A Tau protein (TAU) detection kit and preparation method thereof

Abstract

The present invention belongs to the technical field of kits, and in particular to a Tau protein (TAU) detection kit and a preparation method thereof. The Tau protein (Tau) magnetic particle chemiluminescence detection kit of the present invention adopts a long-arm biotin N-hydroxysuccinimide biotin and an octamer streptavidin binding mode, and the sensitivity is greatly improved, and the interference of endogenous biotin in the sample to the test result can be effectively avoided, and the utilization rate of the antibody can also be effectively improved, and the cost can be reduced; the octamer streptavidin used in the present invention has more high-affinity sites than the conventional tetramer streptavidin, and more biotin linkers can be combined, and the steric hindrance can be reduced by cooperating with N-hydroxysuccinimide biotin, and the sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of kits, and in particular relates to a Tau protein (TAU) detection kit and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a chronic central nervous system degenerative disease that occurs in the elderly or pre-elderly stage, with progressive cognitive dysfunction, memory loss, decreased ability to live daily, and mental and behavioral abnormalities as the main clinical manifestations. Recent statistics show that about 50 million people in the world suffer from Alzheimer's disease or other types of dementia. The World Health Organization predicts that the total number of dementia patients will reach 82 million in 2030 and 152 million in 2050. At present, there are about 9 million patients with Alzheimer's disease in my country, and it is expected that the number will exceed 40 million by 2050. Faced with such a large number of patients, there are very few options for AD treatment. The main reason is that the pathogenesis of AD is not yet fully understood. With the discovery of familial AD, the genetic theory involved in the pathogenesis of AD has gradually attracted people's attention.

[0003] In the past few years, the International Working Group (IWG) and the National Institute on Aging, the Alzheimer's Association, have introduced diagnostic criteria for Alzheimer's disease (AD); by better defining clinical phenotypes and integrating biomarkers into the diagnostic process, thus comprehensively covering all stages of the disease (from asymptomatic to the most severe dementia stage). The most important practical application of these criteria is that they allow for earlier prevention measures in the prodromal stage of the disease and facilitate research on secondary prevention of preclinical AD. Through the research and application of these criteria, a universal standard that can truly detect the presence of AD in the routine field will eventually be developed.

[0004] In 2014, IWG revised the new IWG diagnostic criteria, namely the IWG-2 diagnostic criteria, and published it in the journal Lancet Neurology. This is the latest AD diagnostic criteria to date. The IWG-2 diagnostic criteria for the first time divided AD biomarkers into diagnostic markers and progression markers. Cerebrospinal fluid β-amyloid protein and Tau, amyloid protein positron emission tomography (PET) and AD pathogenic gene carriers are diagnostic markers for AD, while brain structural magnetic resonance imaging (MRI) and 2-deoxy-D-glucose are AD progression markers.

[0005] The main pathological characteristics of AD are neurofibrillary tangles (NFTs) and senile plaques (APs), among which NFTs are mainly caused by excessive phosphorylation of Tau protein. Tau protein is an important microtubule associated protein (MAP) that maintains cell structure by binding to tubulin. In the brains of AD patients, Tau protein is overphosphorylated, and the content of phosphorylated Tau is 3 to 4 times that of normal people. Overphosphorylated Tau can cause microtubule disintegration, thereby destroying cell structure. Studies have found that the expression of tau and p-tau proteins in the cerebrospinal fluid of AD patients is significantly increased, which is related to the degree of dementia.

[0006] Tau is encoded by a gene located on the long arm of chromosome 17, contains 16 exons, is 352 to 441 amino acids long, and has a molecular weight of 45 to 65 kDa. Conventional analysis methods for Tau mainly include neuroimaging, immunoassays, and mass spectrometry. Among them, neuroimaging technology can be used to study the pathological imaging of Tau, immunoassays are used for the analysis of Tau or P-Tau in plasma or CSF, and mass spectrometry is mainly used to study PTM of Tau. Immunoassays are based on the specific binding of antigens and antibodies, including enzyme-linked immunosorbent assays, protein blotting, nanoparticle-based immunopolymerase chain reaction, and immunomagnetic reduction. ELISA is one of the most widely used methods.

