A phosphated tau181 protein magnetic microparticle chemiluminescence detection kit and its use method and application

Abstract

The application belongs to the field of in vitro diagnostic reagents, and provides a phospho-Tau181 protein magnetic microparticle chemiluminescence detection kit, a use method and application thereof. The kit comprises: magnetic beads coated with an anti-phospho-Tau181 protein capture antibody, a chemiluminescence agent-labeled anti-phospho-Tau181 protein detection antibody, a diluent, and a calibrator and a quality control containing a P-Tau181 recombinant protein. The kit provided by the application has a detection sensitivity of 0.6 pg / mL, a cross-reaction rate of less than 0.5%, a single detection time of less than 40 minutes, and can be stably stored for 13 months under the condition of 2-8 DEG C, and can be used for early diagnosis and disease monitoring of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and in particular to a phosphorylated Tau181 protein magnetic microparticle chemiluminescence detection kit and its usage and application. Background Technology

[0002] Alzheimer's disease is a neurodegenerative disease characterized by progressive cognitive impairment and memory loss. Its pathological features are primarily senile plaques formed by the deposition of β-amyloid protein in the brain and neurofibrillary tangles formed by the aggregation of hyperphosphorylated Tau protein. With the increasing aging of the global population, the incidence of Alzheimer's disease is rising annually. It is estimated that there are approximately 50 million dementia patients worldwide, with Alzheimer's disease accounting for more than 60%, placing a heavy medical burden on society and families. In the pathological process of Alzheimer's disease, abnormal hyperphosphorylation of Tau protein is one of the key molecular events. This protein is mainly distributed in neuronal axons and has the function of maintaining microtubule stability. Under normal physiological conditions, the phosphorylation level of Tau protein is strictly regulated; however, under pathological conditions, Tau protein undergoes hyperphosphorylation at specific amino acid sites, causing it to dissociate from microtubules and aggregate to form neurofibrillary tangles, thereby leading to neuronal dysfunction and death. Phosphorylation of threonine at position 181 of the Tau protein has been confirmed as an important biomarker for the early diagnosis and monitoring of Alzheimer's disease. Studies have shown that the levels of this marker in cerebrospinal fluid and blood are positively correlated with the pathological burden of Tau in the brain, and its elevation occurs earlier than the appearance of clinical symptoms, effectively distinguishing Alzheimer's disease from other types of dementia. Therefore, the need for accurate and rapid detection of phosphorylated Tau181 protein is increasingly urgent, and related detection technologies have become a research hotspot in the field of biomedical testing.

[0003] Currently, the most commonly used technique for detecting phosphorylated Tau181 protein is enzyme-linked immunosorbent assay (ELISA). This technique typically uses an ELISA plate coated with an antibody that captures phosphorylated Tau181 protein, along with a biotin-labeled detection antibody, horseradish peroxidase-labeled streptavidin, and a chromogenic substrate. The antigen and antibody specifically bind to form a sandwich complex, which is then catalyzed by the enzyme to develop color, and the absorbance value is measured to calculate the sample concentration. However, conventional enzyme-linked immunosorbent assay (ELISA) kits have many limitations: their detection limits are typically 10 to 20 picograms per milliliter, making it difficult to detect low concentrations of phosphorylated Tau181 protein in early-stage patient samples, thus limiting their application in early disease diagnosis; existing capture and detection antibodies have limited specific binding capacity to target proteins and are prone to cross-reaction with non-phosphorylated Tau proteins, other phosphorylated site isoforms, and homologous proteins, resulting in a high false-positive rate; the entire detection process involves multiple incubation, washing, and color development steps, taking a total of four to six hours, which cannot meet the needs of rapid detection; in addition, enzyme markers and chromogenic substrates are temperature-sensitive, with a shelf life of only 6 months under normal refrigeration conditions, and temperature fluctuations during transportation can easily lead to a decrease in activity, resulting in a coefficient of variation of test results that is often greater than 10%, poor stability, and cumbersome operation that is prone to human error.

