Kit for combining markers P-tau181 and T-tau as well as preparation method and application of kit

Abstract

The invention relates to a kit for combining markers P-tau181 and T-tau as well as a preparation method and application of the kit, and relates to the technical field of immunodetection. The kit comprises a first reagent and a second reagent, the first reagent comprises a magnetic bead working solution and a magnetic microsphere coated with a Tau polyclonal antibody; and the second reagent comprises a labeled antibody diluent and an acridinium ester labeled anti-Tau antibody. According to the invention, high-specificity anti-P-tau181 and anti-T-tau antibodies are adopted, one-time sampling can be realized, and the values of P-tau181 and T-tau proteins in human serum, plasma or urine samples can be detected at the same time; after the kit is adopted for 8 minutes, a result can be obtained, the repeatability is within 5%, the lowest detection limit of P-tau181 is 0.96 pg / ml, and the lowest detection limit of T-tau is 1.08 pg / ml.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassays, and in particular to a kit for combining markers P-tau181 and T-tau, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD), also known as senile dementia, is a severe, primary, degenerative disease of the central nervous system characterized by cognitive impairment and memory loss. It is the most common type of dementia. Epidemiological studies show that the incidence of AD is positively correlated with age, with a 2-3% incidence in those aged 75-80 years and a 12% incidence in those aged 85-90 years. This demonstrates that AD poses a significant health challenge to the elderly. Recent studies have shown that aggregation caused by abnormal phosphorylation of tau protein is more strongly associated with AD than abnormal deposition of β-amyloid protein.

[0003] Tau is a multifunctional cytoskeletal protein, also known as a microtubule-associated protein. The tau gene is located on the long arm of chromosome 17. In normal individuals, six isoforms are expressed due to differences in the splicing of tau mRNA. Tau is a phosphate-containing protein, with two to three phosphate groups in the normal mature brain. Microtubules are composed of tubulin and microtubule-associated proteins, with tau being the most abundant microtubule-associated protein. The cellular function of tau in the normal brain is to bind to tubulin and promote its polymerization to form microtubules. By binding to formed microtubules, tau maintains microtubule stability, reduces tubulin dissociation, and induces microtubule bundling. By binding to tubulin, tau promotes microtubule assembly and stability, playing a crucial role in neurite formation, maintaining cytoskeletal integrity, and axonal transport. Under physiological conditions, Tau protein can be phosphorylated and modified. Abnormal phosphorylation is a key factor in the accumulation of Tau protein. Hyperphosphorylated Tau protein loses its ability to bind to microtubules and easily aggregates, forming neurofibrillary tangles (NFTs), which lead to cytoskeletal abnormalities and cell death. The microtubule system is a component of the neural cytoskeleton and plays a role in various cellular functions. However, Tau protein in the brains of AD patients is abnormally overphosphorylated, with each molecule containing 5 to 9 phosphate groups, and loses its normal biological function.

[0004] In recent years, research teams have discovered that plasma p-tau has a high sensitivity as a marker for diagnosing AD. The phospho-sites of p-tau protein are mostly located at amino acids 181 (p-tau181), 217 (p-tau217), or 231 (p-tau231) of the Tau protein. Clinical studies on neuropathology have found that the expression level of plasma p-tau protein is significantly correlated with the deposition of intracranial Aβ plaques and the degree of Tau neural tangles. Based on this association, the expression level of plasma p-tau protein can be used to distinguish whether a patient's brain has pathological changes of AD. More importantly, elevated expression levels of plasma p-tau protein are only observed in patients with AD. No abnormal expression is found in other Tau-related diseases, such as progressive supranuclear palsy, basal cortical degeneration, or Pick's disease, indicating that plasma p-tau protein has a high specificity for the diagnosis of AD.

[0005] In large-scale clinical trials, plasma p-tau has been shown to accurately distinguish AD dementia from non-AD neurodegenerative disease-related dementia with high accuracy. Plasma p-tau levels in patients with AD dementia are approximately 250% to 600% higher than in patients with non-AD neurodegenerative diseases. Plasma p-tau217 levels are most significantly elevated in AD patients, and their diagnostic efficacy for AD is approaching that of CSF markers and tau PET. Furthermore, in patients with mild cognitive impairment (MCI), plasma p-tau181 and p-tau217 have been shown to accurately predict cognitive decline and conversion to AD dementia within the next 2 to 6 years. In the early stages of AD, plasma p-tau levels gradually increase over time, likely due to increased synthesis and secretion of p-tau by neurons affected by AD pathology. Plasma p-tau217 levels are increased in both the preclinical and prodromal stages of AD. Plasma p-tau levels have broad application prospects in evaluating and monitoring the therapeutic effects of new drugs for AD in clinical trials.

[0006] The levels of P-tau181 and T-tau in the human body reflect the presence of Aβ pathology in the brain and can accurately distinguish Alzheimer's disease (AD) from non-AD neurodegenerative disease-related dementia with high accuracy. Currently, cerebrospinal fluid (CSF) analysis and positron emission tomography (PET) are commonly used to measure P-tau181 and T-tau in the brain. However, CSF testing is invasive and has low patient acceptance, while the high cost of PET limits its application. Therefore, the study of P-tau181 and T-tau in peripheral fluids has attracted considerable attention. P-tau181 and T-tau can be cleared from the brain to the blood via various pathways, such as clearance through the blood-brain barrier and absorption through the CSF. However, due to the low concentrations of P-tau181 and T-tau in plasma, the varying sensitivity of different detection techniques can lead to significant discrepancies in results.