[0007] The ELISA platform is represented by Germany's IBL-International and EU Diagnostics. IBL's Alzheimer's related kits have obtained CE certification, including five indicators: β-Amyloid 1-42, β-Amyloid 1-40, phosphorylated Tau, total Tau and non-phosphorylated Tau protein. EU Diagnostics currently has four products: β-Amyloid 1-42, β-Amyloid 1-40, phosphorylated Tau and total Tau. EU Diagnostics' AD marker antibodies come from the world's leading neurodegenerative disease marker development companies represented by Roche Diagnostics and Japan's Fujirebio. Roche Diagnostics is a leader in the field of AD diagnosis. Roche Diagnostics' β-Amyloid 1-42, phosphorylated Tau-181 and total Tau kits have obtained CE and FDA registration certifications respectively. By measuring the concentrations of β-Amyloid 1-42, phosphorylated Tau-181 and total Tau in human cerebrospinal fluid, it is used for early diagnosis of AD. After acquiring the Belgian company Innogenetics, Japan's Fujirebio obtained the CE certification for β-amyloid protein 1-40 and successfully transferred four AD marker products to its Lumipulse fully automatic chemiluminescence instrument.

[0008] Tau protein can be measured using the double antibody sandwich method. If cerebrospinal fluid is used as a sample, the burden on the patient's body and the risk of physical damage are very high when the sample is collected. In fact, using cerebrospinal fluid as a sample reduces the detection rate, the cost is too high, and the risk is also high. Because the migration of Aβ from cerebrospinal fluid to blood is extremely small, the determination of Aβ in serum requires high reagent sensitivity.

[0009] Previous studies on Tau protein have mainly focused on cerebrospinal fluid. However, it is difficult to obtain samples of cerebrospinal fluid, while it is convenient to obtain samples of peripheral blood. Actively exploring Tau protein in peripheral blood is of great significance for the diagnosis of AD. However, due to the existence of the blood-brain barrier and the large volume of peripheral blood, the concentration of related proteins is greatly diluted, so a highly sensitive detection method must be used. The sensitivity, accuracy and precision of magnetic particle chemiluminescence are greatly improved compared with other detection methods. Its principle is that the enzyme catalyzes the substrate to produce a light signal, and a very small amount of enzyme can induce a large amount of enzymatic reaction. Its accuracy is high enough to trace the location of antigens or antibodies at the cellular or subcellular level, and even quantify them at the microgram or nanogram level.

[0010] A Chinese patent application document (publication number: CN109580955A) discloses a chemiluminescent immunoassay method for separating magnetic microparticles of human Tau protein (TAU). However, the patent states that a competitive reaction mode is adopted to eliminate the influence of HOOK. In the specific implementation, a double antibody sandwich method is used. There are contradictions. In this patent, carboxyl magnetic beads are directly coated with antibodies (EDC is an activator, and 0.5 mg of antibody is added to 10 mg of magnetic beads). Alkaline phosphatase labeled with antibodies (2IT and SMCC are activators) is used as a reaction reagent. Agent, detect the TAU protein content in human serum or plasma samples. The method of antibody direct coating magnetic beads is adopted. Due to the use of different methods, the state of antibody magnetic particle connection cannot be well controlled, and the magnetic particles will often clump and be difficult to disperse, which will affect the test results. Therefore, there are often problems such as large batch differences, stability deviation, and the need for further improvement in specificity. In addition, the utilization rate of antibodies is generally low, and the production cost is very high. The sensitivity of this patent is 0.05ng / mL, and the linear range is not clearly stated in the patent, which is 1.0-200 and 0.5-500ng / mL respectively. Summary of the invention

[0011] The purpose of the present invention is to provide a Tau protein (TAU) detection kit which is simple, highly sensitive, reproducible, easy to operate, low in cost and highly automated in order to solve the above problems in the prior art.

[0012] The object of the present invention can be achieved by the following technical scheme: A Tau protein (TAU) detection kit, characterized in that the kit includes R1 reagent, R2 reagent, and R3 reagent;

[0013] The R1 reagent includes magnetic particles containing octamer streptavidin;

[0014] The R2 reagent includes a Tau antibody labeled with N-hydroxysuccinimide biotin;

[0015] The R3 reagent includes an alkaline phosphatase-labeled Tau antibody.