[0004] Meanwhile, single-molecule immunoassay, an ultrasensitive detection method based on microfluidic chips and single-molecule counting principles, has also been applied to the detection of phosphorylated Tau181 protein. This technology utilizes magnetic beads coated with capture antibodies to capture the target protein, forming an immune complex with biotin-labeled detection antibodies and enzyme-labeled streptavidin. Individual magnetic beads are then separated using a microfluidic chip, and quantification is achieved by detecting the fluorescence signal. Although this technology theoretically has high sensitivity, it still faces significant challenges in practical applications. Currently available antibody products still suffer from insufficient specificity, and cross-reactivity leads to a high false-positive rate. The detection process, including sample incubation, antibody incubation, enzyme-catalyzed luminescence, and fluorescence imaging, still takes four to five hours in total and relies on dedicated microfluidic chips and imaging equipment, making it complex and costly. Furthermore, the galactosidase used in the detection system exhibits significant stability defects with the fluorescent substrate, resulting in catalytic efficiency far lower than conventional horseradish peroxidase. To ensure signal intensity, the micro-unit size needs to be reduced, making it difficult to distinguish the signal from the background, and the repeatability of the detection results remains unsatisfactory. More importantly, since the components in the detection system are premixed, strict time control is required for the magnetic bead loading step into the micropore in order to reduce background signal. This places high demands on the precision of the equipment and the skill level of the operator.

[0005] In summary, existing technologies have significant shortcomings in terms of detection sensitivity, specificity, detection speed, reagent stability, and ease of operation, making it difficult to meet the clinical needs for accurate, rapid, and stable detection of phosphorylated Tau181 protein, and also unsuitable for clinical applications in primary healthcare institutions and emergency settings.

[0006] Therefore, developing a phosphorylated Tau181 protein detection kit that is highly sensitive, highly specific, rapid, stable, and easy to operate is of great clinical application value and social significance for the early diagnosis, disease monitoring, and efficacy evaluation of Alzheimer's disease. Summary of the Invention

[0007] The first aspect of the present invention provides a chemiluminescent detection kit for phosphorylated Tau181 protein magnetic microparticles, the kit comprising:

[0008] Reagent 1: Magnetic beads coated with antiphosphorylated Tau181 protein capture antibody;

[0009] Reagent 2: Chemiluminescent labeled antibody for detecting phosphorylated Tau181 protein;

[0010] Reagent 3: Diluent;

[0011] The components of the diluent include: buffer solution, bovine serum albumin, sodium chloride, Tween-20, PEG, Proclin 300, protein stabilizer, Biolipidure, and SCO2.

[0012] Specifically, the capture antibody and detection antibody are capable of binding to different sites on the phosphorylated Tau181 protein.

[0013] More specifically, based on the antibody's specific characteristics, it can be classified as one or both of polyclonal and monoclonal antibodies. Based on its source, it can be one or more of mouse-derived antibodies, rabbit-derived antibodies, and sheep-derived antibodies, with rabbit-derived antibodies being the most preferred.

[0014] Optionally, the phosphorylated amino acid sequence at position 181 of the capture antibody synthetic peptide 2# is PPAPK(pT)PP (SEQ ID NO: 1); the amino acid sequence without phosphorylated site of the detection antibody synthetic peptide 9# is PPAPKTPP (SEQ ID NO: 2).

[0015] Further optionally, the capture antibody includes a heavy chain CDR region as shown in SEQ ID NO. 3-5 and a light chain CDR region as shown in SEQ ID NO. 6-8;

[0016] The detection antibody includes a heavy chain CDR region as shown in SEQ ID NO. 9-11 and a light chain CDR region as shown in SEQ ID NO. 12-14;

[0017] Capture antibodies:

[0018] CDR-H1: HQDQEGDTD(SEQ ID NO: 3);

[0019] CDR-H2: EPKKVA (SEQ ID NO: 4);

[0020] CDR-H3: EPPSSPKYVSSVTSR (SEQ ID NO: 5);

[0021] CDR-L1: QPSSPA (SEQ ID NO: 6);

[0022] CDR-L2: RSA (SEQ ID NO: 7);

[0023] CDR-L3: QIVYKPVDLSKVTS (SEQ ID NO: 8).

