A kit for detecting alzheimer's disease-related proteins and use thereof

Abstract

The application provides a kit for detecting Alzheimer's disease related protein and application thereof, the kit improves the stability of the Alzheimer's disease related protein in the detection sample by adding sucrose in the fluorescence reagent diluent, adds HEPES in the sample diluent, and adds blocking agents HBR6 and HBR11 in the reaction buffer to improve the specific binding capacity of the related protein, so that the content of the Alzheimer's disease related protein in the sample can be accurately detected through 2.5-step room temperature shaking incubation, and the detection time is greatly reduced; the working concentration of SA-PE, the mixed molar ratio of the antibody and biotin, and the incubation condition are also optimized. The kit provided by the application is simple to operate, improves the detection efficiency, accuracy, specificity and sensitivity, is convenient for clinical detection application, can reduce the cost of reagents and labor, has good repeatability, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of flow cytometry, and particularly relates to a kit for detecting Alzheimer disease-related proteins and application thereof. BACKGROUND

[0002] Alzheimer disease (AD), commonly known as senile dementia, is a progressive central nervous system degenerative disease with insidious onset. The typical histopathological changes of Alzheimer disease are senile plaques caused by β-amyloid (Aβ) deposition, neurofibrillary tangles caused by abnormal phosphorylation of Tau protein, and loss of neurons and synapses. According to the amyloid cascade hypothesis, Aβ pathology is an upstream event of AD, driving the occurrence of neocortical tau pathology and neurodegeneration. Aβ1-42, Aβ1-40, T-Tau and P-Tau are currently widely recognized as AD biomarkers. Studies have shown that the expression of Aβ1-42 and Aβ1-42 / Aβ1-40 is down-regulated, while the expression of T-Tau and p-Tau-181 is up-regulated in the plasma of AD patients.

[0003] Aβ1-42 and Aβ1-40 are produced by amyloid precursor protein (APP) after hydrolysis by BACE and γ-secretase, and can gradually accumulate outside the membrane to form Aβ protein polymers, which have toxic effects on neurons, leading to neuronal degeneration. Compared with Aβ1-40, Aβ1-42 has very high aggregation, and gradually accumulates in the early stage of senile plaque formation. The content of plasma Aβ1-42 and the ratio of Aβ1-42 / Aβ1-40 reflect the Aβ pathology in the brain, and can be used to assess the risk of AD dementia and Mild Cognitive Impairment (MCI). Among them, Aβ1-42 can even decrease before Amyloid-β positron emission tomography (Aβ-PET) abnormalities.

[0004] Tau protein is a microtubule-associated protein (MAPs) with high content in neurons of central nervous system, and its main function is to regulate the stability of axon microtubules. It is also a phosphoprotein with multiple phosphorylation sites, among which the most studied are p-Tau-181 and p-Tau-217. Phosphorylated Tau competes with tubulin to bind normal Tau and other microtubule-associated proteins, leading to microtubule depolymerization and forming paired helical filaments (PHF) in neurons, affecting the normal physiological function of Tau protein. Studies have shown that the concentration of plasma T-Tau increases in Creutzfeldt-Jakob disease (CJD) and frontotemporal lobar dementia (FTD), and is an important predictor of CJD. The levels of plasma p-Tau-181 and p-Tau-217 can reflect the pathological conditions of Aβ and Tau in AD, and can distinguish AD from other neurodegenerative diseases (such as Parkinson's disease and vascular dementia, etc.), and can monitor the progression of AD patients through the levels of plasma p-Tau-181 and p-Tau-217 during the entire clinical process.

[0005] In summary, the combined detection of Aβ1-42, Aβ1-40, p-Tau-181 and p-Tau-217 can improve the accuracy of AD determination. Therefore, by jointly detecting the concentrations of Aβ1-42, Aβ1-40, p-Tau-181 and p-Tau-217 in human plasma, it can be used for the auxiliary diagnosis of AD.

[0006] Currently, the main method for capturing Aβ1-42, Aβ1-40, p-Tau-181 and p-Tau-217 in human blood samples is the double antibody sandwich method, which can be detected by chemiluminescence or flow cytometry. The selectivity and anti-interference ability of chemiluminescence are poor, which means that in complex samples, chemiluminescence may not be able to accurately distinguish specific analytes, or be easily interfered by other components, resulting in inaccurate results. Moreover, using chemiluminescence can only detect Aβ1-42, Aβ1-40, p-Tau-181 and p-Tau-217 individually, and the performance of reagent sensitivity, precision, etc. is poor, and the detection time is long and the operation is complex. Although flow cytometry can detect these markers at the same time, the detection process may be affected by many non-specific interferences, affecting the accuracy.

[0007] The team of the present application once applied for invention patent CN2023111107302 in 2023, which discloses a method for simultaneously detecting the contents of Aβ1-42, Aβ1-40, T-Tau, p-Tau-181 and alpha-synuclein in a sample after two-step incubation at a low temperature of 2-8℃. Although this method significantly improves the sensitivity, accuracy and specificity of Alzheimer's disease-related protein detection, the operation process is complicated, multiple types of reagents are added, and the incubation time is long, resulting in high time cost of detecting samples. Long incubation time also increases uncontrollable factors, affecting the detection results.

[0008] Therefore, there is an urgent need for a new kit and method for detecting Alzheimer's disease-related proteins to solve the problems in the prior art. SUMMARY

[0009] In view of the problems in the prior art, the present application provides a kit for detecting Alzheimer's disease-related proteins and its application. The kit adds sucrose to the fluorescence reagent diluent to improve the stability of detecting Alzheimer's disease-related proteins in human blood, adds HEPES to the sample diluent, and adds blocking agents HBR6 and HBR11 to the reaction buffer to improve the specific binding capacity of the related proteins, thereby accurately detecting the content of Alzheimer's disease-related proteins in the sample through 2.5-step room temperature shaking incubation, greatly reducing the detection time. The present application also optimizes the working concentration of SA-PE, the mixed molar ratio of antibody and biotin, and the incubation conditions. The kit provided by the present application is simple to operate, improves the detection efficiency and accuracy, specificity and sensitivity, and the detection sensitivity of Aβ1-40 reaches 0.154 pg / mL, the detection sensitivity of Aβ1-42 reaches 0.181 pg / mL, the detection sensitivity of p-Tau-181 reaches 0.169 pg / mL, and the detection sensitivity of p-Tau-217 reaches 0.049 pg / mL. In addition, the present application also facilitates clinical detection application, can reduce the cost of reagents and labor, has good repeatability, and has good application prospect.

[0010] In one aspect, the present application provides a kit for detecting Alzheimer's disease-related proteins, which comprises a microsphere solution coupled with antibodies, a biotin-coupled antibody solution and a fluorescence reagent. The fluorescence reagent contains SA-PE diluent, and the SA-PE diluent contains sucrose. The antibodies are one or more of Aβ1-40 antibody, Aβ1-42 antibody, p-Tau-181 antibody and p-Tau-217 antibody.