[0016] The present invention combines long-arm biotin N-hydroxysuccinimide biotin with octamer streptavidin, and the sensitivity is significantly improved compared with the mode of antibody direct coating magnetic beads adopted by other manufacturers. The coating method of the present invention can effectively improve the utilization rate of antibodies and reduce costs. The arm length of long-arm biotin N-hydroxysuccinimide biotin used in the present invention is increased by 40%, and octamer streptavidin has more high-affinity sites than conventional tetramer streptavidin, and can bind more biotin linkers. The combination with long-arm biotin can reduce steric hindrance, so that the sensitivity is greatly improved.

[0017] In the above-mentioned Tau protein (TAU) detection kit, the concentration of the magnetic microparticles containing octamer streptavidin in the R1 reagent is 0.25-1.0 mg / mL.

[0018] In the above-mentioned Tau protein (TAU) detection kit, the magnetic microparticles containing octamer streptavidin are prepared by mixing octamer streptavidin with magnetic particles at a mass ratio of (0.005-0.2):10 and stored in a magnetic bead storage solution.

[0019] Preferably, the magnetic particles are one or more of tosyl magnetic particles, carboxyl magnetic particles and amino magnetic particles.

[0020] Preferably, the preparation method of magnetic microparticles containing octamer streptavidin comprises the following steps: washing the tosyl magnetic particles with a buffer solution, then sequentially adding octamer streptavidin and ammonium sulfate for suspension reaction, adding a blocking agent for suspension reaction, washing with TBS buffer solution, adding magnetic bead preservation solution, and finally diluting and preserving.

[0021] Preferably, the concentration of the magnetic microparticles containing octamer streptavidin after adding the magnetic bead storage solution is 5-15 mg / mL.

[0022] Preferably, the sealing agent contains one or more of 1-10wt% BSA, 1-10wt% casein, 1-10wt% fish skin gelatin, CE210, CE510, DB1130, and PA1080.

[0023] Preferably, the amount of the blocking agent is 100 μL of blocking agent per 10 mg of magnetic particles. CE210 and PA1080 are preferably used.

[0024] Preferably, the TBS buffer contains 0.05-2 wt % of one or more of Tween 20, SDS, and Tween 80.

[0025] In the above-mentioned Tau protein (TAU) detection kit, the pH of the magnetic bead storage solution is 6-9, including: 0.01-0.05moL / L Hepes, 0.1-0.5moL / L sodium chloride, 0.1-10wt% BSA, casein, at least one of bovine γ, 0.1-5wt% Proclin 950, 0.1-5wt% tween20, 0.1-5wt% mannitol, and 0.5-10wt% gelatin.

[0026] The magnetic particle preservation solution of the present invention introduces bovine gamma protein, which has a good effect of reducing background, wherein proclin950 has a good improvement on the dispersibility of magnetic beads, and as an antibacterial agent, it can also have a good improvement effect on the stability of the reagent.

[0027] In the above-mentioned Tau protein (TAU) detection kit, the concentration of the Tau antibody labeled with N-hydroxysuccinimide biotin in the R2 reagent is 0.05-5ug / mL.

[0028] In the above-mentioned Tau protein (TAU) detection kit, the preparation method of the Tau antibody labeled with N-hydroxysuccinimide biotin comprises the following steps:

[0029] S1. Dialyze the Tau antibody against PBS buffer to obtain a Tau antibody solution;

[0030] S2, then dissolving N-hydroxysuccinimide biotin ester in dimethyl sulfoxide to obtain an N-hydroxysuccinimide biotin ester solution;

[0031] S3. Evenly mix the Tau antibody solution and the N-hydroxysuccinimide biotin ester solution, then let the reaction stand, add tris(hydroxymethylaminomethane) buffer to terminate the reaction, and finally dilute and store.

[0032] In the above-mentioned Tau protein (TAU) detection kit, the molar ratio of N-hydroxysuccinimide biotin ester to Tau antibody is (8-12):1.

[0033] Preferably, the pH of the buffer solution for dilution and storage in step S3 is 7-9, and includes 0.01-0.03M diethanolamine, 3-6g / L BSA, 0.5-4.5g / L casein, 45-55mg / L blocking agent, and 0.1-0.3wt% P300 preservative.

