild cognitive impairment (MCI) stage. It can also be used to monitor AD progression and assess treatment efficacy. Combining p-tau231 with other biomarkers can improve the diagnostic accuracy of AD. Several methods exist for detecting p-tau231, including enzyme-linked immunosorbent assay (ELISA), immunoprecipitation mass spectrometry (IP-MS), liquid chromatography-mass spectrometry (LC-MS), single molecule array (Simoa), and chemiluminescence detection. Classic and traditional ELISA methods have the disadvantages of being non-automated, requiring large sample volumes, having low sensitivity, and poor precision. IP-MS and LC-MS, while highly accurate, are expensive and not widely available. Simoa offers the advantages of high throughput and ultrasensitivity, but is expensive and time-consuming. Chemiluminescence-based detection of p-tau231 offers low inter-laboratory and batch variability, is fully automated, highly reliable, and requires minimal time, making it suitable for clinical use. However, there are currently no p-tau231 assay kits for use with the MAGICL6000 fully automated chemiluminescence analyzer. These methods typically require combination with monoclonal antibodies that specifically recognize p-tau231, but the number of commercially available monoclonal antibodies is limited and their sensitivity is low. Summary of the Invention

[0004] The technical solution of the present invention to solve the above technical problems is to provide a chemiluminescent detection kit for phosphorylated Tau-231 protein, which includes an acridinium ester (AE)-labeled tau antibody, a dual magnetic particle reagent and a p-tau231 calibrator; wherein the dual magnetic particle reagent includes a p-tau231 antibody, the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 2.

[0005] Furthermore, the dual magnetic particle reagent includes: carboxyl magnetic particles coated with p-tau231 antibodies and Tosyl magnetic particles coated with p-tau231 antibodies, which are mixed in a ratio of 1:1-3.

[0006] Furthermore, the AE-labeled tau antibody is obtained by labeling the tau antibody with acridinium ester.

[0007] To solve the above technical problems, the present invention also proposes an antibody variable region for use in the p-tau231 antibody in the above-mentioned chemiluminescent detection kit for phosphorylated Tau-231 protein, wherein the CDR1 of the p-tau231 antibody light chain is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV;

[0008] The CDR1 of the p-tau231 antibody heavy chain is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

[0009] To solve the above technical problems, the present invention also provides a method for preparing a dual magnetic particle reagent, which is used to prepare the dual magnetic particle reagent described above, comprising the following steps:

[0010] Carboxyl magnetic particles were pre-treated and activated, then coupled with p-tau231 antibodies, blocked, washed, and stored;

[0011] Tosyl magnetic particles were pretreated, coupled with p-tau231 antibodies, and stored after multiple blocking and washing.

[0012] The carboxyl magnetic particles coated with the p-tau231 antibody and the Tosyl magnetic particles coated with the p-tau231 antibody are mixed in a ratio of 1:1-3 to obtain a double magnetic particle reagent.

[0013] The technical solution of the present invention establishes a chemiluminescence detection kit for phosphorylated Tau-231 protein suitable for the MAGICL6000 fully automatic chemiluminescence analyzer. The kit has high sensitivity, a wide linear range, and broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0015] Figure 1 This is a graph showing the calibration results of the carboxyl magnetic particle antibody, the Tosyl magnetic particle antibody, and the double magnetic particle antibody of the present invention;

[0016] Figure 2 TEM images of magnetic particles in different states of the present invention, including (a) original carboxyl magnetic particles; (b) carboxyl magnetic particles after coupling; (c) Tosyl magnetic particles; (d) Tosyl magnetic particles after coupling;

[0017] Figure 3 The DLS results of magnetic particles in different states of the present invention are shown in Figure 1, wherein A. original carboxyl magnetic particles; B. carboxyl magnetic particles after coupling; C. Tosyl magnetic particles; D. Tosyl magnetic particles after coupling;

[0018] Figure 4 This is a test result diagram of the detection ability of the reagent of the present invention. DETAILED DESCRIPTION

[0019] The present invention provides a chemiluminescent detection kit for phosphorylated Tau-231 protein, an antibody variable region and a preparation method thereof, aiming to design a detection kit with high sensitivity and a wide linear range.