[0007] Currently available testing products, such as colloidal gold immunochromatography, suffer from large batch-to-batch variability and low sensitivity. Its physical adsorption binding method can easily cause antigen / antibody detachment from the gold particle surface, resulting in unstable labels and limited qualitative or semi-quantitative results. Enzyme-linked immunosorbent assays (ELISAs) are complex and time-consuming, requiring specialized personnel. Furthermore, numerous operational factors can influence the process, leading to false-positive and false-negative results. Chemiluminescence methods offer high sensitivity, good specificity, excellent stability, and are non-toxic to the environment and the human body. However, currently available products only test for a single sample type and can only measure marker concentrations in plasma samples. This single-indicator assay is time-consuming and has poor reproducibility. Rapidly and accurately obtaining results for the diagnosis and treatment of Alzheimer's disease, as well as accurate results to guide subsequent treatment, are pressing challenges in clinical diagnostic research. The development of a chemiluminescence detection kit for the combined detection of P-tau181 and T-tau, characterized by high sensitivity, short reaction time, simple operation, and high interference resistance, is highly desirable. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a kit for the combined detection of P-tau181 and T-tau proteins, as well as its preparation method and application. The goal is to provide a chemiluminescent detection kit for the combined detection of P-tau181 and T-tau proteins with high sensitivity, short reaction time, simple operation, and high interference resistance.

[0009] The technical solution of the present invention to solve the above technical problems is as follows:

[0010] In a first aspect, a kit for the combined markers P-tau181 and T-tau is provided, the kit comprising a first reagent and a second reagent; the first reagent comprises a magnetic bead working solution and magnetic microspheres coated with Tau polyclonal antibodies, the magnetic microspheres coated with Tau polyclonal antibodies include magnetic microspheres coated with P-tau181 polyclonal antibodies and magnetic microspheres coated with T-tau polyclonal antibodies; the second reagent comprises a labeled antibody diluent and an acridinium ester-labeled anti-Tau antibody, the acridinium ester-labeled anti-Tau antibody include an acridinium ester-labeled anti-P-tau181 antibody and an acridinium ester-labeled anti-T-tau antibody.

[0011] The principle of the present invention is as follows: the concentration of P-tau181 and T-tau is detected by chemiluminescent immunosandwich method, high-specificity anti-P-tau181 polyclonal antibody and T-tau polyclonal antibody are coupled with magnetic microspheres, and acridinium ester is used to label high-specificity anti-P-tau181 and anti-T-tau antibodies respectively. The sample and the magnetic microspheres coated with P-tau181 polyclonal antibody, the magnetic microspheres coated with T-tau polyclonal antibody, and the acridinium ester-labeled high-specificity anti-P-tau181 and acridinium ester-labeled high-specificity anti-T-tau antibodies are added to the reaction tube respectively. After incubation, the P-tau in the sample is detected. P-tau181 and T-tau proteins bind to magnetic beads coated with polyclonal antibodies against P-tau181 and T-tau, and simultaneously bind to acridinium ester-labeled, highly specific anti-P-tau181 and T-tau antibodies, forming an antibody-antigen-antibody sandwich complex. After the reaction is complete, a magnetic field attracts the beads and washes away unbound material. Chemiluminescent substrates (pre-excitation solution / chemiluminescent substrate A solution, excitation solution / chemiluminescent substrate B solution) are added to the respective tubes to generate chemiluminescence. The number of photons produced by the reaction is measured by a photomultiplier tube, which is proportional to the analyte concentration in the sample. The amount of analyte in the sample is determined using a calibration curve, and the instrument automatically calculates the ratio of P-tau181 to T-tau proteins based on the sample concentration.

[0012] The beneficial effects of the present invention are as follows: highly specific anti-P-tau181 and anti-T-tau antibodies are used, and the values of P-tau181 and T-tau proteins in human serum, plasma or urine samples can be detected simultaneously with a single sampling; a magnetic bead working solution and a labeled antibody diluent are used to further improve the sensitivity and stabilizer of the kit; the kit of the present invention can produce results within 8 minutes, with a repeatability within 5%, a minimum detection limit of 0.96 pg / ml for P-tau181, and a minimum detection limit of 1.08 pg / ml for T-tau; at the same time, the kit of the present invention is more stable during transportation and storage, and can be kept stable for 2 years at 2-8°C for a long time. It can be kept stable for 13 days after an aging test at 37°C. Calibrators and quality control products that are matched with the reagents do not need to be freeze-dried and are in liquid form, ready for use, and can be kept stable for 2 years at 2-8°C for a long time.

[0013] On the basis of the above technical solution, the present invention can also be improved as follows.

[0014] Furthermore, the mass ratio of the P-tau181 polyclonal antibody to the magnetic microspheres coated with the P-tau181 polyclonal antibody is 1-5:50-100;

[0015] The mass ratio of the T-tau polyclonal antibody to the magnetic microspheres in the magnetic microspheres coated with the T-tau polyclonal antibody is 1-5:50-100.