[0034] Preferably, the blocking agent is one or more of anti-biotin antibody, P-heterophil antibody blocking agent, and MAB33 IgG1 / IgG1 multimer.

[0035] In the above-mentioned Tau protein (TAU) detection kit, the concentration of the alkaline phosphatase-labeled Tau antibody in the R3 reagent is 0.03-5 ug / mL.

[0036] In the above-mentioned Tau protein (TAU) detection kit, the preparation method of the alkaline phosphatase-labeled Tau antibody comprises the following steps:

[0037] S1, reacting the chitosan solution with the sodium periodate solution in the dark, and adding ethylene glycol to terminate the reaction after dialysis;

[0038] S2, then add alkaline phosphatase and Tau antibody and stir evenly, then add sodium borohydride to react, and finally add Tris buffer to terminate the reaction;

[0039] S3. After purification by gel filtration, dilute with enzyme diluent and store.

[0040] In the above-mentioned Tau protein (TAU) detection kit, the mass ratio of Tau antibody to chitosan and alkaline phosphatase is 1:(80-120):(15-30).

[0041] The R3 reagent labeling end of the present invention uses an antibody to connect with a polyhydroxy compound chitosan and alkaline phosphatase, and the effect of improving sensitivity is achieved by controlling the feeding ratio. At the same time, reduced glutathione, sugars and surfactants are introduced into the buffer, which greatly improves the stability of the enzyme.

[0042] Preferably, the pH of the enzyme dilution solution in step S3 is 7-9, and includes: 0.01-0.05 moL / L Hepes, 0.1-0.5 moL / L sodium chloride, 1-10 wt‰ BSA, 1-10 wt‰ gelatin, 1-10 wt‰ trehalose, 1-10 wt‰ mannitol, 1-10 wt‰ reduced glutathione, 45-55 mg / L blocking agent, and 0.2 wt% P300 preservative.

[0043] Preferably, the blocking agent is one or more of anti-biotin antibody, P-heterophil antibody blocking agent, and MAB33 IgG1 / IgG1 multimer.

[0044] Preferably, the Tau protein (TAU) detection kit of the present invention further comprises a Tau calibrator, wherein the Tau calibrator comprises a protein solution having a Tau antigen concentration of 2.0, 50, 500, 5000, and 10000 pg / mL, respectively.

[0045] The Tau protein (TAU) detection kit of the present invention also includes a Tau quality control product, which includes a protein solution with Tau antigen concentrations of 20, 200, and 2000 pg / mL, respectively.

[0046] More preferably, the Tau calibrator protein solution includes: 6 g / L Tris, 50 g / L BSA, 8 g / L sodium chloride, 0.1 mL 1 Mol / L magnesium chloride, 0.1 mL 0.1 Mol / L zinc chloride, and 2 g / L P300.

[0047] The present invention also provides a method for using the above-mentioned Tau protein (TAU) detection kit, which comprises the following steps: reacting the sample to be tested with R2 reagent and R3 reagent for 5-15 minutes, then adding R1 reagent to react for 3-10 minutes to form a sandwich complex magnetic suspension, placing the sandwich complex magnetic suspension in a magnetic field for separation, adding chemiluminescent substrate solution after washing, and detecting the intensity of luminescent photons.

[0048] Compared with the prior art, the kit of the present invention has the following advantages:

[0049] 1. The Tau protein (Tau) magnetic particle chemiluminescence detection kit of the present invention adopts a long-arm biotin N-hydroxysuccinimide biotin and an octamer streptavidin combination mode, which greatly improves the sensitivity, and can effectively avoid the interference of endogenous biotin in the sample on the detection result, and can also effectively improve the utilization rate of the antibody and reduce the cost; the octamer streptavidin used in the present invention has more high-affinity sites than the conventional tetramer streptavidin, and can bind more biotin linkers. The combination with N-hydroxysuccinimide biotin can reduce steric hindrance and improve the sensitivity.