[0024] Antibody detection:

[0025] CDR-H1:GTTAEEAGI(SEQ ID NO:9);

[0026] CDR-H2: KARMVS (SEQ ID NO: 10);

[0027] CDR-H3: SIPGFPAEGAIPLPVDF (SEQ ID NO: 11);

[0028] CDR-L1: SVQIVY(SEQ ID NO: 12);

[0029] CDR-L2: PES (SEQ ID NO: 13);

[0030] CDR-L3: KPQPSSPAVTSRTGSSG (SEQ ID NO: 14).

[0031] In certain embodiments of the present invention, the heavy chain variable region sequence of the capture antibody (P-Tau181-C12) is shown in SEQ ID NO.15; the light chain variable region sequence of the capture antibody is shown in SEQ ID NO.16.

[0032] P-Tau181-C12 heavy chain variable region:

[0033] EEAGIGDTPSLEDEAAGHVTQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRHAPELLKHQLLGTLTCTVSGIDLSTYAMGWVRQAP (SEQ ID NO: 15);

[0034] P-Tau181-C12 light chain variable region:

[0035] DRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDTQTASPVSAAVGGTDIVMTQ(SEQ ID NO: 16);

[0036] In certain specific embodiments of the present invention, the heavy chain variable region sequence of the detection antibody (P-Tau181-D8) is shown in SEQ ID NO.17; and the light chain variable region sequence of the capture antibody is shown in SEQ ID NO.18.

[0037] P-Tau181-D8 Heavy Chain Variable Region:

[0038] GSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 17);

[0039] P-Tau181-D8 Light Chain Variable Region:

[0040] AKGQDAPLEFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRSSAK (SEQ ID NO: 18).

[0041] Specifically, the magnetic beads have a particle size of 0.5-5 μm and a working concentration of 0.05-1 mg / mL.

[0042] Preferably, the diameter of the magnetic bead is 1-3 μm.

[0043] More preferably, the diameter of the magnetic bead is 1.5-3 μm.

[0044] Preferably, the working concentration of the magnetic beads is 0.1 mg / mL to 1 mg / mL.

[0045] More preferably, the working concentration of the magnetic beads is 0.1 mg / mL to 0.5 mg / mL.

[0046] Specifically, the chemiluminescent agent includes at least one of acridine ester, alkaline phosphatase, horseradish peroxidase, luminol, isoluminol, or terpyridine ruthenium.

[0047] More specifically, the working concentration of the chemiluminescent agent is 0.1 μg / mL to 5 μg / mL.

[0048] Preferably, the working concentration of the chemiluminescent agent is 0.5 μg / mL to 5 mg / mL.

[0049] More preferably, the working concentration of the chemiluminescent agent is 0.5 μg / mL to 2 mg / mL.

[0050] Specifically, the diluent comprises: bovine serum albumin: 0.25-3 wt%; sodium chloride: 0.1-0.9 wt%; Tween-20: 0.05-0.1 wt%; PEG: 0.25-3 wt%; Proclin 300: 0.05-0.2 wt%; protein stabilizer: Biolipidure: 0.03-0.08 wt%; SCO2: 0.05-0.15 wt%.

[0051] Specifically, the kit also includes calibrators and quality control samples.

[0052] More specifically, the calibrators and quality control samples contain 0-100 pg / mL P-Tau181 recombinant protein, phosphate matrix; bovine serum albumin: 0.25-3 wt%; sodium chloride: 0.1-0.9 wt%; Tween-20: 0.05-0.1 wt%; PEG: 0.25-3 wt%; Proclin 300: 0.05-0.2 wt%; Biolipidure: 0.03-0.08 wt%; SCO2: 0.05-0.15 wt%.