[0011] The present application proves through a large number of researches that the purchased SA-PE solution is not stable, and when it is used to prepare a fluorescent reagent, the final detection result is easily affected due to unstable phycoerythrin (PE) coupling effect. The saccharide has viscosity, can wrap up protein molecules, form a carbohydrate vitreous body similar to glass ice in structure, block the chain forging movement of macromolecular substances, prevent protein extension and precipitation, and maintain the stability of the three-dimensional structure of protein molecules, thereby playing a protection role. The addition of sucrose to the SA-PE diluent for preparing the fluorescent reagent can play a stabilizing and protecting role on streptavidin (SA) and phycoerythrin (PE), and further improve the stability and accuracy of the reagent kit detection.

[0012] In some modes, the components of the fluorescent reagent include NaCl, sucrose, Tris, Proclin300 preservative and SA-PE.

[0013] Further, the reagent kit further comprises a reaction buffer containing blocking agents HBR6 and HBR11.

[0014] HBR is a heterophilic antibody blocking reagent (Heterophilic Antibody Blocking Reagent), which is mainly composed of core active components (such as animal-derived IgG, artificially synthesized polypeptide / antibody) and auxiliary components (buffer, BSA stabilizer, preservative, etc.). Its main role is to bind to heterophilic antibodies in the sample in advance, occupy their binding sites, avoid cross-reactions between heterophilic antibodies and detection reagents, thereby improving the accuracy of the detection result and reducing the occurrence of false positives. Heterophilic antibodies are endogenous autoantibodies that can non-specifically bind to animal immunoglobulins, which are secreted by the human immune system stimulated by known or unknown antigenic substances. Especially through close contact with animals, eating contaminated food, receiving treatment from monoclonal animal antibody preparations, infection, blood transfusion, etc. can become the cause of the production of heterophilic antibodies. The sample detected by the reagent kit of the present application is human blood sample, which contains heterophilic antibodies and can interfere with the detection. Its interference principle is that heterophilic antibodies can bind to the Fc (crystallizable fragment) and Fab (antigen binding fragment) epitopes of immunoglobulins, so the fluorescent microspheres of the conjugated antibody cannot capture the antigen to be detected in the blood sample, resulting in false positives. The blocking agents HBR6 and HBR11 used in the present application are purchased from Scantibodies company, which can prevent the binding of heterophilic antibodies in the sample to the fluorescent microspheres of the conjugated antibody, and can block the sites without biological regulation to reduce non-specific binding. At the same time, after adding the blocking agent to the reaction buffer during the detection process, the detection accuracy is significantly improved.

[0015] The present application screens different blocking agents on the market, and finally selects a combination of 5% HBR6 and 5% HBR11 as the blocking agent added in the reaction buffer, which can effectively eliminate false positives and prevent non-specific binding.

[0016] In some ways, the components of the reaction buffer include NaCl, Tris, BSA, HBR6, HBR11, Proclin30 preservative, Tween-20.

[0017] The light incubation time of the five proteins Aβ1-42, Aβ1-40, T-Tau, p-Tau-181, and α-synuclein in CN2023111107302 is as long as 18.5 hours, resulting in high time cost of the detection sample. Long incubation time also increases uncontrollable factors, affecting the accuracy of the detection result. Moreover, an incubation agent must be added before incubation, and the incubation condition is 2-8℃ oscillation incubation. The raw materials for preparing the incubation agent include sorbitol, polyvinylpyrrolidone, Triton X-100, sodium citrate, sodium hyaluronate, and Tris. The subsequent washing may not be complete, which may affect the accuracy and sensitivity of the detection result. The present application adds the blocking agents HBR6 and HBR11 in the reaction buffer and sucrose in the SA-PE diluent for preparing the fluorescent reagent, so that the incubation agent is no longer needed, and a 2.5-step normal temperature incubation method is realized. The total incubation time is only 2.5 hours at the shortest, which not only shortens the sample detection time, but also improves the accuracy, specificity, and sensitivity of the detection of Aβ1-42, Aβ1-40, p-Tau-181, and p-Tau-217.

[0018] Further, the fluorescent reagent further contains SA-PE, and the concentration of the SA-PE diluted by the SA-PE diluent is 0.25-2 μg / mL.

[0019] In some ways, the concentration of the SA-PE diluted by the SA-PE diluent is 1 μg / mL.

[0020] Further, the kit further includes a sample diluent, and the sample diluent contains HEPES.

[0021] HEPES is an amphoteric ion buffer agent containing both a weak acidic group (sulfonic acid group) and a weak basic group (piperazine nitrogen atom), which can accept and give protons in a certain pH range, better buffer the pH fluctuation of the external environment, and maintain the stability of the protein.

[0022] In some ways, the components of the sample diluent include NaCl, Proclin300, and HEPES.

[0023] Furthermore, the molar ratio of antibody to biotin in the biotin-coupled antibody solution is 1:(10-60).

[0024] In some embodiments, the ratio of antibody to biotin is preferably 1:40.

[0025] Furthermore, the Aβ1-40 antibody is a mouse anti-human antibody, the Aβ1-42 antibody is a mouse anti-human antibody, the p-Tau-181 antibody is a mouse anti-human antibody, and the p-Tau-217 antibody is a rabbit anti-human antibody.

[0026] Furthermore, the kit also includes a washing buffer.

[0027] In some embodiments, the wash buffer comprises KH2PO4, K2HPO4·12H2O, NaCl, KCl, BSA, Proclin 300 preservative, and Tween-20.

[0028] Furthermore, the kit also includes calibrators and quality control products.

[0029] The present invention also optimizes the detection steps of the kit for detecting Alzheimer's disease-related proteins. After the blood sample is incubated in 2.5 steps, it is placed in a flow cytometer for detection, which greatly reduces the detection time and improves the detection efficiency.

[0030] The detection principle of the kit is that fluorescent microsphere-coupled antibody proteins and biotin-coupled antibody proteins jointly capture the target protein in the test sample. The biotin-coupled antibody protein then binds to streptavidin-coupled phycoerythrin, forming an immune complex. During detection, the immune complex is illuminated by two beams of excitation light of different wavelengths emitted by a flow cytometer. The fluorescence intensity of the different fluorescent microspheres determines the type of detection indicator, and the fluorescence intensity of phycoerythrin determines the content of each detection indicator.

[0031] In another aspect, the present invention provides a method for detecting an Alzheimer's disease-related protein, using the above-mentioned kit for detection, comprising the following steps:

[0032] (1) Add reaction buffer and antibody-coupled microsphere solution to the sample tube and mix well;

[0033] (2) Add the sample to be tested into the sample tube, mix well and incubate;

[0034] (3) After washing, add biotin-conjugated antibody solution to the sample tube, mix well and incubate;

[0035] (4) Add fluorescent reagent to the sample tube, mix well and incubate;

[0036] (5) Detect the content of Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 on a flow cytometer.