[0016] Furthermore, the mass ratio of acridinium ester to anti-P-tau181 antibody in the acridinium ester-labeled anti-P-tau181 antibody is 5-9:30-50;

[0017] The mass ratio of acridinium ester to anti-T-tau antibody in the acridinium ester-labeled anti-T-tau antibody is 5-9:30-50.

[0018] Furthermore, each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH=7.4.

[0019] Furthermore, each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

[0020] Furthermore, the kit also includes a calibrator, a quality control product and a chemiluminescent substrate; the chemiluminescent substrate is a pre-excitation solution / chemiluminescent substrate A solution and an excitation solution / chemiluminescent substrate B solution.

[0021] In a second aspect, a method for preparing a kit for combining the markers P-tau181 and T-tau comprises the following steps:

[0022] Preparation of the first reagent:

[0023] Activating the magnetic microspheres with an activator to obtain activated magnetic beads; adding P-tau181 polyclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with the P-tau181 polyclonal antibodies; and storing the magnetic microspheres coated with the P-tau181 polyclonal antibodies in a magnetic bead working solution;

[0024] And / or, activating the magnetic microspheres with an activator to obtain activated magnetic beads; adding T-tau polyclonal antibodies to the activated magnetic beads, performing a coupling reaction to obtain magnetic microspheres coated with the T-tau polyclonal antibodies, and storing the magnetic microspheres coated with the T-tau polyclonal antibodies in a magnetic bead working solution;

[0025] Preparation of the second reagent:

[0026] The anti-P-tau181 antibody is subjected to a labeling reaction with acridine to obtain an antibody labeling mixture; the antibody labeling mixture is subjected to ultrafiltration to obtain an acridinium ester-labeled anti-P-tau181 antibody, and the acridinium ester-labeled anti-P-tau181 antibody is stored in a labeled antibody diluent;

[0027] And / or, the anti-T-tau antibody is subjected to a labeling reaction with acridine to obtain an antibody labeling mixture; the antibody labeling mixture is ultrafiltered to obtain an acridinium ester-labeled anti-T-tau antibody, and the acridinium ester-labeled anti-T-tau antibody is stored in a labeled antibody diluent.

[0028] Furthermore, the activators are all EDC; the mass ratio of the activators to the magnetic beads is 10:0.5-2;

[0029] The activation parameters are: room temperature, 20 min to 40 min;

[0030] The coupling reaction parameters are: room temperature, 3h to 8h.

[0031] The ratio of the magnetic microspheres coated with the P-tau181 polyclonal antibody to the magnetic bead working solution only needs to meet practical requirements. The ratio of the magnetic microspheres coated with the T-tau polyclonal antibody to the magnetic bead working solution only needs to meet practical requirements.

[0032] Furthermore, the parameters of the labeling reaction are: 37°C, 20 min to 40 min;

[0033] The ultrafiltration parameters are: 14000xg, 5-15min.

[0034] Among them, the dosage ratio of the acridinium ester-labeled anti-P-tau181 antibody and the labeled antibody diluent only needs to meet actual requirements; the dosage ratio of the acridinium ester-labeled anti-T-tau antibody and the labeled antibody diluent only needs to meet actual requirements.

[0035] In the third aspect, a kit for the combined detection of the markers P-tau181 and T-tau is used to prepare a detection product for the Alzheimer's disease markers P-tau181 and T-tau. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a calibration curve diagram of the reagent P-tau181 of the present invention;

[0037] Figure 2 This is a T-tau calibration curve diagram of the reagent of the present invention;

[0038] Figure 3 This is the linear correlation diagram of the reagent P-tau181 of the present invention;

[0039] Figure 4 This is a linear correlation diagram of the reagent T-tau of the present invention. DETAILED DESCRIPTION

[0040] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0041] Example

[0042] 1. Materials and reagents.

[0043] (1) Antigen and Antibody:

[0044] Tau polyclonal magnetic microsphere-coated antibody, anti-P-tau181 labeled antibody, and anti-T-tau labeled antibody were all purchased from Beijing Baixinyi Biotechnology Co., Ltd. at a concentration of 10 mg / mL; P-tau181 antigen and T-tau antigen were purchased from Beijing Aibosheng Biotechnology Co., Ltd. at a concentration of 5 mg / mL.

[0045] (2) Other reagents:

[0046] Acridinium ester (AE) was purchased from Helison (Xiamen) Biotechnology Co., Ltd., and carboxyl magnetic beads were purchased from Nanjing Ruibeixi Biotechnology Co., Ltd. at a concentration of 10 mg / mL; pre-excitation solution / excitation solution (pre-excitation solution / chemiluminescent substrate A solution, excitation solution / chemiluminescent substrate B solution) were purchased from Guangzhou Hongrunkang Technology Development Co., Ltd.; disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), glucose, sodium chloride, sucrose, 3-morpholinepropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methylglycine (Tricine), sodium caseinate, hydrazine yellow, fruit green, lactose, Tween-20, Triton-100, glycerol, tris(hydroxymethyl)aminomethane hydrochloride (Tris), 2-(N-morpholine)ethanesulfonic acid Conventional chemical reagents such as MES, ethylene glycol, hydrochloric acid, sodium periodate, and sodium borohydride were purchased from Sinopharm Chemical Reagent Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), bovine serum albumin (BSA), Proclin-300, and polyethylene glycol 6000 were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; amaranth (standard solution) was purchased from Tianjin Guangfu Fine Chemical Research Institute; sorbitan polyoxyethylene ether tetraoleate, methyl glucoside sesquistearate (CAS: 68936-95-8), diethylene glycol monolaurate (CAS: 141-20-8), and polyoxypropylene stearate (CAS: 25190-52-7) were purchased from Shanghai Kao Co., Ltd.