[0020] Main experimental instruments and reagents:

[0021] A. Experimental instruments;

[0022] MAGICL6000 fully automatic chemiluminescence analyzer (GeneBio), digital display ordinary ultrasonic cleaning machine (Xinzhi Bio), etc.

[0023] B. Experimental reagents;

[0024] Tau antibody and p-tau231 antigen were purchased from Renduan Biotechnology, p-tau231 antibody was from Jilin University, bovine serum albumin was purchased from Roche, carboxyl magnetic particles and tosyl magnetic particles were purchased from Jidan Biotechnology, and acridinium ester was purchased from Shanghai Maxway.

[0025] The top block of the chemiluminescent detection kit for phosphorylated Tau-231 protein proposed by the present invention will be described in the following specific examples:

[0026] In the technical solution of this embodiment, Figure 1 As shown, a chemiluminescent detection kit for phosphorylated Tau-231 protein is provided, the kit comprising an AE-labeled tau antibody, a dual magnetic particle reagent and a p-tau231 calibrator; wherein the dual magnetic particle reagent comprises a p-tau231 antibody, the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 2.

[0027] Furthermore, the dual magnetic particle reagent includes:

[0028] Carboxyl magnetic particles coated with p-tau231 antibody and Tosyl magnetic particles coated with p-tau231 antibody.

[0029] Furthermore, the AE-labeled tau antibody is obtained by labeling the tau antibody with acridinium ester.

[0030] Furthermore, the CDR1 of the p-tau231 antibody light chain is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV;

[0031] The CDR1 of the p-tau231 antibody heavy chain is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

[0032] Example 1: A chemiluminescent detection kit for phosphorylated Tau-231 protein, comprising an AE-labeled tau antibody, a dual-magnetic particle reagent, and a p-tau231 calibrator; wherein the dual-magnetic particle reagent comprises a p-tau231 antibody, whose light chain variable region amino acid sequence is shown in SEQ ID NO: 1, and whose heavy chain variable region amino acid sequence is shown in SEQ ID NO: 2.

[0033] The amino acid sequence of the light chain variable region of the p-tau231 antibody is:

[0034] AQVLTQTPSSVSAAVGGTVTINCQASQS

[0035] VYNNNWLSWFQRKPGQPPKLLIHSVSTLYSGVSSQFKGSGSGTQFTLTIS

[0036] DVQCDDAATYYCLGDFDCSSADCGVFGGGTEVVVR.

[0037] The full chain is AQVLTQTPSSVSAAVGGTVTINCQASQS

[0038] VYNNNWLSWFQRKPGQPPKLLIHSVSTLYSGVSSQFKGSGSGTQFTLTIS

[0039] DVQCDDAATYYCLGDFDCSSADCGVFGGGTEVVVR

[0040] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC;

[0041] The amino acid sequence of the heavy chain variable region of the p-tau231 antibody is:

[0042] QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN

[0043] GMNWVRQAPGKGLEWIAYIKGGGNAYYASWAKGRFTISKSSTTVDLKITT<苟富贵,勿相忘。

[0044] PTTEDTATYFCATSGSDYWGPGTLVTVSS。

[0045] The full chain is QSLEESGGRLVTPGTPLTLTCTVSGIDLSRN

[0046] GMNWVRQAPGKGLEWIAYIKGGGNAYYASWAKGRFTISKSSTTVDLKITT

[0047] PTTEDTATYFCATSGSDYWGPGTLVTVSS

[0048] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTV

[0049] APSTCSKPTCP

[0050] PPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFKCKVHNKALPAPIEKTISKAR

[0051] It should be noted that there is an inappropriate sentence "苟富贵,勿相忘。" in the provided text which seems to be an incorrect addition. I have translated it as is while keeping this anomaly. If this is not intentional, please correct the original text for a more accurate translation.GQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK.

[0052] The CDR1 of the p-tau231 antibody light chain is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV;

[0053] The CDR1 of the p-tau231 antibody heavy chain is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

[0054] 1. Preparation of AE-labeled tau antibody:

[0055] Labeling: tau antibody and acridinium ester at a molar ratio of 1:25, incubated in a 37°C water bath for 1 hour;

[0056] Blocking: add 5M Tris and incubate in a 37°C water bath for 30 minutes;

[0057] Desalting and storage: Pass through a desalting plate, collect the filtrate, add phosphate and glycerol, and then divide into aliquots and store at -20℃.