[0037] It is found through research that, in addition to maintaining protein stability, adding sucrose to the SA-PE diluent can keep the conjugated antibody microspheres in a suspended state during incubation, reduce the probability of agglomeration and cross-linking of the conjugated antibody microspheres, and thus achieve the effect of promoting the reaction. On the one hand, sucrose increases the density and viscosity of the solution, helping the fluorescent microspheres to maintain stable suspension in the solution, and on the other hand, it can wrap the conjugated antibodies on the microspheres to form a carbohydrate vitreous body similar in structure to glass ice, blocking the chain forging movement of macromolecular substances and preventing protein extension and precipitation. Therefore, adding sucrose to the SA-PE diluent can allow the reactants to fully react, improving the accuracy of detection.

[0038] Further, the incubation condition in step (2) is incubation at 4-37℃ under light protection and oscillation for 1-6 hours; and the incubation condition in step (3) is incubation at 4-37℃ under light protection and oscillation for 1-6 hours.

[0039] In some modes, the incubation condition in step (2) is incubation at 25℃ under light protection and oscillation for 1 hour; and the incubation condition in step (3) is incubation at 25℃ under light protection and oscillation for 1 hour.

[0040] Further, the incubation condition in step (4) is incubation at 4-37℃ under light protection and oscillation for 0.5 hours.

[0041] In actual application, the incubation temperature in step (4) in the range of 4-37℃ can make the detection result more accurate.

[0042] In some modes, the incubation temperature in step (4) is most preferably 25℃.

[0043] In another aspect, the application also provides the use of the SA-PE diluent and the blocking agent composition in the preparation of a kit for shortening the incubation time of Alzheimer's disease-related protein detection, wherein the SA-PE diluent comprises sucrose, and the blocking agent composition comprises HBR6 and HBR11.

[0044] In some modes, experiments prove that, by adding sucrose to the SA-PE diluent for preparing fluorescent reagents and adding the blocking agent composition HBR6 and HBR11 to the reaction buffer, the incubation time in the detection process can be effectively shortened, and the total incubation time is only 2.5 hours at the shortest, and normal temperature (25℃) incubation is achieved.

[0045] In still another aspect, the present application provides a use of a blocking agent composition comprising HBR6 and HBR11 for preparing a reagent for improving the specific binding ability of detecting Alzheimer's disease related proteins in human blood.

[0046] In some ways, by preparing six different reaction buffers by combining HBR6, HBR11, HBR9 and HBR2 at a concentration of 5% in pairs, respectively detecting solutions containing no AD markers but containing interfering substances, the detection results prove that the blocking agent composition of 5% HBR6 and 5% HBR11 can effectively reduce the detection value of interfering substances; further detecting AD related proteins in blood samples by using the six different reaction buffers, and comparing the detection results with the mass spectrometry detection results, it is proved that the blocking agent composition of 5% HBR6 and 5% HBR11 can effectively reduce the influence of interfering substances on the detection of AD markers, thereby improving the accuracy of detection.

[0047] The present application has the following beneficial effects:

[0048] 1. By adding sucrose and adjusting the proportion relationship in the SA-PE diluent, the stability and accuracy of the detection are improved;

[0049] 2. By adding the blocking agent composition of HBR6 and HBR11 in the reaction buffer, the occurrence of affinity antibody interference is minimized, thereby reducing the occurrence of false positives, enhancing the specific binding ability, and thereby improving the accuracy of detection;

[0050] 3. A 2.5-step incubation method is provided, which shortens the total incubation time of detection to 2.5h;

[0051] 4. The working concentration of SA-PE, the mixing molar ratio of antibody and biotin, and the incubation conditions are optimized, which effectively improves the accuracy, specificity and sensitivity of detection, the detection sensitivity of Aβ1-40 reaches 0.154pg / mL, the detection sensitivity of Aβ1-42 reaches 0.181pg / mL, the detection sensitivity of p-Tau-181 reaches 0.169pg / mL, and the detection sensitivity of p-Tau-217 reaches 0.049pg / mL;

[0052] 5. The present application reduces the cost of reagents and labor for clinical detection application, has high accuracy, strong stability, high sensitivity, good repeatability, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 The schematic diagram of the detection principle of the kit of Example 1 is shown in the figure;

[0054] Figure 2Linear evaluation results for Aβ1-40 of Example 1;

[0055] Figure 3 Linear evaluation results for Aβ1-42 of Example 1;

[0056] Figure 4 Linear evaluation results for p-Tau-181 of Example 1;

[0057] Figure 5 Linear evaluation results for p-Tau-217 of Example 1. DETAILED DESCRIPTION

[0058] The following further describes the embodiments of the present application, it should be noted that the following embodiments are intended to facilitate the understanding of the present application, and do not have any limiting effect on the present application, all the features disclosed in the embodiments of the present application, or the steps in all the disclosed methods or processes, except for the mutually exclusive features and / or steps, can be combined in any way.

[0059] Example 1: Preparation of kit, detection process and evaluation

[0060] 1. Preparation of kit

[0061] (1) Preparation of fluorescent microsphere solution coupled with antibody

[0062] The four kinds of fluorescent microspheres and the antibodies coupled therewith are shown in Table 1. The A1-A4 fluorescent microspheres in Table 1 are 5 μm carboxyl fluorescent microspheres with different fluorescent intensities, the Aβ1-40 antibody, the Aβ1-42 antibody and the p-Tau-181 antibody are all mouse anti-human monoclonal antibodies, and the p-Tau-217 antibody is a rabbit monoclonal antibody.

[0063] Table 1, fluorescent microspheres and the antibody proteins coupled therewith

[0064]

[0065] Preparation of Aβ1-40 antibody coupled fluorescent microspheres: take 5 μm carboxyl fluorescent microspheres A1 5x10 6The A1 fluorescent microspheres were washed twice with PBST buffer (phosphate buffer containing 0.05% Tween-20, pH 7.4, purchased from Sigma). 100 μg of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, purchased from Sigma) and 50 μg of NHS (N-hydroxysulfosuccinimide sodium salt, purchased from Aladdin) were added to the washed A1 fluorescent microspheres and left for 30 minutes to activate the fluorescent microspheres. 100 μg of mouse anti-human Aβ1-40 antibody was added and rotated at room temperature for 5 hours to wash the fluorescent microspheres to remove excess antibody. After washing the A1 fluorescent microspheres to remove excess mouse anti-human Aβ1-40 antibody, 5% skim milk powder was added for blocking for 30 minutes. After removing the blocking solution, Tris buffer (pH 7.2) was added for storage.