[0047] Polypropylene fumarate used the compound from the article "Wu Yongchao, Zheng Qixin, Guo Xiaodong, et al. Synthesis, cross-linking, biomechanics and in vitro degradation detection of polypropylene fumarate [J]. Bio-Orthopedic Materials and Clinical Research, 2003."; isodecyl citrate monoester and lauryl citrate monoester used the compounds from the article "Huang Di et al. Study on solution properties of new surfactant citric acid monoester and its application in gel oil. Modern Food Science and Technology 34.5 (2018): 10."; surfactant Coltide HSi used the compound from the article "Li Linping. Application of a new surfactant in laundry detergent. China Detergent Industry 8 (2014): 3."

[0048] (3) Instruments:

[0049] SMART 500S fully automatic chemiluminescence immunoassay analyzer produced by Chongqing Cosmay Biotechnology Co., Ltd. (Yu Medical Device Registration No. 20192220077).

[0050] 2. Experimental method.

[0051] 2.1 Reagent preparation:

[0052] (1) Magnetic bead coating buffer:

[0053] Add 9.762 g of MES, 0.1 mL of Triton-100, and 5.00 g of disodium alkyl polyglycoside sulfosuccinate, and dilute to 1 L with purified water.

[0054] (2) Magnetic bead working solution:

[0055] HEPES (4-hydroxyethylpiperazineethanesulfonic acid) 11.91 g, sodium chloride 9.00 g, BSA 25.00 g, trehalose 100.00 g, glucose 80.00 g, Tween-20 1 mL, Proclin 300 1 mL, polyethylene glycol 20000 80.00 g; alkyl polyglycoside glycerol ether 7.2 g, adjust the pH to 7.4, and dilute to 1 L with purified water.

[0056] (3) Luminescent labeling buffer:

[0057] 13.435 g of disodium hydrogen phosphate dodecahydrate, 0.8265 g of sodium dihydrogen phosphate dihydrate, 2.3 g of α-sulfo fatty acid methyl ester (mono) sodium salt, 0.6 g of fatty alcohol sulfate (ester), and dilute to 1 L with purified water.

[0058] (4) Luminescent blocking buffer:

[0059] 12.1 g of tris(hydroxymethyl)aminomethane, 9.0 g of sodium chloride, 10.0 g of lysine, 100 g of BSA, 1.5 g of lauryl citrate, 5.0 g of sorbitan polyoxyethylene ether tetraoleate, and dilute to 1 L with purified water.

[0060] (5) Acridinium ester labeled antibody dilution solution:

[0061] 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate, and 0.01 g of amaranth were diluted to 1 L with purified water.

[0062] (6) Calibrator diluent:

[0063] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5.00 g of hydrazine yellow, 5.00 g of fruit green, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0064] (7) Quality control diluent:

[0065] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5.00 g of hydrazine yellow, 5.00 g of fruit green, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0066] (8) Working cleaning fluid:

[0067] 9.76 g of N-tris(hydroxymethyl)methylglycine (Tricine), 0.70 mL of hydrochloric acid, 9.00 g of sodium chloride, 5.0 g of monolauryl phosphate MAP, 2.5 mL of Triton 100, 40 g of surfactant Coltide HSi and 1 mL of Proclin 300 were added, and the volume was adjusted to 1 L with purified water.

[0068] 2.2 Preparation of magnetic microspheres:

[0069] (1) Magnetic bead washing: Place 10 mL of magnetic beads on a magnetic rack. After all the magnetic beads are adsorbed, discard the supernatant. Add 20 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 10 min. Place the magnetic beads on a magnetic rack. After all the magnetic beads are adsorbed, discard the supernatant. Repeat this washing process twice and discard the supernatant.

[0070] (2) Activation of magnetic beads: Weigh 10.00 mg of EDC and dissolve it in 1 mL of magnetic bead coating buffer. Once fully dissolved, add it to the magnetic beads. Simultaneously add 49.00 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 30 min. Then, repeat step 1.2.2.1 to wash the magnetic beads and discard the supernatant.

[0071] (3) Magnetic Bead Coating: Add 5 mg of anti-Tau magnetic microsphere coating antibody to the magnetic beads, then add 50 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 5 h. Then, repeat step 1.2.2.1 to wash the magnetic beads and discard the supernatant.

[0072] (4) Magnetic bead storage: Add 500 mL of magnetic bead working solution to the magnetic beads at 30 rpm / min, mix at room temperature for 30 min, and store at 2-8°C.