[0050] 2. The magnetic particle preservation solution of the present invention has a good effect of reducing the background by introducing bovine gamma protein. Proclin950 not only significantly improves the dispersibility of magnetic beads, but also has a good improvement effect on the stability of reagents as an antibacterial agent. Moreover, the antibody-labeled alkaline phosphatase technology of the present invention can effectively increase the labeling amount of alkaline phosphatase on a single molecule by using the polyhydroxyl groups on chitosan. The labeled end uses antibodies to connect with polyhydroxy chitosan and alkaline phosphatase. By controlling the feed ratio, multiple alkaline phosphatase molecules can be connected to one chitosan molecule to achieve the effect of improving sensitivity. At the same time, the introduction of reduced glutathione, sugars and surfactants in the buffer has a very good improvement on the stability of the enzyme.

[0051] 3. The present invention can significantly improve the sensitivity of the reagent by controlling the reaction ratio of the antibody and the enzyme, and has a high antibody utilization rate, which is very effective in cost saving.

[0052] 4. The Tau protein (TAU) kit of the present invention has high sensitivity and specificity, a wider linear range, better process repeatability, good stability, easy implementation, and convenient detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The luminescence standard curve was tested for the calibrator of Example 1.

[0054] Figure 2 The correlation between the test results of the kit prepared in Example 1 and the Abcam kit (CE certified).

[0055] Figure 3 The state diagram of magnetic beads with single-arm biotin + tetrameric streptavidin in conventional buffer;

[0056] Figure 4 This is a diagram of the state of the magnetic beads of single-arm biotin + tetrameric streptavidin in the magnetic bead storage solution of Example 1.

[0057] Figure 5 This is a diagram of the state of magnetic beads containing double-arm biotin + octamer streptavidin in a conventional buffer.

[0058] Figure 6 This is a diagram of the state of magnetic beads in the magnetic bead storage solution of Example 1. DETAILED DESCRIPTION

[0059] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the accompanying drawings, but the present invention is not limited to these embodiments.

[0060] The raw materials used in the following examples are as follows:

[0061] Embodiment 1:

[0062] Preparation of R1 reagent:

[0063] S1. Add 10 mg of tosyl magnetic particles into an EP tube;

[0064] S2, EP tubes were placed on a magnetic rack and washed three times with coupling buffer; the coupling buffer was 0.1M Hepes buffer with a pH of 8.0;

[0065] S3. Add 0.05 mg of octamer streptavidin and 3 M ammonium sulfate in an amount equal to 1 times the total volume of the magnetic particles, and then suspend at 37° C. for 18 h.

[0066] S4, add 0.1mL blocking agent and suspend at 37℃ for 18h, then wash with TBS buffer and replace with magnetic bead storage solution, the final concentration of magnetic particles is 10mg / mL, and finally dilute to 0.5mg / mL for storage;

[0067] The blocking agent used was CE210 from JSR, and 100 μL was added per 10 mg of magnetic particles. The TBS buffer contained 0.1 wt% Tween20;

[0068] The pH value of the magnetic bead storage solution is 8, and it includes: 0.02 mol / L Hepes, 0.2 mol / L sodium chloride, 1 wt% BSA, 0.1 wt% bovine gamma, 0.1 wt% Proclin 950, 0.1 wt% tween 20, 1 wt% mannitol, and 1 wt% gelatin.

[0069] Preparation of R2 reagent:

[0070] S1. Take 1 mg of Tau antibody and dialyze it overnight with pH 7.4 PBS buffer at 2-8°C to prepare a Tau antibody solution with a concentration of 1 mg / mL;

[0071] S2, dissolving N-hydroxysuccinimide biotin ester in dimethyl sulfoxide to prepare an N-hydroxysuccinimide biotin ester solution, wherein the molar concentration of the N-hydroxysuccinimide biotin ester is 10 mM;

[0072] S3, adding the N-hydroxysuccinimide biotin ester solution to the Tau antibody solution and mixing evenly;

[0073] S4. Then place it at 26°C for 30 minutes, add 1.0M Tris buffer to terminate the reaction, place it at 2-8°C for 10 minutes, and finally dilute it to a concentration of 1.0ug / mL for storage.