[0066] Preparation of fluorescent microspheres coupled with Aβ1-42 antibodies: 5×10 A2 fluorescent microspheres 6 The A2 fluorescent microspheres were washed twice with PBST buffer, 100 μg of EDC and 50 μg of NHS were added to the washed A2 fluorescent microspheres and allowed to stand for 30 minutes to activate the fluorescent microspheres, 100 μg of mouse anti-human Aβ1-42 antibody was added and rotated at room temperature for 5 hours to wash the fluorescent microspheres to remove excess antibodies, the A2 fluorescent microspheres were washed to remove excess mouse anti-human Aβ1-42 antibodies, and then 5% skim milk powder was added for blocking for 30 minutes. After removing the skim milk powder, Tris buffer with a pH of 7.2 was added for storage;

[0067] Steps for preparing p-Tau-181 antibody-coupled fluorescent microspheres: Take 5×10 A3 fluorescent microspheres 6 The A3 fluorescent microspheres were washed twice with PBST buffer, 100 μg of EDC and 50 μg of NHS were added to the washed A3 fluorescent microspheres and left for 30 minutes to activate the fluorescent microspheres, 100 μg of mouse anti-human p-Tau-181 antibody was added and rotated at room temperature for 5 hours to wash the fluorescent microspheres to remove excess antibodies, the A3 fluorescent microspheres were washed to remove excess mouse anti-human p-Tau-181 antibodies, and then 5% skim milk powder was added for blocking for 30 minutes. After removing the skim milk powder, Tris buffer with a pH of 7.2 was added for storage;

[0068] Steps for preparing p-Tau-217 antibody-coupled fluorescent microspheres: Take 5×10 A4 fluorescent microspheres 6One, add PBST buffer solution to wash 2 times, add 100 μg of EDC and 50 μg of NHS in the A4 fluorescent microspheres after washing to activate the fluorescent microspheres for 30 minutes, add 100 μg of rabbit anti-human p-Tau-217 antibody to rotate the reaction for 5 hours at room temperature to clean the fluorescent microspheres to remove the excess antibody, add 5% skimmed milk powder after cleaning the A4 fluorescent microspheres to remove the excess rabbit anti-human p-Tau-217 antibody for 30 minutes, remove the skimmed milk powder, and store in Tris buffer solution with pH 7.2;

[0069] Take 98 mL of Tris buffer solution with pH 7.2, according to the detection index of the kit, add 0.5 mL of Aβ1-40, Aβ1-42, p-Tau-181, p-Tau-217 antibody coupled fluorescent microspheres respectively, and prepare the fluorescent microsphere solution of the coupled antibody.

[0070] (2) Preparation of biotin-coupled antibody solution

[0071] Preparation of antibody-biotin complex, the preparation steps are as follows:

[0072] Take 100 μg of the mouse anti-human Aβ1-40 antibody, mouse anti-human Aβ1-42 antibody, mouse anti-human p-Tau-181 antibody and rabbit anti-human p-Tau-217 antibody in (1), respectively, add biotin according to the molar ratio of antibody to biotin 1:40, incubate at room temperature for 5 hours, and obtain the biotin-coupled anti-human Aβ1-40 antibody, biotin-coupled anti-human Aβ1-42 antibody, biotin-coupled anti-human p-Tau-181 antibody and biotin-coupled anti-human p-Tau-217 antibody, remove the unbound biotin, and dilute the biotin-coupled anti-human Aβ1-40 antibody, biotin-coupled anti-human Aβ1-42 antibody, biotin-coupled anti-human p-Tau-181 antibody and biotin-coupled anti-human p-Tau-217 antibody to 4 μg / mL respectively with 0.01 mol / L PBS solution;

[0073] Take 25 mL of the above concentration of 4 μg / mL biotin-coupled anti-human Aβ1-40 antibody, biotin-coupled anti-human Aβ1-42 antibody, biotin-coupled anti-human p-Tau-181 antibody and biotin-coupled anti-human p-Tau-217 antibody respectively, then the concentration of each biotin-labeled antibody is 1 μg / mL.

[0074] (3) Preparation of fluorescent reagent

[0075] Preparation of SA-PE dilution solution: 9 g NaCl, 50 g sucrose, 6.057 g Tris were dissolved in 1000 mL pure water, 0.1% mass concentration of Proclin300 preservative was added, and the pH was adjusted to 7.4 for standby. SA-PE was purchased from thermofisher, and SA-PE was diluted to 1 μg / mL using SA-PE dilution solution for standby.

[0076] (4) Preparation of reaction buffer

[0077] 9 g NaCl, 6.057 g Tris were dissolved in 1000 mL pure water, 1% mass concentration of BSA, 5% mass concentration of HBR6 (purchased from Scantibodies company, item number 3KC542) and 5% of HBR11 (purchased from Scantibodies company, 3KC565), 0.1% mass concentration of Proclin300 preservative, 0.1% mass concentration of Tween-20 were added, and the pH was adjusted to 7.4 for standby.

[0078] (5) Preparation of washing buffer

[0079] Prepare the washing buffer (10×), dissolve 2.4 g KH2PO4, 36.32 g K2HPO4·12H2O, 8 g NaCl, 2 g KCl in 1000 mL pure water, add 25 g of BSA, 0.1% mass concentration of Proclin300 preservative, 0.5% mass concentration of Tween-20, mix for standby, and dilute it to 1× with pure water.

[0080] (6) Preparation of sample dilution solution

[0081] 9 g NaCl, 2.38 g HEPES were dissolved in 1000 mL pure water, 0.1% mass concentration of Proclin300 preservative was added, and the pH was adjusted to 7.4 for standby.

[0082] (7) Preparation of calibrators

[0083] Freeze-dried powder Aβ1-40, Aβ1-42, p-Tau-181, p-Tau-217 were diluted to different concentrations using the sample dilution solution to establish the calibration curve when detecting. The concentration of Aβ1-40 was 1250 pg / mL, the concentration of Aβ1-42 was 625 pg / mL, the concentration of p-Tau-181 was 100 pg / mL, and the concentration of p-Tau-217 was 40 pg / mL.

[0084] (8) Preparation of quality control

[0085] The calibrator solution in the above (7) is prepared into low-value quality control 1 and high-value quality control 2 with the sample diluent to judge the performance of the detection result, and the concentrations of the low-value quality control 1 and the high-value quality control 2 are shown in Table 2.

[0086] Table 2, concentration of low-value quality control 1 and high-value quality control 2

[0087]

[0088] 2. Detection process

[0089] The detection process is as follows:

[0090] (1) Add 25 μL of reaction buffer and 25 μL of microsphere solution of conjugated antibody to the sample tube, mix thoroughly, and shake for more than 30 seconds;

[0091] (2) Add 75 μL of sample to the sample tube, mix well after shaking, and incubate at room temperature (25°C) for 1 h in the dark (shaking amplitude is 600-900 r / min). Add 1 mL of 1x washing buffer to the sample tube, resuspend the microspheres by vortexing, centrifuge at 400g for 5 min, and carefully remove the supernatant;

[0092] (3) Add 25 μL of detection antibody reagent to the sample tube, mix well after shaking, and incubate at room temperature (25°C) for 1 h in the dark (shaking amplitude is 600-900 r / min);

[0093] (4) Add 25 μL of fluorescence reagent to the sample tube, mix well after shaking, and incubate at room temperature in the dark for 0.5 h (shaking amplitude is 600-900 r / min). Add 1 mL of 1x washing buffer to the sample tube, resuspend the microspheres by vortexing, centrifuge at 400g for 5 min, and carefully remove the supernatant;

[0094] (5) According to the sample requirements, add 150-300 μL of 1x washing buffer to the tube, resuspend the microspheres by vortexing, and detect the contents of Aβ1-42, Aβ1-40, p-Tau-181, and p-Tau-217 on a flow cytometer.