[0073] 2.3 Preparation of Luminescent Markers: (Taking P-tau181 Luminescent Marker Working Solution as an Example)

[0074] (1) Antibody labeling:

[0075] Take 1uL of acridinium ester mother solution (5mM) and add 9uL of DMF (N,N-dimethylformamide) to prepare the acridinium ester working solution (acridinium ester mother solution: anhydrous dimethylformamide = 1:9).

[0076] Add 50 μg of anti-P-tau181 labeled antibody to a centrifuge tube, add luminescent labeling buffer to 300 μL (the volume here increases with the amount of antibody), add 30 μL of acridine working solution, and shake in a 37°C incubator for 30 minutes;

[0077] Add 100 μL of luminescent blocking solution and shake in a 37°C incubator for 30 minutes;

[0078] (2) Ultrafiltration of antibody after labeling:

[0079] Rinse: Pipette 500 μL of luminescent labeling buffer into the sample reservoir, close the lid, and centrifuge at 10,000 x g for 10 minutes. Aspirate the liquid from the filtrate receiver. Repeat the rinse cycle.

[0080] Ultrafiltration: Add labeled antibodies to the sample pool, cover the lid, and centrifuge at 14,000 x g for 10 minutes to concentrate the labeled antibodies in the sample pool;

[0081] Washing: Add 200 μL of acridinium ester labeled antibody diluent to the sample pool, centrifuge at 14,000 x g for 20 minutes, and concentrate the labeled antibody in the sample pool after washing;

[0082] Collection: Add 100 μL of acridinium ester-labeled antibody diluent to the sample pool, pipette and reconstitute (pipetting and reconstitute at least 30 times to avoid creating bubbles), transfer to a 1.5 ml centrifuge tube, add acridinium ester-labeled antibody diluent to 1 ml, and store at -20°C. (Luminescent marker storage solution, concentration: 0.05 mg / ml).

[0083] (3) Preparation of luminescent marker working solution:

[0084] Take 5 mL of the luminescent marker stock solution stored at -20°C in the dark, add 495 mL of acridinium ester labeled antibody diluent, mix at 30 rpm / min, 2-8°C in the dark for 30 minutes, and then store at 2-8°C in the dark.

[0085] The preparation of T-tau luminescent marker was the same as described above.

[0086] 2.4 Preparation of calibrators:

[0087] Take 0.5 μL of each of P-tau181 and T-tau antigens and add them to 999 μL of Calibrator Diluent. Mix thoroughly at room temperature to prepare a 2.5 μg / mL stock solution. Then, use Calibrator Diluent to serially dilute the stock solution to six concentration points: 1500 ng / mL, 750 ng / mL, 250 ng / mL, 50 ng / mL, 25 ng / mL, and 0 ng / mL. Store at 2-8°C.

[0088] 2.5 Preparation of quality control products:

[0089] Take 0.5 μL of each of P-tau181 and T-tau antigens and add them to 999 μL of Calibrator Diluent. Mix at room temperature to prepare a 2.5 μg / mL stock solution. Then, dilute the stock solutions to 200 ng / mL for Control Level 1 and 600 ng / mL for Control Level 2 using Control Diluent. Store at 2-8°C.

[0090] 2.6 Detection method:

[0091] (1) Testing procedures:

[0092] When loading the reagent bottle onto the machine for the first time, it should be gently turned over 30 times before opening, and the reagent bottle should be observed to ensure that the magnetic beads are completely suspended. If the magnetic beads are still attached to the bottom of the reagent bottle, continue to turn the reagent bottle until they are completely suspended. If they still cannot be suspended, the bottle of reagent cannot be used.

[0093] The system requires a sample volume of 10 μL per measurement. Before applying for a test, you should prepare all the materials required for the test and carefully read the chemiluminescence immunoassay user manual to obtain information on system operating procedures, sample management, usage precautions, maintenance, and other information. The main steps for applying for a test are as follows:

[0094] ⑴ Enter the sample application interface and enter the sample number, sample rack number, position number, sample type, remarks and other information according to the test requirements;

[0095] ⑵Select the test item, select the sample type in the item options, enter the number of repetitions and other information;

[0096] ⑶ After preparing the test sample and placing it correctly, click the start button to start the test. The chemiluminescence immunoassay analyzer will perform the following operations in sequence:

[0097] ①The sample processing system transfers the sample rack to the sample suction position;

[0098] ② The cuvette loading and scheduling system loads the cuvette from the loading area to the sample loading position;

[0099] ③The sample injection system completes the sample injection and cleaning of the sample needle;

[0100] The reagent processing system provides the reagents required for the test, mixes each reagent, and sends it to the reagent aspiration position for aspiration;

[0101] The sample injection system completes the reagent injection and cleaning of the reagent needle;

[0102] ④Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0103] ⑤After the incubation is completed, the magnetic separation system uses a cleaning solution to clean and separate the reaction mixture;

[0104] ⑥ The substrate system injects preheated substrate solution into the reaction cup after magnetic separation, mixes, incubates, and waits for light detection;

[0105] ⑦The light measurement reaction system sends each reaction cup to the signal collection position to collect signals and calculate the luminescence value;

[0106] ⑧Calculate the content of the analyte in the sample.