[0053] A second aspect of the present invention provides a method for detecting phosphorylated Tau181 protein, characterized in that the method uses any of the above-described kits.

[0054] Specifically, the method includes the following steps:

[0055] S1. Mix the sample to be tested with reagent 1 and reagent 3, and incubate for 15-20 minutes;

[0056] S2. After cleaning, add reagent 2 and incubate for 3-5 minutes;

[0057] S3. After cleaning, add excitation solution and pre-excitation solution, and detect the chemiluminescence signal;

[0058] S4. Calculate the concentration of phosphorylated Tau181 protein in the sample.

[0059] More specifically, the sample to be tested includes one or more of cerebrospinal fluid, blood, serum, and plasma.

[0060] A third aspect of the invention provides the use of a kit in detecting diseases associated with phosphorylated Tau181 protein.

[0061] Specifically, the diseases include Alzheimer's disease, mild cognitive impairment, frontotemporal dementia, Lewy body dementia, vascular dementia, Parkinson's disease dementia, and Down syndrome.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] 1. Extremely high detection sensitivity: The detection limit is reduced to 0.6 pg / mL, which can accurately detect low concentrations of phosphorylated Tau181 protein in early patient samples.

[0064] 2. High specificity: The cross-reactivity rate is less than 0.5%, far lower than the 5%-8% of existing technologies, which can effectively avoid interference from non-phosphorylated Tau protein and homologous proteins and reduce the false positive rate.

[0065] 3. Fast testing speed: The total testing time is only 40 minutes, which is 6-9 times more efficient than the existing technology of 4-6 hours, meeting the needs of rapid batch testing.

[0066] 4. Good stability: The kit has a shelf life of up to 13 months when stored at 4℃, with a relative deviation of less than ±15% and repeatability of <5%.

[0067] 5. Simple operation: Reduces incubation steps and time; washing can be automated by a plate washing machine, minimizing human error and ensuring high accuracy of test results. Attached Figure Description

[0068] Figure 1 The detection flowchart of the chemiluminescence reagent kit provided by the present invention.

[0069] Figure 2 The standard curve diagram of the reagent kit provided by this invention.

[0070] Figure 3 The correlation between the chemiluminescence reagent kit provided by this invention and the results of clinical sample detection by single-molecule immunoassay. Detailed Implementation

[0071] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0072] Example 1

[0073] Acridinium ester (Xindesheng), paramagnetic beads (JSR), P-Tau181-C12, P-Tau181-D8, P-Tau181 recombinant protein (Ebosheng), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (Thermo Fisher Scientific), N-hydroxysuccinimide (NHS) (Thermo Fisher Scientific), desalting column (Thermo Fisher Scientific).

[0074] Magnetic bead coupling solution (50mM MES, pH=6.0), magnetic bead washing solution (PBST solution), acridine ester coupling solution (PBS), diluent (phosphate matrix; bovine serum albumin (BSA): 2wt%; sodium chloride: 0.5wt%; Tween-20: 0.1wt%; PEG: 0.5wt%; Proclin 300 (preservative): 0.1wt%; protein stabilizer: 0.05% Biolipidure, 0.1% SCO2 dissolved in 50mM PBS, pH=7.4), magnetic bead blocking solution (2% BSA), acridine ester termination solution (0.9% NaCl, 3% glycine dissolved in 50mM PBS, pH=7.4).

[0075] 1. Preparation of Reagent 1

[0076] (1) Take 20 μL of 100 mg / mL carboxyl magnetic beads (purchased from JSR, with a diameter of 3 μm), wash 3 times with magnetic bead coupling solution, and remove the magnetic bead coupling solution.