[0074] Preparation of R3 reagent:

[0075] S1. Take 5 mg of chitosan, dissolve it in pure water to 50 mg / mL, add 1.0 mL of 0.03 M sodium periodate solution, and react in the dark. After dialysis, add 16 μL of ethylene glycol, react at room temperature for 1 hour, and terminate the reaction.

[0076] S2. Dilute the oxidized chitosan with 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 solution to a concentration of 20 mg / mL. Take 1 mL of the solution, add 0.2 mg of Tau antibody, and add 4 mg of alkaline phosphatase and stir evenly.

[0077] S3, adding 0.2 mL 1M sodium borohydride per mL of the reaction solution, reacting at room temperature for at least 6 hours, adding 0.2 mL 1M Tris per mL of the reaction solution to terminate the reaction, and reacting at room temperature for 2 hours;

[0078] S4, purifying the protein-chitosan conjugate from the unconjugated protein and chitosan by gel filtration using Sephacryl S-200, purifying the conjugate, and storing it at 2-8°C for future use;

[0079] S5. Dilute the purified conjugate with enzyme diluent to a final concentration of 1.0 μg / mL;

[0080] Enzyme dilution pH 7.2, 0.02moL / L Hepes, 0.1moL / L sodium chloride, 10‰ BSA, 5‰ gelatin, 5‰ trehalose, 2‰ mannitol, 1‰ reduced glutathione, 50mg / L blocking agent, 0.2% P300 preservative.

[0081] Preparation of calibrators and quality control products:

[0082] Take 0.6g Tris, 5g BSA, 0.8g sodium chloride, 0.1ml 1mol magnesium chloride, 0.1ml 0.1mol zinc chloride, and 0.2g P300 and add them to a container, then add 100ml purified water and stir evenly to obtain a buffer solution.

[0083] Tau antigen was prepared into calibrators with concentrations of 2.0, 50, 500, 5000, and 10000 pg / mL using a buffer solution.

[0084] Tau antigen was prepared into quality control products with concentrations of 20, 200, and 2000 pg / mL using buffer.

[0085] Application Example 1:

[0086] 20 μL of the sample to be tested was reacted with 50 μL of R2 reagent and 50 μL of R3 reagent for 10 min, and then 50 μL of R1 reagent was added to react for 5 min to form a sandwich complex magnetic suspension. The sandwich complex magnetic suspension was placed in a magnetic field for separation, and after washing, a chemiluminescent substrate solution was added to detect the chemiluminescent photon intensity.

[0087] The calibration samples of 0pg / mL, 2.0pg / mL, 50pg / mL, 500pg / mL, 5000pg / mL, and 10000pg / mL were tested in parallel, and the average luminescence value was calculated, and the standard curve was fitted using a four-parameter equation. The luminescence values ​​of the calibration samples are shown in Table 1.

[0088] Table 1: Luminescence value results of the kit calibrator test in Example 1

[0089]

[0090] Figure 1 The luminescence standard curve of the calibration product in Example 1 is tested by combining Table 1 and Figure 1 The results showed that the Tau protein (TAU) kit had a good reaction gradient for the calibrator and the curve fitting degree R2 was close to 1.

[0091] Blank limit performance test:

[0092] The luminescence value (RLU) of the zero-value calibrator was measured in parallel 20 times, and its mean (M) and standard deviation (SD) were calculated to obtain the RLU value corresponding to M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the concentration-luminescence value (RLU) results between the zero-concentration calibrator and the adjacent calibrator. The M+2SD luminescence value was substituted into the above equation to obtain the corresponding concentration value, which was not higher than the blank detection limit (0.5 pg / mL). The results are shown in Table 2.

[0093] Table 2: Blank limit performance test results of the test kit in Example 1

[0094]

[0095] As can be seen from Table 2, the blank limit of the kit prepared in Example is 0.066 pg / mL, which meets the requirement of not higher than 0.5 pg / mL.

[0096] Linearity test:

[0097] Take a high-concentration sample close to the upper limit of the linear range and a low-concentration sample close to the lower limit of the linear range, and mix them into at least 5 dilution concentrations according to a certain ratio. Test each dilution concentration twice and calculate the mean of the test results. Take the dilution concentration as the independent variable and the mean of the test results as the dependent variable to obtain the linear regression equation and calculate the correlation coefficient r. The results are shown in Table 3.