[0058] 2. Preparation of calibrators:

[0059] The p-tau231 antigen was prepared into seven calibrators with concentrations of S0 (0 pg / mL), S1 (5 pg / mL), S2 (25 pg / mL), S3 (50 pg / mL), S4 (125 pg / mL), S5 (250 pg / mL), and S6 (500 pg / mL) using PBS containing 5% BSA as the calibrator diluent and stored at 2-8°C.

[0060] 3. Preparation of double magnetic particle antibodies:

[0061] (1) Preparation of carboxyl magnetic particle antibodies:

[0062] Pretreatment: 1.5 μm carboxyl magnetic microparticles were dispersed in 50 mM / L MES to a final concentration of 1% w / v. 5 mg / L 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added and incubated at 37°C, 220 rpm, for 30 minutes. After magnetic separation, the activated carboxyl magnetic microparticles were resuspended in MES.

[0063] Coupling: p-tau231 antibody was diluted with MES to a final concentration of 0.02% w / v and slowly added to the carboxyl magnetic microparticles. Coupling was performed at 37°C, 220 rpm, for 3 hours. After magnetic separation, the particles were resuspended in 2-morpholinoethanesulfonic acid (MES).

[0064] Blocking: add 40 μL of 10% bovine serum albumin solution and block at 37°C, 220 rpm, for 90 minutes;

[0065] Washing: Magnetic separation was performed and the supernatant was discarded. The suspension was ultrasonicated in 4 mL of 20 mm Tris containing 0.05% Tween 20 in a water bath for 5 min, and repeated 3 times.

[0066] Storage: Finally, the carboxyl magnetic particles coated with p-tau231 antibody were distributed in 50 mmol MES buffer.

[0067] (2) Preparation of Tosyl magnetic particle antibodies:

[0068] Pretreatment: 2 μm Tosyl magnetic particles were dispersed in 100 mM sodium tetraborate (Na2B4O7) to a final concentration of 1% w / v;

[0069] Coupling: Dilute the p-tau231 antibody with Na2B4O7 to a final concentration of 0.02% w / v and slowly add it to the carboxyl magnetic microparticles. Coupling is carried out at 37°C and 220 rpm for 8 hours. After magnetic separation, the Na2B4O7 is resuspended and dispersed.

[0070] Blocking: add 1 mL of 10% bovine serum albumin solution and block at 37°C, 220 rpm for 8 hours;

[0071] Washing: Discard the supernatant after magnetic separation, wash with 2 mL of 20 mM Tris containing 0.05% Tween 20, and repeat the blocking and washing steps 3 times;

[0072] Storage: Finally, the carboxyl magnetic particles coated with p-tau231 antibody were distributed in 50 mM MES buffer.

[0073] (3) Preparation of dual magnetic particle antibodies:

[0074] The carboxyl magnetic particles coated with the p-tau231 antibody and the Tosyl magnetic particles coated with the p-tau231 antibody were mixed in a ratio of 1:1 to obtain a double magnetic particle antibody.

[0075] To ensure the successful preparation of magnetic particle antibodies, dynamic light scattering and Zeta potential analysis are used to reveal the physical properties of magnetic particle antibodies; transmission electron microscopy can show the dispersion state of magnetic particles before and after immunization.

[0076] Figure 2 The TEM images shown show that the original magnetic particles are in good shape; after being coupled with the antibody, the magnetic particles remain stable, evenly dispersed, and are wrapped by a polymer shell, confirming that the antibody is successfully labeled.

[0077] Figure 3 Particle size measurements show that the carboxyl magnetic particles increased from 1546 nm to 2621 nm, and the tosyl magnetic particles increased from 2147 nm to 2599 nm, indicating successful antibody conjugation. The Polymer Dispersion Index (PDI) was below 0.100, indicating that all solutions were stable and monodisperse, which helps reduce interparticle interactions and improve stability.

[0078] 4. Accuracy analysis of the kit in Example 1:

[0079] Precision is a key indicator of the effectiveness of in vitro diagnostic reagents. Using the same batch of reagents, we performed 20 replicates of samples at both medium and high concentrations, and calculated the mean, standard deviation, and coefficient of variation. As shown in Table 1 below, the intra-batch precision of this kit is less than 7%.