[0077] (2) Weigh a certain amount of EDC and NHS, dissolve them to 20 mg / ml, add them to the solution in (1), activate them on a constant temperature shaker at 25°C for 30 min, after activation, wash them 3 times with magnetic bead coupling solution, remove the magnetic bead coupling solution, and add 500 μL of magnetic bead coupling solution to resuspend them.

[0078] (3) Take 20 μg of P-Tau181-C12 capture antibody and add it to the magnetic beads in (2). Mix well and incubate at 25°C in a constant temperature shaker for 2 hours at a speed of 1000 rpm / min.

[0079] (4) After incubation, add 20mM PBS buffer for washing, mix well on a vortex mixer and place on a magnetic rack for magnetic separation, and wash 3 times with PBST.

[0080] (5) Add 100 μL of 10 mg / ml blocking solution (2% BSA) to the magnetic beads, incubate on a constant temperature shaker for 1 h at a speed of 1000 rpm / min, and remove the supernatant after magnetic separation.

[0081] (6) Add magnetic bead preservation solution, with a magnetic bead storage concentration of 2 mg / mL, and store at 4℃. This is reagent 1.

[0082] 2. Preparation of Reagent 2

[0083] (1) Take 60 μg of P-Tau181-D8 detection antibody, add acridine ester conjugate, and dilute the antibody concentration to 0.3 mg / mL. Then calculate the amount of acridine ester to be added based on the amount of antibody, so that the molar ratio of antibody to acridine ester is (1:20). Add 5.33 μL of 0.5 M acridine ester to the antibody, and incubate in the dark on a constant temperature shaker for 2 h at a speed of 1000 rpm / min.

[0084] (2) After incubation, add 30 μL of acridine ester stop solution and incubate in the dark on a constant temperature shaker for 30 min at a speed of 1000 rpm / min.

[0085] (3) Desalting column pretreatment: Place the desalting column in a high-speed centrifuge and centrifuge at 4000 rpm for 1 min to remove the liquid. Then add 400 μL PBS and centrifuge at 4000 rpm for 1 min. Repeat twice.

[0086] (4) After the termination, add the reagent to the desalting column after the pretreatment to remove excess acridine ester, at 4000 rpm for 2 min.

[0087] (5) Add the preservation solution. The storage concentration of acridine ester is 150 μg / mL. Store at 4°C protected from light.

[0088] 3. Preparation of Reagent 3:

[0089] Phosphate matrix; Bovine serum albumin (BSA): 2wt%, sodium chloride 0.5wt%, Tween-20 0.1wt%, PEG 0.5wt%, Proclin 300 (preservative) 0.1wt%, protein stabilizers are 0.05% Biolipidure and 0.1% SCO2.

[0090] 4. Preparation of calibrators and quality control materials

[0091] The preparation method of P-Tau181 calibrators and quality control samples is as follows: using a phosphate buffer containing phosphate matrix, bovine serum albumin (BSA) 2wt%, sodium chloride 0.5wt%, Tween-20 0.1wt%, PEG 0.5wt%, Proclin 300 (preservative) 0.1wt%, and trehalose 2wt%, P-Tau181 antigen purchased from Ibrahim was prepared into solutions of 0, 5, and 100 pg / mL to obtain calibrators 0, 1, and 2 respectively; solutions of 10 and 50 pg / mL were prepared to obtain quality control samples Q1 and Q2 respectively.

[0092] 4. Detection method using a chemiluminescence detection kit for phosphorylated Tau181 protein magnetic microparticles.

[0093] The reaction procedure for the phosphorylated Tau181 protein magnetic microparticle chemiluminescence detection kit is as follows: Figure 1 As shown. Sample volume: 50 μL; R1 reagent: 40 μL; R2 reagent: 100 μL; R3 reagent: 50 μL; Reaction time: 37℃, 20 min (first incubation) + 5 min (second incubation).