[0098] Table 3: Example 1 Kit Linearity Test Results

[0099]

[0100]

[0101] It can be seen from Table 3 that within the concentration range of 1 to 10000 pg / ml, the correlation coefficient r>0.99, and the detection range of the kit prepared in Example 1 is 1 to 10000 pg / ml.

[0102] Precision test:

[0103] Take one copy of the high value quality control product, the middle value quality control product and the low value quality control product, repeat the test 10 times, calculate the average value and standard deviation. According to the formula: coefficient of variation CV = (standard deviation / average value) × 100%, calculate the precision, and the results are shown in Table 4.

[0104] Table 4: Precision test results of the kit of Example 1

[0105] QCL QCM QTC 1 21.3 197.83 1990.5 2 20.65 189.4 1977.29 3 20.08 191.73 1882.59 4 20.46 186.28 1922.34 5 20.22 200.74 2039.51 6 20.92 198.26 1979.17 7 21.1 192.93 2033.52 8 21.19 197.37 2028.82 9 20.77 204.13 1934.62 10 20.15 202.52 1917.14 X 20.684 196.119 1970.55 SD 0.445750304 5.840118815 54.60829058 CV 2.16％ 2.98％ 2.77％

[0106] As can be seen from Table 4, the CV of the high-value quality control product is 2.77%; the CV of the middle-value quality control product is 2.98%; and the CV of the low-value quality control product is 2.16%, indicating that the kit prepared in Example 1 has good precision.

[0107] Stability test:

[0108] The reagents were stored at 37°C and 2-8°C for follow-up testing. The reagents stored at 37°C were tested twice for luminescence value (RLU) on day 0, day 3, day 7, and day 9. The average value was calculated, and the relative deviation was calculated from the result on day 0. The results are shown in Table 5.

[0109] Table 5: Stability test results of the kit of Example 1 on day 0, day 3, day 7 and day 9

[0110]

[0111] It can be seen from Table 5 that all relative deviations of the kit prepared in Example 1 are <10%, indicating that the kit prepared in Example 1 has good stability.

[0112] The reagents stored at 2-8°C were tested for luminescence value (RLU) twice at month 0, month 6, month 9, month 12 and month 14. The average value was calculated, and the relative deviation was calculated compared with the result at month 0. The results are shown in Table 6.

[0113] Table 6: Kit stability test results at 0th, 6th, 9th, 12th and 14th months

[0114]

[0115] It can be seen from Table 6 that all relative deviations of the kit prepared in Example 1 are <15%, indicating that the kit prepared in Example 1 has good stability.

[0116] Table 7: Comparison results of the kits of Example 1 and Comparative Example 1

[0117]

[0118] Figure 2 This is a state diagram of magnetic beads of single-arm biotin + tetramer streptavidin in conventional buffer. It can be seen from the figure that the state of the magnetic particles is poor and there is a more obvious aggregation state. When stored in this buffer, the reagent sensitivity is low and the stability is poor.

[0119] Figure 3 This is a diagram of the state of the magnetic beads of single-arm biotin + tetramer streptavidin in the magnetic bead storage solution of Example 1; it can be seen from the figure that the state of the magnetic particles has been greatly improved, the magnetic particles are dispersed, and there is no obvious aggregation. The reagent sensitivity is significantly improved compared with condition -1, and the stability is also significantly improved.

[0120] Figure 4 This is a state diagram of the magnetic beads of double-arm biotin + octamer streptavidin in a conventional buffer solution. It can be seen from the figure that the state of the magnetic particles is poor and there is a more obvious aggregation state. When stored in this buffer solution, the sensitivity of the reagent is significantly improved, but the stability of the reagent is not good.

[0121] Figure 5 This is a diagram of the state of the magnetic beads in the magnetic bead storage solution of Example 1; it can be seen from the figure that the state of the magnetic particles has been greatly improved, the magnetic particles are dispersed, there is no obvious aggregation, the reagent sensitivity has been significantly improved, and the stability has been significantly improved.

[0122] Table 8: Comparison of enzyme labeling methods and buffers

[0123]

[0124]

[0125] The reaction method and buffer used in the present invention significantly improve stability and sensitivity.