[0095] Figure 1The figure is a schematic diagram of the relationship between the specific antibody, microspheres, detection antibody and phycoerythrin in this embodiment. Among them, 1 is a fluorescent microsphere, 2 is an antibody protein, 3 is the target protein in the sample to be tested, 4 is an antibody protein, 5 is biotin, 6 is streptavidin, and 7 is phycoerythrin. The antibody protein 2 coupled to the fluorescent microsphere 1 and the antibody protein 4 coupled to the biotin 5 jointly capture the target protein 3, and then the biotin 5 coupled to the antibody protein 4 is combined with the streptavidin 6 coupled to the phycoerythrin 7, and they together form an immune complex. During the detection, two beams of excitation light of different wavelengths emitted by the flow cytometer illuminate the immune complex, and the type of detection index is determined by the fluorescence intensity of the different fluorescent microspheres 1, and the content of each detection index is determined by the fluorescence intensity of the phycoerythrin 7.

[0096] 3. Make a calibration curve

[0097] Calibrators 1250 pg / mL Aβ1-40, 625 pg / mL Aβ1-42, 100 pg / mL p-Tau-181, and 40 pg / mL p-Tau-217 were diluted two-fold to eight different concentrations (xi). The test results were obtained according to the detection process described in this example. Each concentration was tested three times, and the mean value (yi) of the test results was calculated to create a calibration curve.

[0098] 4. Performance Evaluation

[0099] (1) Linear evaluation

[0100] The linearity of the standard curve prepared in 3 above was evaluated. The linear regression equation was obtained with the dilution concentration (xi) as the independent variable and the mean value of the test result (yi) as the dependent variable. The correlation coefficient (R) of the linear regression was calculated.

[0101] Figure 2 This is the linear evaluation result of Aβ1-40 in the embodiment of the present invention, and its linear regression equation is y=1.042x-6.3501, R 2 =0.9997; Figure 3 This is the linear evaluation result of Aβ1-42 in the embodiment of the present invention. Its linear regression equation is y=0.9733x+0.5117, R 2 =1; Figure 4 The linear evaluation result of p-Tau-181 in the embodiment of the present invention is shown in FIG. 1 , wherein the linear regression equation is y=1.0087x-0.144, and R 2 =0.9999; Figure 5 This is the linear evaluation result of p-Tau-217 in the embodiment of the present invention, and its linear regression equation is y=0.9454x+0.1494, R 2 =0.9999. R of the four linear regression equations 2All greater than 0.99, indicating good linearity.

[0102] (2) Blank limit and detection limit evaluation

[0103] Blank limit and detection limit detection method: 1 flow cytometer; 2 reagent batches (1 and 2); 3 test days; 5 blank samples, 5 low concentration level samples; each sample was measured 4 times. The number of blank sample and low concentration level sample test results was 60 respectively, and the classic method was used to evaluate the blank limit (LoB) and detection limit (LoD). Default α = β = 0.05. The blank sample test results were non-normal distribution, and the low concentration sample test results were normal distribution. The blank limit of Aβ1-40 was 0.154 pg / mL, the blank limit of Aβ1-42 was 0.181 pg / mL, the blank limit of p-Tau-181 was 0.169 pg / mL, and the blank limit of p-Tau-217 was 0.049 pg / mL.

[0104] Blank limit verification method: zero concentration calibrator was used as sample for detection, according to the detection process described in this embodiment, 20 times of repeated determination was carried out, the concentration value of 20 times of measurement results was obtained according to the curve equation of the calibrator used in the kit, the average value (M) and the standard deviation (SD) were calculated, and M+2SD was obtained. Compared with the blank limit. The blank limit verification results are shown in Table 3.

[0105] Table 3, blank limit verification results

[0106]

[0107] According to the results in Table 3, the M+2SD values of Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 are all lower than the blank limit value, which shows that the setting of the blank limit of the kit is basically reasonable.

[0108] Detection limit verification method: 5 low concentration samples (the low concentration is approximately equal to the detection limit, which is slightly higher than the blank limit) were detected, each sample was detected 5 times, and the detection results were sorted according to the size. The number of detection results lower than the blank limit (the blank limits of Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 were 0.154 pg / mL, 0.181 pg / mL, 0.169 pg / mL and 0.049 pg / mL respectively) should be less than or equal to 3. The detection limit verification results are shown in Table 4.

[0109] Table 4, detection limit verification results

[0110]

[0111] According to Table 4, the number of parallel five detection results of each sample Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 less than or equal to 3 is less than or equal to 3, which indicates that the detection limit of the kit is basically reasonable.

[0112] (3) repeatability evaluation

[0113] According to the detection process provided in the embodiment, the Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 samples of two concentration levels of high-value quality control 2 and low-value quality control 1 are detected respectively, each repeated detection is 10 times, the average value M and the standard deviation SD of 10 results are calculated, and the coefficient of variation CV is calculated. The detection results are shown in Table 5.

[0114] Table 5, repeatability test results

[0115]

[0116] According to the results in Table 5, the repeatability coefficients of variation CV of Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 high-value and low-value calibrators are all within 10%. Although there is a certain discreteness, but from the confidence limit specified by the standard deviation, this discreteness is reliable, which indicates that the kit has good stability.

[0117] (4) batch difference evaluation

[0118] Take three batches of kits, according to the detection process provided in the embodiment, detect the same 1 reference sample repeatedly, the reference sample is quality control 2, repeat detection 10 times, calculate the average value M and the standard deviation SD of 30 measurement results, calculate the coefficient of variation CV, and the batch difference detection results are shown in Table 6.

[0119] Table 6, batch difference detection results

[0120]

[0121] According to the results in Table 6, the batch difference CV of Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 of three batches is within 10%, which indicates that the batch difference of the kit is very small.

[0122] From the above linear evaluation, blank limit and detection limit evaluation, repeatability evaluation, batch difference evaluation, it can be seen that the kit provided by the application has wide linear range, high sensitivity, strong stability, accurate determination results and small batch difference.