[0080] Table 1 Precision analysis:

[0081]

[0082] 5. Accuracy analysis of the kit in Example 1:

[0083] Physiological saline and p-tau231 standards of different concentrations were added to the homemade samples to prepare basic samples and recovery samples. Each sample was tested three times, and the recovery rate was calculated. The results are shown in Table 2. The recovery rates of the three samples were 92.00%, 107.00%, and 102.5%, respectively, with an average recovery rate of 100.5%. The p-tau231 recovery rates of the three experimental samples were all between 90% and 110%, which met clinical requirements and proved that the detection accuracy of the reagent was good.

[0084] Table 2 Accuracy analysis:

[0085]

[0086] 6. Sensitivity analysis of the kit in Example 1:

[0087] The following terms are generally used to describe the detection capability or sensitivity of a reagent: Limit of blank (LOB), Limit of detection (LOD) and Limit of quantitation (LOQ). Their specific meanings are as follows:

[0088] LoB refers to the maximum value among the blank sample series results;

[0089] LoD refers to the lowest concentration of the analyte that can be detected by the method;

[0090] LoQ is the actual concentration at which the analyte can be reliably detected with an uncertainty that meets the laboratory's established goals.

[0091] Theoretically, the relationship between the blank limit, detection limit and quantification limit is LoB<LoD≤LoQ.

[0092] Referencing EP17-A2, "Evaluation of the Capability of Clinical Laboratory Measurement Procedures," published by the American Clinical Laboratory Standards Association, five blank samples were tested 20 times, yielding a total of 100 test results. A normal distribution analysis was performed. If the data conformed to a normal distribution, LoB = μB + 1.645σB. The LOD calculation method is similar to the LOB.

[0093] Five low-concentration samples were tested 20 times using the same reagent under the same conditions, and the coefficient of variation was calculated. The total error was set to 30%. Based on the coefficient of variation = 1 / 3 of the total error, the lowest detection concentration with a coefficient of variation ≤ 10% was defined as LoQ.

[0094] The results are shown in Table 3: The blank limits for all five samples were less than 3 pg / mL, resulting in a LoB of 3 pg / mL. The 100 test results conformed to a normal distribution, with a calculated overall standard deviation (σS) of 0.23, resulting in a limit of detection (LoD) of 3.4 pg / mL.

[0095] Table 3 Measurement results of blank samples:

[0096]

[0097] The same reagent was used to test 5 low-concentration samples (4 pg / mL, 5 pg / mL, 6 pg / mL, 7 pg / mL, and 8 pg / mL) 20 times. The 100 test results were in accordance with the normal distribution, and the coefficient of variation of each sample was calculated. Figure 4 As shown in the figure, the bar graph is the detection mean of samples with different concentrations, and the broken line is the coefficient of variation of the detection results of samples with different concentrations. When the sample concentration is ≥ 5 pg / mL, the coefficient of variation CV of the sample is less than 10%, which is in line with the clinical diagnostic reagent specifications, that is, the limit of quantification LoQ is 5 pg / mL.

[0098] 7. Linear analysis of the kit of Example 1:

[0099] A high-concentration sample near the upper limit of the linear range of 500 pg / mL and a low-concentration sample near the lower limit of the linear range were mixed into 8 dilution concentrations. The reagent was tested separately. Each dilution concentration was tested 3 times and the average value was taken. The deviation from the theoretical value and the linear correlation coefficient were calculated. The results are shown in Table 4, indicating that the kit has good linearity in the linear range of 5-500 pg / mL.

[0100] Table 4 Linear analysis

[0101]

[0102] Example 2: A method for preparing a dual magnetic particle reagent, comprising the following steps:

[0103] (1) Preparation of carboxyl magnetic particle antibodies:

[0104] Pretreatment: 1.5 μm carboxyl magnetic microparticles were dispersed in 50 mM / L MES to a final concentration of 1% w / v. 5 mg / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added and incubated at 37°C, 220 rpm, for 30 minutes. After magnetic separation, the activated carboxyl magnetic microparticles were resuspended in MES.

[0105] Coupling: p-tau231 antibody was diluted with MES to a final concentration of 0.02% w / v and slowly added to the carboxyl magnetic microparticles. Coupling was carried out at 37°C and 220 rpm for 3 hours. After magnetic separation, the particles were resuspended in MES.