[0126] Figure 6This is the correlation between the test results of the kit prepared in Example 1 and the Abcam kit (CE certified). From the figure, it can be seen that the linear regression equation of the two reagents is Y=0.9524X+3.0844, and the correlation coefficient is R2=0.9944. This shows that the Tau protein kit prepared in Example 1 has good consistency with the control reagent in the test sample.

[0127] In summary, the Tau protein (Tau) magnetic particle chemiluminescence detection kit of the present invention adopts the combination mode of long-arm biotin N-hydroxysuccinimide biotin and octamer streptavidin, which greatly improves the sensitivity, effectively avoids the interference of endogenous biotin in the sample on the detection result, and can effectively improve the utilization rate of antibodies and reduce costs; the octamer streptavidin used in the present invention has more high-affinity sites than conventional tetramer streptavidin, can bind more biotin linkers, and can reduce steric hindrance and improve sensitivity when combined with N-hydroxysuccinimide biotin.

[0128] The parts of the technical scope of the present invention that are not exhausted by the midpoint value of the embodiment and the new technical solutions formed by the equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of protection claimed by the present invention, and unless otherwise specified, there is no unique and irreplaceable combination between all the parameters involved in the solutions of the present invention.

[0129] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0130] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. A Tau protein detection kit, characterized in that: The kit includes R1 reagent, R2 reagent and R3 reagent; The R1 reagent includes magnetic particles containing octamer streptavidin; The R2 reagent includes a Tau antibody labeled with N-hydroxysuccinimide biotin; The R3 reagent includes an alkaline phosphatase-labeled Tau antibody; The method for preparing an alkaline phosphatase-labeled Tau antibody comprises the following steps: S1, reacting the chitosan solution with the sodium periodate solution in the dark, and adding ethylene glycol to terminate the reaction after dialysis; S2, then add alkaline phosphatase and Tau antibody and stir evenly, then add sodium borohydride to react, and finally add Tris buffer to terminate the reaction; S3, after purification by gel filtration, dilute with enzyme diluent for storage; The mass ratio of Tau antibody to chitosan and alkaline phosphatase is 1:(80-120):(15-30).

2. A Tau protein detection kit according to claim 1, characterized in that: The concentration of magnetic microparticles containing octamer streptavidin in the R1 reagent is 0.25-1.0 mg / mL.

3. A Tau protein detection kit according to claim 1 or 2, characterized in that: The magnetic microparticles containing octamer streptavidin are prepared by mixing octamer streptavidin with magnetic particles at a mass ratio of (0.005-0.2):10 and stored in a magnetic bead storage solution.

4. A Tau protein detection kit according to claim 3, characterized in that: The magnetic bead storage solution has a pH of 6-9 and includes: 0.01-0.05 mol / L Hepes, 0.1-0.5 mol / L sodium chloride, 0.1-10wt% BSA, casein, one or more of bovine gamma, 0.1-5wt% Proclin 950, 0.1-5 wt% tween20, 0.1-5wt% mannitol and 0.5-10wt% gelatin.

5. A Tau protein detection kit according to claim 1, characterized in that: The concentration of N-hydroxysuccinimide-biotin labeled Tau antibody in R2 reagent is 0.05-5ug / mL.

6. A Tau protein detection kit according to claim 1 or 5, characterized in that: The preparation method of the Tau antibody labeled with N-hydroxysuccinimide biotin comprises the following steps: S1. Dialyze the Tau antibody against PBS buffer to obtain a Tau antibody solution; S2, then dissolving N-hydroxysuccinimide biotin ester in dimethyl sulfoxide to obtain an N-hydroxysuccinimide biotin ester solution; S3. Evenly mix the Tau antibody solution and the N-hydroxysuccinimide biotin ester solution, then let the reaction stand, add tris(hydroxymethylaminomethane) buffer to terminate the reaction, and finally dilute and store.

7. A Tau protein detection kit according to claim 6, characterized in that: The molar ratio of N-hydroxysuccinimide biotin ester to Tau antibody was (8-12):

1.

8. A Tau protein detection kit according to claim 1, characterized in that: The concentration of alkaline phosphatase-labeled Tau antibody in R3 reagent is 0.03-5ug / mL.

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