[0123] Example 2, influence of different sugars on detection

[0124] The difference between this embodiment and embodiment 1 is the variety and concentration of the saccharide in the SA-PE diluent. The varieties and concentrations of the saccharide are 3% sucrose, 5% sucrose, 10% sucrose, 5% trehalose, 5% glucose, and 5% fructose, respectively. The detection stability of the SA-PE diluent after long-term storage at 2-8℃ is tested. The SA-PE diluent containing different varieties and concentrations of the saccharide is placed at 2-8℃ for 1 month, 3 months, 6 months, 9 months, 12 months, and 18 months, respectively, and then is used to prepare a fluorescent reagent for detection. The sample for detection is quality control 1. Each quality control is detected three times, and the average value is calculated to obtain the relative deviation from the theoretical value. The results are shown in Table 7.

[0125] Table 7, detection results of the stability experiment of different varieties and concentrations of the saccharide

[0126]

[0127]

[0128] According to the results in Table 7, the effects of the SA-PE diluent prepared by 3%, 5%, and 10% sucrose on detection after being placed at 2-8℃ for different time are compared. The detection results of 3% sucrose have a large deviation from the theoretical value, while the detection results of 5% and 10% sucrose are close to the theoretical value, and their relative deviations are close, both of which are controlled within ±5%. The effects of the SA-PE diluent prepared by 5% sucrose, 5% trehalose, 5% glucose, and 5% fructose on detection after being placed at 2-8℃ for different time are compared. The relative deviation of the detection results of the SA-PE diluent prepared by 5% sucrose is always controlled within ±5% with the extension of the storage time, while the relative deviations of the detection results of the SA-PE diluent prepared by the other groups become larger and larger with the extension of the storage time. This indicates that 5% sucrose can significantly improve the stability of the SA-PE diluent and further improve the accuracy of detection. Considering the detection performance and cost, 5% sucrose is selected to be added to the SA-PE diluent, so that the SA-PE diluent can be stored at 2-8℃ for more than 18 months.

[0129] Example 3, effect of the blocking agent in the reaction buffer on detection

[0130] When no blocking agent is added, there will be a more serious non-specific binding problem. In order to reduce the false positive or false negative detection results caused by non-specific binding in the sample, improve the accuracy and specificity of the detection. It is necessary to add blocking agent in the reaction buffer, different blocking agents HBR2 (purchased from Scantibodies company, item number 3KC535), HBR6 (purchased from Scantibodies company, item number 3KC542), HBR9 (purchased from Scantibodies company, 3KC564), HBR11 (purchased from Scantibodies company, 3KC565) are combined, as shown in Table 8 below. In this embodiment, the kit provided in Example 1 is used to detect the sample close to the blank limit (PBS buffer containing no AD related markers) with interfering substances (rheumatoid factor 1500 IU / mL, HAMA 1000 ng / mL), while detecting Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 four markers, wherein the reaction buffer is added with different blocking agent compositions as shown in Table 8, the influence of different blocking agent compositions on the detection results of the blank limit is investigated, each group is repeated three times, the average value is calculated, and the experimental results are shown in Table 8.

[0131] Table 8, comparison of different blocking agent compositions for interference detection

[0132]

[0133] According to the analysis of the results in Table 8, the detection results of different blocking agent compositions for interference are compared, and the combination of 5% HBR6 and 5% HBR11 reduces the occurrence of interference to the minimum, thereby reducing the occurrence of false positives, improving specific binding, and further improving the accuracy of detection.

[0134] Further, the different blocking agent compositions in Table 8 above are used to detect Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217 four AD markers in non-specific samples (blood samples), and the influence of different blocking agents on the detection results is investigated, each group is repeated three times, the average value is calculated, and the relative deviation of the detection results compared with the mass spectrometry detection results is calculated, and the detection results are shown in Table 9.

[0135] Table 9, detection results of different blocking agents for non-specific sample detection

[0136]

[0137] According to the analysis of the results in Table 9, the relative deviation of the detection results of different blocking agent compositions compared with the mass spectrometry detection values is compared, and only the detection results of the blocking agent composition of 5% HBR6+5% HBR11 are controlled within ±5%, and the detection accuracy is higher.

[0138] Example 4, the effect of adding blocking agent to the reaction buffer and adding SA-PE diluent to sucrose on detection

[0139] 1. Two-step incubation method

[0140] The incubation method described in CN2023111107302 is a two-step incubation method, specifically: 25 μL of reaction buffer, 25 μL of microsphere solution of conjugated antibody are added to the sample tube, mixed uniformly, and shaken for more than 30 seconds on a shaker; 75 μL of sample is added to the sample tube; 25 μL of biotin-conjugated antibody and 25 μL of incubation agent are added to the sample tube, the incubation agent contains sorbitol, polyvinylpyrrolidone, Triton X-100, sodium citrate, sodium hyaluronate, and tris(hydroxymethyl) aminomethane; the sample tube is incubated at 2-8°C in the dark for 18 hours, with a shaking frequency of 500 r / min; 1000 μL of 1x washing buffer is added to the sample tube, the microspheres are resuspended by vortexing, mixed uniformly, shaken for more than 30 seconds on a shaker, then centrifuged at 300g for 5 minutes, and the supernatant is discarded; 150-300 μL of 1x washing buffer is added to the sample tube, the microspheres are resuspended by vortexing, mixed uniformly, shaken for more than 30 seconds on a shaker, and the fluorescence type and fluorescence signal intensity are detected on a flow cytometer.

[0141] The above incubation method needs to add an incubation agent, and the incubation agent has a complex composition, including sorbitol, polyvinylpyrrolidone, Triton X-100, sodium citrate, sodium hyaluronate, and Tris, and subsequent washing may not be completely removed, which may affect the detection results. The incubation condition of the incubation agent is 2-8°C, because low temperature can reduce non-specific binding, stabilize fluorescent markers, and maintain the activity of biomolecules. However, low-temperature incubation means that relatively strict conditions need to be controlled. The present team conceives that the blocking agent has a similar effect to the incubation agent. In addition, the blocking agent helps to provide a relatively stable chemical environment for biomolecules at room temperature, reduces protein denaturation caused by environmental changes, and helps to maintain the activity of biomolecules and the progress of the reaction at room temperature. Therefore, 5% HBR6 and 5% HBR11 blocking agents are added to the reaction buffer instead of the incubation agent. In addition, the present team conceives that sucrose can not only maintain protein stability, but also keep the coupled antibody microspheres in a suspended state during incubation, reducing the probability of aggregation and cross-linking of the coupled antibody microspheres, thereby achieving the effect of promoting the reaction. Therefore, sucrose is added to the SA-PE diluent for preparing fluorescent reagents. In order to verify whether the addition of HBR6 and HBR11 blocking agents in the reaction buffer can replace the incubation agent to achieve room temperature incubation, and whether the addition of sucrose in the SA-PE diluent for preparing fluorescent reagents can improve the detection stability and thus improve the accuracy, the following five groups are set:

[0142] The first group: according to the incubation method described in CN2023111107302;

[0143] The second group: the incubation temperature of 2-8°C in the incubation method described in CN2023111107302 is changed to room temperature (25°C);

[0144] The third group: the incubation agent in the incubation method described in CN2023111107302 is replaced by 5% HBR6 and 5% HBR11 blocking agents, and the incubation temperature of 2-8°C is changed to room temperature (25°C);

[0145] The fourth group: 5% sucrose is added to the SA-PE diluent for preparing fluorescent reagents in the incubation method described in CN2023111107302;

[0146] The fifth group: the incubation agent in the incubation method described in CN2023111107302 is replaced by 5% HBR6 and 5% HBR11 blocking agents, and the incubation temperature of 2-8°C is changed to room temperature (25°C), and 5% sucrose is further added to the SA-PE diluent for preparing fluorescent reagents.