[0107] (2) Calibration:

[0108] Perform a calibration test using the included kit and calibrator. Before starting the kit calibration, scan the master curve in the kit's 2D barcode and import it into the system.

[0109] The chemiluminescence immunoassay analyzer uses the results of calibrator tests to adjust the master curve and generate the calibration curve for the current system. The instrument operating software includes a calibration check function that automatically verifies the validity of the calibration curve. If calibration fails, consider the instrument settings, status, location of the calibrators (place low-value calibrators first, then high-value calibrators), and expiration dates.

[0110] A valid calibration curve is required before all tests. Recalibration is required in the following situations:

[0111] ① Use a new batch of test kit;

[0112] ② The same batch of test kits has been used on the analyzer for more than 30 days;

[0113] ③After instrument maintenance;

[0114] ④The quality control value exceeds the specified range;

[0115] (3) Quality Control:

[0116] To ensure the reliability of test results, it is recommended that both high and low level quality control products be tested once every 24 hours. In addition, quality control testing is also recommended after each calibration test, reagent batch change, maintenance and troubleshooting.

[0117] The test values of quality control samples should be within the specified range. If they are outside the specified range, the user should check the detection system, such as the location, expiration date, storage method, calibration process, instrument performance and status, and recalibrate if necessary. If the results are still outside the range after retesting, please contact customer service.

[0118] (4) Calculation:

[0119] Using stored calibration data, the system software automatically determines the sample test results using a weighted four-parameter logarithmic curve (4PLC, Y-weighted) mathematical method. Results are given in pg / mL.

[0120] 3. Experimental results.

[0121] 3.1 Establishment of calibration curve:

[0122] The establishment of the calibration curve is shown in Table 1 and Table 2. Figure 1 and Figure 2 .

[0123] Table 1 P-tau181 calibration results of the kit of the present invention

[0124]

[0125] Table 2 T-tau calibration results of the kit of the present invention

[0126]

[0127]

[0128] 3.2 Minimum detection limit.

[0129] Using PBS buffer as a blank sample, the detection and comparison reagents prepared in the present invention were simultaneously measured 20 times. The test strip test and comparison values prepared in the present invention, plus twice the standard deviation, were substituted into the fitting curve for regression. The results showed that the detection kit prepared in the present invention had a minimum detection limit of 0.96 pg / ml for P-tau181 and 1.08 pg / ml for T-tau. The comparison reagent had a minimum detection limit of 2.32 pg / ml for P-tau181. The results are shown in Table 3.

[0130] Table 3 Results of minimum detection limits of reagents

[0131]

[0132] 3.3 Repeatability.

[0133] The test kit of the present invention was tested repeatedly for two levels of quality control 10 times, and the CV was calculated. The results showed that the quality control level 1CV of P-tau181 of the test kit of the present invention was 4.217%, and the quality control level 2CV was 3.07%; the quality control level 1CV of T-tau was 4.38%, and the quality control level 2CV was 3.16%; it can be seen that the preparation method of the test strip prepared by the present invention has good repeatability. The results are shown in Tables 4 and 5.

[0134] Table 4 P-tau181 repeatability results

[0135]

[0136]

[0137] Table 5 T-tau repeatability results

[0138]

[0139] 3.4 Linear.

[0140] A high-value sample containing a P-tau181 concentration of 160 pg / ml and a T-tau concentration of 1250 pg / ml was diluted with PBS buffer, mixed and diluted by 2 times, and the dilution ratio was 8 gradients and 10 gradients (xi) to test the test strips prepared by the present invention. The sample of each dilution concentration was tested 3 times, and the mean value (yi) of the measurement results was calculated respectively. The linear regression equation and linear regression coefficient were calculated with the dilution concentration (xi) as the independent variable and the mean value (yi) of the measurement result as the dependent variable. The results showed that for the kit of the present invention, the linear correlation coefficient (r) of P-tau181 in the range of [1.25-160] pg / ml was not less than 0.9990, and the linear correlation coefficient (r) of T-tau in the range of [2.44-1250] pg / ml was not less than 0.9990. It can be seen that the preparation method of the test strips prepared by the present invention has good linearity. The results are shown in Tables 6 to 7 and Figures 3 and 4 .

[0141] Table 6 Linearity test results of the inventive reagent P-tau181

[0142]

[0143] Table 7 T-tau linearity test results of the invention reagent

[0144]

[0145]

[0146] 3.5 accuracy.

[0147] The high concentration reference substance (liquid A) was added to the low concentration reference substance (liquid B), and the volume ratio between the high concentration reference substance (liquid A) and the low concentration reference substance (liquid B) was not greater than 1:9. Each sample was measured 3 times and the average value was taken. According to the calculation results of formula (1), it can be seen that the recovery rates of P-tau181 and T-tau in the detection preparation method prepared by the present invention are respectively , and the accuracy is good. The results are shown in FIG.

[0148] Tables 8 and 9.

[0149]

[0150] Where: R is the recovery rate; V is the volume of solution A added; V0 is the volume of serum sample B; c is the detection concentration of serum sample after adding solution A; c0 is the detection concentration of serum sample B; c s is the concentration of solution A.

[0151] Table 8 Accuracy test results of the inventive reagent P-tau181

[0152]

[0153]

[0154] Table 9 T-tau accuracy test results of the invention reagent

[0155]

[0156] 3.6 Stability.