[0094] The prepared P-Tau181-C12 capture antibody-coated magnetic bead component (working concentration 0.2 mg / mL), the P-Tau181-D8 detection antibody-labeled acrid ester component (working concentration 0.5 μg / mL), and the dilution solution were placed in reagent bottles. Calibrators 0, 1, and 2 and quality control samples Q1 and Q2 were placed in the reagent rack. The reagent bottles and reagent rack were placed in the fully automated chemiluminescence analyzer. The instrument automatically aspirates the sample, reagent 1, and reagent 3, mixes and incubates at 37°C for 15 min, washes 3 times, adds reagent 2, mixes and incubates at 37°C for 5 min, washes 3 times, and forms an antibody-antigen-antibody complex. This complex emits light under the action of the excitation and pre-excitation solutions, and is detected by the fully automated chemiluminescence immunoassay analyzer to obtain relative luminescent units (RLUs). The relative luminescent units are proportional to the content of the analyte P-Tau181.

[0095] Example 2

[0096] The relative luminescence intensities (RLUs) detected by the kit in a fully automated chemiluminescence analyzer are shown in Table 1. Based on the values ​​in Table 1, a fitting was performed to obtain... Figure 2 The standard curve of phosphorylated Tau181 protein concentration versus luminescence value is shown.

[0097] Table 1. Calibration results of the P-Tau181 reagent kit

[0098]

[0099] Comparative Example 1

[0100] The magnetic beads had a particle size of 1 μm and were purchased from JSR. Except for the different particle size, the other conditions were the same as in Example 1.

[0101] Comparative Example 2

[0102] The magnetic beads had a particle size of 1.5 μm and were purchased from JSR. Except for the different particle size, the other conditions were the same as in Example 1.

[0103] Comparative Example 3

[0104] The magnetic beads had a particle size of 2.7 μm and were purchased from JSR. Except for the different particle size, the other conditions were the same as in Example 1.

[0105] Comparative Example 4

[0106] The magnetic beads had a particle size of 4.5 μm and were purchased from JSR. Except for the different particle size, the other conditions were the same as in Example 1.

[0107] Comparative Example 5

[0108] The magnetic beads had a particle size of 5 μm and were purchased from JSR. Except for the different particle size, the other conditions were the same as in Example 1.

[0109] Comparative Example 6

[0110] Twenty monoclonal capture antibodies (P-Tau181-C1-20) were synthesized according to synthetic peptide 2# and paired with P-Tau181-D8 respectively; other conditions were the same as in Example 1.

[0111] Comparative Example 7

[0112] Twenty monoclonal capture antibodies (P-Tau181-D1-20) were synthesized according to synthetic peptide 9# and paired with P-Tau181-C12 respectively; other conditions were the same as in Example 1.

[0113] Comparative Example 8

[0114] The diluent was a Tris matrix, and other conditions were the same as in Example 1.

[0115] Comparative Example 9

[0116] The diluent was MES matrix, and other conditions were the same as in Example 1.

[0117] Comparative Example 10

[0118] The diluent was a MOPES matrix, and other conditions were the same as in Example 1.

[0119] Comparative Example 11

[0120] The diluent was HEPES matrix, and other conditions were the same as in Example 1.

[0121] Comparative Example 12

[0122] The diluent was a glycine matrix, and other conditions were the same as in Example 1.

[0123] The main difference between Comparative Examples 1-5 and Example 1 lies in the magnetic bead size. The sensitivity and coefficient of variation (CV) values ​​(%) of Example 1 and Comparative Examples 1-5 are shown in Table 2.

[0124] Table 2 Sensitivity and CV values ​​of Example 1 and Comparative Examples 1-5

[0125]

[0126] The main difference between Comparative Examples 8-12 and Example 1 lies in the magnetic bead size. The sensitivity and coefficient of variation (CV) values ​​(%) of Example 1 and Comparative Examples 8-12 are shown in Table 3.

[0127] Table 3. Sensitivity and CV values ​​of Example 1 and Comparative Examples 8-12

[0128]

[0129] The main difference between Comparative Example 6 and Example 1 lies in the pairing of different capture and detection antibodies P-Tau181-D8 to test the specificity of Example 1 and Comparative Example 6, as shown in Table 4.