[0147] The five groups of incubation methods described above were used to detect Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217 quality control 1, each quality control was detected three times and the average value was taken, and the detection results are shown in Table 10.

[0148] Table 10, the effect of blocking agent instead of incubation agent on detection

[0149]

[0150] According to the data analysis in Table 10, the comparison of the first and second groups of data shows that according to the incubation method described in CN2023111107302, directly changing the low temperature incubation temperature of 2-8℃ to room temperature (25℃) will result in inaccurate detection results; but comparing the first, second and third groups of data, it shows that replacing the incubation agent with 5% HBR6 and 5% HBR11 blocking agent can change the low temperature incubation temperature of 2-8℃ to room temperature (25℃), and the detection results are not affected; comparing the first and fourth groups of data shows that adding 5% sucrose to the SA-PE diluent for preparing fluorescent reagents can improve the detection stability and thus improve the accuracy; the accuracy of the fifth group of data is higher than that of the first to fourth groups, which shows that using 5% HBR6 and 5% HBR11 blocking agent instead of incubation agent achieves normal temperature (25℃) incubation, and adding 5% sucrose to the SA-PE diluent for preparing fluorescent reagents further improves the detection accuracy.

[0151] Therefore, only by replacing the incubation agent in CN2023111107302 with 5% HBR6 and 5% HBR11 blocking agent and adding 5% sucrose to the SA-PE diluent for preparing fluorescent reagents, can the low temperature incubation condition of 2-8℃ be changed to room temperature (25℃) incubation while further improving the detection accuracy.

[0152] 2、2.5-step incubation method

[0153] Through the above Experiment 1, the method in CN2023111107302 has been optimized, but the microsphere reagent coupled with antibody and the biotin-coupled antibody reagent are still incubated together. In order to make the fluorescent microspheres coupled with antibody and the biotin-coupled antibody more fully bind to the sample to be tested, the above optimized two-step incubation method in CN2023111107302 is split into a 2.5-step incubation method, and the specific steps of the 2.5-step incubation method are as follows:

[0154] (1) The first step of incubation: 25 μL of reaction buffer (containing 5% HBR6 and 5% HBR11 blocking agent) and 25 μL of microsphere solution of conjugated antibody are added to the sample tube, mixed uniformly, and shaken for more than 30 seconds on a shaker; 75 μL of sample is added to the sample tube, mixed uniformly after shaking, and incubated at room temperature (25°C) for 9 hours in the dark with shaking at 500 r / min;

[0155] (2) The second step of incubation: 1 mL of 1x washing buffer is added to the sample tube, the microspheres are resuspended by vortexing, centrifuged at 400 g for 5 min, and the supernatant is carefully removed; 25 μL of detection antibody reagent is added to the sample tube, mixed uniformly after shaking, and incubated at room temperature (25°C) for 9 hours in the dark with shaking at 500 r / min;

[0156] (3) The second step of incubation: 25 μL of fluorescent reagent (containing 5% sucrose) is added to the sample tube, mixed uniformly after shaking, and incubated at room temperature for 0.5 hours (shaking amplitude at 600-900 r / min) in the dark; 1 mL of 1x washing buffer is added to the sample tube, the microspheres are resuspended by vortexing, centrifuged at 400 g for 5 min, and the supernatant is carefully removed; according to the sample requirements, 150-300 μL of 1x washing buffer is added to the tube, the microspheres are resuspended by vortexing, mixed uniformly, shaken for more than 30 seconds on a shaker, and the fluorescence type and fluorescence signal intensity are detected on a flow cytometer.

[0157] Further, to verify whether the 2.5-step incubation method can further improve the detection accuracy, the following experiments are performed.

[0158] The following four groups are set up:

[0159] The first group: detected according to the above-mentioned two-step incubation method in 1 (replace the incubation agent described in CN2023111107302 with 5% HBR6 and 5% HBR11 blocking agent, incubate at room temperature (25°C), and add 5% sucrose to the SA-PE diluent prepared for the fluorescent reagent);

[0160] The second group: detected according to the above-mentioned 2.5-step incubation method;

[0161] The third group: detected according to the above-mentioned 2.5-step incubation method, but the fluorescent reagent in step (3) does not contain 5% sucrose;

[0162] The fourth group: detected according to the above-mentioned 2.5-step incubation method, but the 5% HBR6 and 5% HBR11 blocking agent are replaced with the incubation agent in CN2023111107302.

[0163] The four sets of detection methods described above were used to detect the quality control 1 of Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217, each quality control was detected three times and the average value was taken, and the detection results are shown in Table 11.

[0164] Table 11, the influence of different 2.5-step incubation conditions on detection

[0165]

[0166] According to the data analysis in Table 11, comparing the first and second sets of detection data shows that splitting the two-step incubation method optimized in the above 1 into a 2.5-step incubation method can further improve the detection accuracy; comparing the second to fourth sets of detection data shows that, on the one hand, in the 2.5-step incubation method, if the blocking agents of 5% HBR6 and 5% HBR11 are not used instead of the incubation agent, normal temperature (25°C) incubation cannot be achieved; on the other hand, adding 5% sucrose to the SA-PE diluent for preparing the fluorescent reagent can further improve the detection accuracy.

[0167] In summary, the two-step incubation method described in CN2023111107302 is improved in this embodiment, 5% HBR6 and 5% HBR11 blocking agents are used instead of the incubation agent, and 5% sucrose is added to the SA-PE diluent for preparing the fluorescent reagent, and a new 2.5-step incubation method is also provided. Under the combined action of the above three changes, the incubation temperature changes from 2-8°C to normal temperature (25°C), and the detection accuracy is further improved.

[0168] Example 5, condition screening of 2.5-step room temperature shaking incubation

[0169] It has been proved in Example 4 that adding blocking agent compositions HBR6 and HBR11 to the reaction buffer and adding sucrose to the SA-PE diluent for preparing the fluorescent reagent can significantly improve the detection accuracy, and achieve incubation at room temperature (25°C) by 2.5-step incubation method, but the total incubation time still needs 18.5h, which cannot meet the detection needs of some urgent samples.