[0106] Blocking: add 40 μL of 10% bovine serum albumin solution and block at 37°C, 220 rpm, for 90 minutes;

[0107] Washing: Magnetic separation was performed and the supernatant was discarded. The suspension was ultrasonicated in 4 mL of 20 mm Tris containing 0.05% Tween 20 in a water bath for 5 min, and repeated 3 times.

[0108] Storage: Finally, the carboxyl magnetic particles coated with p-tau231 antibody were distributed in 50 mmol MES buffer.

[0109] (2) Preparation of Tosyl magnetic particle antibodies:

[0110] Pretreatment: 2 μm Tosyl magnetic particles were dispersed in 100 mM Na2B4O7 to a final concentration of 1% w / v;

[0111] Coupling: p-tau231 antibody was diluted with Na2B4O7 to a final concentration of 0.02% w / v and slowly added to the carboxyl magnetic microparticles. Coupling was carried out at 37°C and 220 rpm for 8 hours. After magnetic separation, the Na2B4O7 was resuspended and dispersed.

[0112] Blocking: add 1 mL of 10% bovine serum albumin solution and block at 37°C, 220 rpm for 8 hours;

[0113] Washing: After magnetic separation, discard the supernatant and wash with 2 mL of 20 mM Tris containing 0.05% Tween 20. Repeat the blocking and washing steps 3 times;

[0114] Storage: Finally, the carboxyl magnetic particles coated with p-tau231 antibody were distributed in 50 mM MES buffer.

[0115] (3) Preparation of dual magnetic particle antibodies:

[0116] The carboxyl magnetic particles coated with the p-tau231 antibody and the Tosyl magnetic particles coated with the p-tau231 antibody were mixed in a ratio of 1:1 to obtain a double magnetic particle antibody.

[0117] Example 3: An antibody variable region for use in the p-tau231 antibody in the chemiluminescent detection kit for phosphorylated Tau-231 protein as described in Example 1, wherein the CDR1 of the p-tau231 antibody light chain is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV;

[0118] The CDR1 of the p-tau231 antibody heavy chain is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

[0119] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A chemiluminescent detection kit for phosphorylated Tau-231 protein, characterized in that: The kit includes an AE-labeled tau antibody, a dual-magnetic particle reagent, and a p-tau231 calibrator; wherein the dual-magnetic particle reagent includes a p-tau231 antibody, whose light chain variable region amino acid sequence is shown in SEQ ID NO: 1, and whose heavy chain variable region amino acid sequence is shown in SEQ ID NO: 2; the CDR1 of the p-tau231 antibody light chain is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV; The CDR1 of the p-tau231 antibody heavy chain is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

2. The chemiluminescent detection kit for phosphorylated Tau-231 protein according to claim 1, characterized in that: The dual magnetic particle reagent includes: carboxyl magnetic particles coated with p-tau231 antibodies and Tosyl magnetic particles coated with p-tau231 antibodies, which are mixed in a ratio of 1:1-3.

3. The chemiluminescent detection kit for phosphorylated Tau-231 protein according to claim 1, characterized in that: The AE-labeled tau antibody is obtained by labeling the tau antibody with acridinium ester.

4. An antibody variable region for use in the p-tau231 antibody in the chemiluminescent detection kit for phosphorylated Tau-231 protein according to any one of claims 1 to 3, characterized in that: The CDR1 of the p-tau231 antibody light chain is QSVYNNNW, CDR2 is SVS, and CDR3 is LGDFDCSSADCGV; The CDR1 of the p-tau231 antibody heavy chain is GIDLSRNG, CDR2 is IKGGGNA, and CDR3 is ATSGSDY.

5. A method for preparing a double magnetic particle reagent, for preparing the double magnetic particle reagent according to any one of claims 1 to 3, characterized in that: The following steps are involved: Carboxyl magnetic particles were pre-treated and activated, then coupled with p-tau231 antibodies, blocked, washed, and stored; Tosyl magnetic particles were pretreated, coupled with p-tau231 antibodies, and stored after multiple blocking and washing. The carboxyl magnetic particles coated with the p-tau231 antibody and the Tosyl magnetic particles coated with the p-tau231 antibody are mixed in a ratio of 1:1-3 to obtain a double magnetic particle reagent.

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