[0130] Table 4. Specificity of Example 1 and Comparative Example 6

[0131]

[0132] The main difference between Comparative Example 7 and Example 1 lies in the pairing of different detection antibodies with the capture antibody P-Tau181-C12 to test the specificity of Example 1 and Comparative Example 7, as shown in Table 5.

[0133] Table 5. Specificity of Example 1 and Comparative Example 7

[0134]

[0135] Example 1: Reagent Kit Sensitivity Analysis

[0136] The following terms are generally used to describe the detection capability of a reagent, i.e., sensitivity: Limit of blank (LOB) and Limit of detection (LOD), and their specific meanings are as follows:

[0137] LB refers to the maximum value in the blank sample series results;

[0138] LoD refers to the lowest concentration of the analyte that can be detected by the method.

[0139] Theoretically, the relationship between the limit of blank, limit of detection, and limit of quantitation is LoB < LoD. Referring to EP17-A2, "Assessment of the Detection Capability of Clinical Laboratory Measurement Procedures," published by the Clinical Laboratory Standards Institute (CLSI), 5 blank samples were repeatedly tested 20 times, yielding a total of 100 test results. Normal distribution analysis was performed. If the data conforms to a normal distribution, then LoB = μB + 1.645σB. The calculation method for LOD is similar to that for LOB.

[0140] Five samples were tested 20 times each, and the blank limit for all five samples was less than 0.4 pg / mL, with a LOB of 0.4 pg / mL. The 100 test results followed a normal distribution, and the total standard deviation (σS) was calculated to be 0.088, resulting in a detection limit (LoD) of 0.6 pg / mL. The test results are shown in Table 6.

[0141] Table 6. Test Results of Blank Samples

[0142]

[0143] Example 2: Specificity analysis of the reagent kit

[0144] Specificity analysis of the phosphorylated Tau181 kit was performed by detecting non-phosphorylated Tau protein, phosphorylated Tau217 protein, non-phosphorylated Tau181 protein, and Aβ1-42 protein (with an interferant concentration of 1000 pg / mL). The cross-reactivity rate was calculated and was within ±5%. The results are shown in Table 7.

[0145] Table 7. Specificity and Cross-reactivity of the P-Tau181 Reagent Kit

[0146]

[0147] Example 3: Detection of clinical samples using the P-Tau181 magnetic microparticle chemiluminescence reagent kit.

[0148] Plasma gradient samples with predefined values ​​were procured from external sources, and the concentration of P-Tau181 protein in the samples was determined using the Simoa single-molecule immunoassay method. The specific concentrations are shown in Table 8. The samples determined by the single-molecule immunoassay method were then used to detect the P-Tau181 protein concentration using the kit from Example 1 on a fully automated chemiluminescence analyzer. The results are shown in Table 4.

[0149] Table 8. Clinical Sample Concentration and Chemiluminescence Detection Results

[0150]

[0151] A scatter plot of P-Tau181 concentration for each sample was drawn, with the concentration detected by single-molecule immunoassay on the x-axis and the concentration detected by chemiluminescence on the y-axis. Figure 3 The correlation coefficients of P-Tau 181 concentration values ​​between the two detection methods were calculated. The correlation coefficients between the two detection methods were R=0.9841 and R2=0.9684, indicating a strong correlation between the detection results and the Simoa single-molecule immunoassay.

[0152] Example 4: Precision analysis of the reagent kit

[0153] Precision is a key indicator of the effectiveness of in vitro diagnostic reagents, including repeatability and inter-batch variation. Repeatability involves using the same batch of reagents to simultaneously measure samples at medium and high concentrations, repeating the measurements 10 times, and calculating the mean, standard deviation, and coefficient of variation. The intra-batch repeatability of this kit is <5%, as shown in Table 9 below; the inter-batch variation among the three batches of reagents is <5%, as shown in Table 10 below.