[0170] Because increasing the incubation temperature can shorten the incubation time, the above 25°C is the result of increasing the incubation temperature from the original low-temperature incubation (2-8°C), so it is guessed whether the incubation time has been saturated and whether the incubation time can be shortened. To verify the above guess and explore the optimal sample volume, incubation temperature range and time at room temperature, the following experiments are set up:

[0171] The incubation temperature is 4°C, 25°C and 37°C, the total incubation time is 2.5h, 4.5h, 6.5h and 18.5h, and the sample loading amount is 25μL, 75μL and 100μL respectively. The total incubation time is the sum of the incubation time of the 2.5-step incubation method in Example 4, and the total incubation time is set as shown in Table 12 below:

[0172] Table 12, total incubation time

[0173]

[0174] 4°C sets four experimental groups with total incubation time of 2.5h, 4.5h, 6.5h and 18.5h respectively, while 25°C and 37°C each temperature sets three experimental groups with total incubation time of 2.5h, 4.5h and 6.5h respectively.

[0175] The quality control 2 is named as calibrator C1, which is diluted by 2 times to calibrators C2-C8, and C0 is a sample diluent for blank control. The C1 concentration of Aβ1-40 is 1250pg / mL, the C1 concentration of Aβ1-42 is 625pg / mL, the C1 concentration of p-Tau-181 is 100pg / mL, and the C1 concentration of p-Tau-217 is 40pg / mL. The fluorescence signal values of Aβ1-40 and Aβ1-42 in C1, C2, C4, C7 and C8 are detected, and the fluorescence signal values of p-Tau-181 and p-Tau-217 in C1, C3, C5, C7 and C8 are detected. Each sample is detected three times to take the average value, and the loading amount is selected. In addition, four samples with known concentrations are selected for detection, wherein the Aβ1-40 concentration of sample 1 is 150.12pg / mL, the Aβ1-42 concentration of sample 2 is 75.06pg / mL, the p-Tau-181 concentration of sample 3 is 25pg / mL, and the p-Tau-217 concentration of sample 4 is 10pg / mL. The detection results are shown in Tables 13-16 below.

[0176] Table 13, detection results of Aβ1-40 at different incubation temperatures and incubation times

[0177]

[0178] Table 14, detection results of Aβ1-42 at different incubation temperatures and incubation times

[0179]

[0180] Table 15, detection results of p-Tau-181 at different incubation temperatures and incubation times

[0181]

[0182] Table 16, detection results of p-Tau-217 under different incubation temperatures and incubation times

[0183]

[0184] According to the result analysis of Tables 13-16, all the fluorescence signal values are converted into concentrations. When the sample loading amount is 75 μL, the detection value is closest to the theoretical value, so the sample loading amount is selected as 75 μL.

[0185] When the sample loading amount is 75 μL, the detection results of the total incubation time of 2.5 h, 4.5 h, 6.5 h, and 18.5 h at low temperature 4°C are compared. The fluorescence signal values are converted into concentration values. The detection concentration of the incubation time of 18.5 h has the smallest deviation from the theoretical value, indicating that the total incubation time at low temperature is 18.5 h. The detection results of the total incubation time of 2.5 h, 4.5 h, and 6.5 h at 25°C and 37°C are compared. The fluorescence signal values are converted into concentration values. The detection results at the two temperatures have little difference, and the relative deviation values are controlled within 10%. However, the relative deviation of the detection at 25°C is smaller, and the total incubation time of 2.5 h, 4.5 h, and 6.5 h has no significant difference, and the relative deviation values of the detection results are controlled within 5%. In order to save detection time, the incubation at 25°C for 2.5 h is preferred. The detection results of the total incubation time of 18.5 h at low temperature 4°C and the total incubation time of 2.5 h at room temperature 25°C are compared. The fluorescence signal values are converted into concentration values. The detection result of the total incubation time of 2.5 h at room temperature 25°C has smaller deviation and is more accurate. Therefore, when the sample loading amount is 75 μL, the total incubation time of the 2.5-step incubation method can be shortened by increasing the incubation temperature, and the accuracy of the detection is not affected.

[0186] Based on the above analysis, the sample loading amount of the 2.5-step incubation method is preferably 75 μL, the incubation temperature is preferably 25°C, and the total incubation time is preferably 2.5 h.

[0187] Example 6, optimization of the mixing molar ratio of antibody and biotin and the SA-PE concentration in the fluorescence reagent

[0188] The difference between the present example and Example 1 is that the molar ratio of the antibody to biotin and the concentration of SA-PE in the fluorescent reagent are changed. The molar ratio of the antibody to biotin is 1:10, 1:20, 1:30, 1:40, 1:50, and 1:60, respectively, and the concentration of SA-PE in the fluorescent reagent is 0.25 μg / mL, 0.50 μg / mL, 1.00 μg / mL, and 2.00 μg / mL, respectively. The different molar ratios of the antibody to biotin combined with different concentrations of SA-PE in the fluorescent reagent are used to detect the concentration of AD markers in the sample, and the sample is the quality control 1 in Example 1. Each quality control is detected for 3 times to take the average value, and the relative deviation of the detection result from the theoretical value is calculated, as shown in Table 17 below.

[0189] Table 17, detection results of different molar ratios of the antibody to biotin combined with different concentrations of SA-PE

[0190]

[0191] According to the analysis of the results in Table 17, when the molar ratio of the antibody to biotin is 1:40 and the concentration of SA-PE is 1 μg / mL, the detection accuracy is higher, and the relative deviation of the detection results of the four AD markers can be controlled within ±5%.

[0192] Although the present application is disclosed as above, the present application is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be subject to the scope defined by the claims.

Claims

1. A kit for detecting Alzheimer's disease-related proteins, characterized in that: The Alzheimer's disease-related proteins are Aβ1-40, Aβ1-42, p-Tau-181 and p-Tau-217; the kit consists of a microsphere solution of coupled antibodies, a biotin-coupled antibody solution, a fluorescent reagent and a reaction buffer; the fluorescent reagent contains SA-PE diluent and SA-PE, the SA-PE diluent contains sucrose, and the concentration of the sucrose is 5%; the reaction buffer contains blocking agents HBR6 and HBR11; the SA-PE is diluted with SA-PE The concentration after dilution is 0.25-2 μg / mL; the molar ratio of antibody to biotin in the biotin-conjugated antibody solution is 1:(10-60); the antibodies are Aβ1-40 antibody, Aβ1-42 antibody, p-Tau-181 antibody and p-Tau-217 antibody; the Aβ1-40 antibody is a mouse anti-human antibody, the Aβ1-42 antibody is a mouse anti-human antibody, the p-Tau-181 antibody is a mouse anti-human antibody, and the p-Tau-217 antibody is a rabbit anti-human antibody.

2. The kit according to claim 1, wherein The concentration of HBR6 is 5%, and the concentration of HBR11 is 5%; the concentration of SA-PE after dilution with SA-PE diluent is 1 μg / mL; and the molar ratio of antibody to biotin in the biotin-coupled antibody solution is 1:40.

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