[0157] 3.6.1 Long-term stability at 2-8°C:

[0158] (1) Three batches of the chemiluminescent immunoassay kit for the high-sensitivity combined detection of Alzheimer's disease markers P-tau181 and T-tau of the present invention were stored in a finished product warehouse at 2-8°C in a dry environment. The temperature of the finished product warehouse was monitored using a thermometer and required to be between 2-8°C to ensure compliance with the product storage conditions during the stability study.

[0159] (2) Three batches of the test kit of the present invention stored in a finished product warehouse at 2-8°C were randomly sampled for testing at month 0, month 6, month 12, month 18, month 24, and month 25, respectively. The test items were: appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, calibrator value assignment accuracy, calibrator homogeneity, target value validity of quality control products, and quality control product homogeneity.

[0160] (3) Judgment of test content and results

[0161] ①Appearance

[0162] a. All components of the test kit should be complete and intact, with no leakage of liquid; the Chinese packaging label should be clear and not worn;

[0163] b. The magnetic bead reagent component is a liquid containing brown solid particles. The magnetic beads are not caking, and the liquid is free of flocs and foreign matter.

[0164] c. Other reagent components are clear and uniform liquids without precipitation or flocs.

[0165] ② Accuracy: The recovery rate should be within the range of 85% to 115%;

[0166] ③ Detection limit: P-tau181 no more than 1.25 pg / mL; T-tau no more than 2.44 pg / mL;

[0167] ④ Linearity: The detection range of the kit is P-tau181 [1.25-160] pg / mL, and the correlation coefficient R of the linear interval should be no less than 0.9900; T-tau [2.44-1250] pg / mL, and the correlation coefficient R of the linear interval should be no less than 0.9900;

[0168] ⑤ Repeatability: coefficient of variation CV is not greater than 5%;

[0169] ⑥ Inter-batch difference: inter-batch coefficient of variation CV is not greater than 8%;

[0170] ⑦ Accuracy of calibrator value assignment: The relative deviation of the results of the calibration sample detected by the chemiluminescence immunoassay analyzer calibrated with the calibrator is ±8%;

[0171] ⑧ Homogeneity of calibration material: homogeneity ≤ 8%

[0172] ⑨ Target value validity of quality control products: When quality control products are tested using a chemiluminescence immunoassay analyzer calibrated with the calibrator, the results should be within the target value range;

[0173] ⑩ Homogeneity of quality control products: Homogeneity ≤ 8%.

[0174] (4) Three batches of the kit of the present invention were stored at 2-8°C for 25 months, and all performance characteristics met the requirements. This indicates that the kit is stable when stored at 2-8°C for 25 months. Therefore, the kit of the present invention is valid for 24 months when stored at 2-8°C. The results are detailed in Tables 10-12.

[0175] Table 10 Real-time stability test results (first batch)

[0176]

[0177] Table 11 Real-time stability test results (second batch)

[0178]

[0179]

[0180] Table 12 Real-time stability test results (third batch)

[0181]

[0182]

[0183] 3.6.2 37℃ aging stability:

[0184] (1) The main raw material of the detection kit of the present invention is the coated antibody. Since temperature has a great influence on the activity of the antibody, the higher the temperature, the more serious the attenuation of the antibody activity. The accelerated stability test design adopts the destruction test at 37°C. The stability study is carried out by examining the appearance, detection limit, accuracy, linearity, repeatability, batch difference, calibration value assignment accuracy, calibration uniformity, target value validity of the quality control product, and quality control product uniformity performance indicators, which are required to be no less than the product design requirements.

[0185] (2) Three batches of test kits, 4 boxes each, totaling 12 boxes, were placed in a 37°C incubator according to the commercial packaging for accelerated testing. The full performance (appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, accuracy of calibrator value assignment, uniformity of calibrator, validity of target values of quality control products, and uniformity of quality control products) of the test kits were tested on day 0, day 5, day 10, and day 13, respectively.

[0186] (3) Judgment of test content and results: consistent with the long-term stability at 2-8℃.

[0187] (4) After accelerating the test kit at 37°C for 13 days, the performance of the three batches of the test kit of the present invention met the requirements, indicating that the performance of the test kit is stable within 13 days of accelerating the test kit at 37°C. The results are shown in Tables 13 to 15.

[0188] Table 13 Accelerated stability test results (first batch)

[0189]

[0190]

[0191] Table 14 Accelerated stability test results (second batch)

[0192]

[0193]

[0194] Table 15 Accelerated stability test results (third batch)

[0195]

[0196] In summary, the kit of the present invention adopts the magnetic microparticle chemiluminescence-double antibody sandwich method. Compared with other methodologies such as enzyme-linked immunosorbent assay and fluorescent immunochromatography, it is simple to operate and can be tested on the machine directly after adding the sample. It can also detect two indicators, P-tau181 and T-tau, simultaneously. In addition, through the independently developed acridinium ester working solution, magnetic bead working solution and working cleaning solution, after adding the sample, the incubation time of the magnetic beads and acridinium ester with the sample is greatly shortened. It only takes 3 minutes to reach the optimal reaction time. The subsequent cleaning process only requires 3 cleanings, each for 30 seconds, to remove unreacted impurities. The entire test only takes 360 seconds. Most of the time required for similar chemiluminescence product detection reagents on the market is about 900 seconds. Therefore, the detection time is greatly shortened, and the purpose of real-time detection is achieved, which fully meets the diagnosis and treatment needs of time-sensitive diseases and provides patients with accurate test results.