[0154] Table 9. Repeatability results of the phosphorylated Tau181 magnetic microparticle chemiluminescence reagent kit

[0155]

[0156] Table 10. Inter-batch variation results of the phosphorylated Tau181 magnetic microparticle chemiluminescence reagent kit

[0157]

[0158] Example 5: Stability analysis of the reagent kit

[0159] The reagent kit was placed in a medical refrigerator at 2-8℃. Unopened reagents were collected at 0, 1, 2, 3, 9, 12, and 13 months from the date of placement for testing. The relative deviations of the luminescence values ​​at 1, 2, 3, 9, 12, and 13 months compared to 0 months were calculated. The reagent kit was stably stored for 13 months with relative deviations within ±15%. The results are shown in Table 11.

[0160] Table 11. Stability results of the phosphorylated Tau181 magnetic microparticle chemiluminescence reagent kit

[0161]

[0162] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chemiluminescent detection kit for phosphorylated Tau181 protein magnetic microparticles, characterized in that, The kit includes: Reagent 1: Magnetic beads coated with antiphosphorylated Tau181 protein capture antibody; Reagent 2: Chemiluminescent labeled antibody for detecting phosphorylated Tau181 protein; Reagent 3: Diluent; The capture antibody includes a heavy chain CDR region as shown in SEQ ID NO. 3-5 and a light chain CDR region as shown in SEQ ID NO. 6-8; The detection antibody includes a heavy chain CDR region as shown in SEQ ID NO. 9-11 and a light chain CDR region as shown in SEQ ID NO. 12-14; The diluent includes: buffer solution, bovine serum albumin, sodium chloride, Tween-20, PEG, Proclin 300, protein stabilizer, Biolipidure, and SCO2.

2. The reagent kit according to claim 1, characterized in that, The heavy chain variable region sequence of the capture antibody is shown in SEQ ID NO.15; the light chain variable region of the capture antibody is shown in SEQ ID NO.

16.

3. The reagent kit according to claim 1, characterized in that, The heavy chain variable region of the detection antibody is shown in SEQ ID NO. 17, and the light chain variable region of the detection antibody is shown in SEQ ID NO.

18.

4. The reagent kit according to claim 1, characterized in that, The diluent comprises: bovine serum albumin: 0.25-3 wt%; sodium chloride: 0.1-0.9 wt%; Tween-20: 0.05-0.1 wt%; PEG: 0.25-3 wt%; Proclin 300: 0.05-0.2 wt%; Biolipidure: 0.03-0.08 wt%; SCO2: 0.05-0.15 wt%.

5. The reagent kit according to claim 1, characterized in that, The magnetic beads have a particle size of 0.5-4.5 μm and a working concentration of 0.05-1 mg / mL.

6. The reagent kit according to claim 1, characterized in that, The chemiluminescent agent includes at least one of acridine ester, alkaline phosphatase, horseradish peroxidase, luminol, isoluminol, or terpyridine ruthenium.

7. A method for detecting phosphorylated Tau181 protein, characterized in that, The method uses the kit described in any one of claims 1-6.

8. The method according to claim 7, characterized in that, The method includes the following steps: S1. Mix the sample to be tested with reagent 1 and reagent 3, and incubate for 15-20 minutes; S2. After cleaning, add reagent 2 and incubate for 3-5 minutes; S3. After cleaning, add excitation solution and pre-excitation solution, and detect the chemiluminescence signal; S4. Calculate the concentration of phosphorylated Tau181 protein in the sample.

9. The use of the kit according to any one of claims 1-6 in the detection of diseases related to phosphorylated Tau181 protein.

10. The application according to claim 9, characterized in that, The diseases mentioned include Alzheimer's disease, mild cognitive impairment, frontotemporal dementia, Lewy body dementia, vascular dementia, Parkinson's disease dementia, and Down syndrome.

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