[0197] The repeatability of the kit of the present invention is within 5%, the minimum detection limit of P-tau181 is 0.96 pg / ml, and the minimum detection limit of T-tau is 1.08 pg / ml. At the same time, the kit of the present invention is more stable during transportation and storage. It can be kept stable for 2 years at 2-8°C for a long time. It can be kept stable for 13 days in an aging test at 37°C. The calibrators and quality control products matched with the reagents do not need to be freeze-dried and are in liquid form, ready for use. They can be kept stable for 2 years at 2-8°C for a long time.

[0198] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit for combining the markers P-tau181 and T-tau, characterized in that: The kit includes a first reagent and a second reagent; the first reagent includes a magnetic bead working solution and magnetic microspheres coated with Tau polyclonal antibodies, and the magnetic microspheres coated with Tau polyclonal antibodies include magnetic microspheres coated with P-tau181 polyclonal antibodies and magnetic microspheres coated with T-tau polyclonal antibodies; the second reagent includes a labeled antibody diluent and an acridinium ester-labeled anti-Tau antibody, and the acridinium ester-labeled anti-Tau antibody includes an acridinium ester-labeled anti-P-tau181 antibody and an acridinium ester-labeled anti-T-tau antibody.

2. A kit for combining the markers P-tau181 and T-tau according to claim 1, characterized in that: The mass ratio of the P-tau181 polyclonal antibody to the magnetic microspheres coated with the P-tau181 polyclonal antibody is 1-5:50-100; The mass ratio of the T-tau polyclonal antibody to the magnetic microspheres in the magnetic microspheres coated with the T-tau polyclonal antibody is 1-5:50-100.

3. The kit for combining the markers P-tau181 and T-tau according to claim 1, characterized in that: The mass ratio of acridinium ester to anti-P-tau181 antibody in the acridinium ester-labeled anti-P-tau181 antibody is 5-9:30-50; The mass ratio of acridinium ester to anti-T-tau antibody in the acridinium ester-labeled anti-T-tau antibody is 5-9:30-50.

4. The kit for combining the markers P-tau181 and T-tau according to claim 1, characterized in that: Each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH=7.

4.

5. The kit for combining the markers P-tau181 and T-tau according to claim 1, characterized in that: Each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

6. The kit for combining the markers P-tau181 and T-tau according to claim 1, characterized in that: The kit further comprises a calibrator, a quality control product and a chemiluminescent substrate; the chemiluminescent substrate is a pre-excitation solution / chemiluminescent substrate A solution and an excitation solution / chemiluminescent substrate B solution.

7. A method for preparing a kit for combining the markers P-tau181 and T-tau according to any one of claims 1 to 6, characterized in that: The steps include: Preparation of the first reagent: Activating the magnetic microspheres with an activator to obtain activated magnetic beads; adding P-tau181 polyclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with the P-tau181 polyclonal antibodies; and storing the magnetic microspheres coated with the P-tau181 polyclonal antibodies in a magnetic bead working solution; And / or, activating the magnetic microspheres with an activator to obtain activated magnetic beads; adding T-tau polyclonal antibodies to the activated magnetic beads, performing a coupling reaction to obtain magnetic microspheres coated with the T-tau polyclonal antibodies, and storing the magnetic microspheres coated with the T-tau polyclonal antibodies in a magnetic bead working solution; Preparation of the second reagent: The anti-P-tau181 antibody is subjected to a labeling reaction with acridine to obtain an antibody labeling mixture; the antibody labeling mixture is subjected to ultrafiltration to obtain an acridinium ester-labeled anti-P-tau181 antibody, and the acridinium ester-labeled anti-P-tau181 antibody is stored in a labeled antibody diluent; And / or, the anti-T-tau antibody is subjected to a labeling reaction with acridine to obtain an antibody labeling mixture; the antibody labeling mixture is ultrafiltered to obtain an acridinium ester-labeled anti-T-tau antibody, and the acridinium ester-labeled anti-T-tau antibody is stored in a labeled antibody diluent.

8. The method for preparing a kit for combining the markers P-tau181 and T-tau according to claim 7, characterized in that: The activators are all EDC; the mass ratio of the activators to the magnetic beads is 10:0.5-2; The activation parameters are: room temperature, 20 min to 40 min; The coupling reaction parameters are: room temperature, 3h to 8h.

9. The method for preparing a kit for combining the markers P-tau181 and T-tau according to claim 7, characterized in that: The parameters of the labeling reaction are: 37°C, 20min-40min; The ultrafiltration parameters are: 14000xg, 5-15min.

10. Use of a kit for combining markers P-tau181 and T-tau, characterized in that: The kit for jointly detecting the markers P-tau181 and T-tau according to any one of claims 1 to 6 is used to prepare a detection product for the Alzheimer's disease markers P-tau181 and T-tau.

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