Kit for detecting Alzheimer's disease related protein and application thereof

Abstract

The invention provides a kit for detecting Alzheimer's disease related protein and application thereof, the kit is characterized in that sucrose is added into a fluorescent reagent diluent to improve the stability of Alzheimer's disease related protein in a detection sample, and HEPES is added into the sample diluent to improve the stability of the Alzheimer's disease related protein in the detection sample. Blocking agents HBR6 and HBR11 are added into a reaction buffer solution to improve the specific binding capacity of the related protein, so that the content of the Alzheimer disease related protein in a sample is accurately detected through 2.5-step room-temperature oscillation incubation, and the detection time is greatly shortened; the invention also optimizes the working concentration of SA-PE, the mixed molar ratio of antibody to biotin and incubation conditions. The kit provided by the invention is simple to operate, not only improves the detection efficiency, the detection accuracy, the specificity and the sensitivity, but also facilitates clinical detection application, can reduce the reagent and labor cost, is good in repeatability, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of flow cytometry, and particularly relates to a kit for detecting Alzheimer's disease-related proteins and its application. Background Art

[0002] Alzheimer disease (AD), commonly known as senile dementia, is a central nervous system degenerative disease with insidious onset and progressive development. The typical histopathological changes of Alzheimer's disease are senile plaques caused by the deposition of amyloid-β protein (Aβ), neurofibrillary tangles caused by abnormal phosphorylation of Tau protein, and the 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 AD biomarkers at home and abroad. Research shows that the expressions of Aβ1-42 and Aβ1-42 / Aβ1-40 in the plasma of AD patients are down-regulated, while the expressions of T-Tau and p-Tau-181 are up-regulated.

[0003] Aβ1-42 and Aβ1-40 are produced by the hydrolysis of amyloid precursor protein (APP) by BACE and γ-secretase, and can gradually accumulate outside the membrane to form Aβ protein polymers, which have a toxic effect on neurons, leading to neuronal degeneration. Compared with Aβ1-40, Aβ1-42 has a very high aggregability and has gradually accumulated in the early stage of senile plaque formation. The plasma Aβ1-42 content and the Aβ1-42 / Aβ1-40 ratio reflect the Aβ pathology in the brain and can be used to early evaluate the disease risk of AD dementia and mild cognitive impairment (MCI). Among them, Aβ1-42 even decreases before the abnormality of amyloid-β positron emission tomography (Aβ-PET).

[0004] Tau protein is a microtubule-associated protein (MAPs) that is highly abundant in neurons of the central nervous system. Its main function is to regulate the stability of axonal microtubules. It is also a phosphoprotein with multiple phosphorylation sites, among which p-Tau-181, p-Tau-217, etc. are the most studied. Phosphorylated Tau competes with tubulin for binding to normal Tau protein and other microtubule-associated proteins, leading to microtubule depolymerization and the formation of paired helical filaments (PHF) in neurons, affecting the normal physiological function of Tau protein. Studies have shown that plasma T-Tau concentration increases in Creutzfeldt-Jakob disease (CJD) and frontotemporal lobar dementia (FTD), and it is an important predictor of CJD; while the plasma levels of p-Tau-181 and p-Tau-217 can reflect the pathological conditions of Aβ and Tau in AD, distinguish Alzheimer's disease from other neurodegenerative diseases (such as Parkinson's disease and vascular dementia, etc.), and the disease progression of AD patients can be monitored through the plasma levels of p-Tau-181 and p-Tau-217 throughout the 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 diagnosis. 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 method or flow cytometry, etc. The chemiluminescence method has poor selectivity and anti-interference ability, which means that in complex samples, the chemiluminescence method may not be able to accurately distinguish specific analytes, or is easily interfered by other components, resulting in inaccurate results; moreover, using the chemiluminescence method can only detect Aβ1-42, Aβ1-40, p-Tau-181, and p-Tau-217 individually, and the performance of reagents such as sensitivity and precision is poor, and the detection time is long and the operation is complex. Although flow cytometry can detect these several markers simultaneously, the detection process may be affected by many non-specific interferences, affecting the accuracy.

[0007] The research team of the present invention applied for a patent for invention CN2023111107302 in 2023, which discloses a method for simultaneously detecting the contents of Aβ1-42, Aβ1-40, T-Tau, p-Tau-181, and α-synuclein in a sample after two-step incubation at a low temperature of 2-8°C. Although this method significantly improves the sensitivity, accuracy, and specificity of the detection of Alzheimer's disease-related proteins, its operation process is cumbersome, the types of added reagents are numerous, and the incubation time is long, resulting in a very high time cost for detecting samples. The long incubation time also increases uncontrollable factors and affects the detection results.

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

[0009] Aiming at the problems existing in the prior art, the present invention provides a kit for detecting Alzheimer's disease-related proteins and its application. The kit improves the stability of detecting Alzheimer's disease-related proteins in human blood by adding sucrose to the fluorescent reagent diluent, adds HEPES to the sample diluent, and adds blockers HBR6 and HBR11 to the reaction buffer to improve the specific binding ability of related proteins, thereby enabling accurate detection of the content of Alzheimer's disease-related proteins in a sample through 2.5-step room-temperature shaking incubation, greatly reducing the detection time; the present invention also optimizes the working concentration of SA-PE, the mixed molar ratio of the antibody and biotin, and the incubation conditions. The kit provided by the present invention is simple to operate, improves the detection efficiency, accuracy, specificity, and sensitivity. 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 invention is also convenient for clinical detection applications, can reduce the costs of reagents and labor, has good repeatability, and has a good application prospect.

[0010] On the one hand, the present invention provides a kit for detecting Alzheimer's disease-related proteins, which includes a microsphere solution conjugated with an antibody, a biotin-conjugated antibody solution, and a fluorescent reagent; the fluorescent reagent contains an SA-PE diluent, and the SA-PE diluent contains sucrose; the antibody is one or more of an Aβ1-40 antibody, an Aβ1-42 antibody, a p-Tau-181 antibody, and a p-Tau-217 antibody.

[0011] It has been proven by a large number of studies in the present invention that the externally purchased SA-PE solution is not stable. When it is used to prepare the fluorescent reagent, the coupling effect of phycoerythrin (PE) is prone to be unstable, which affects the final detection result. Carbohydrates are viscous and can wrap protein molecules to form a carbohydrate vitreous body similar to glass ice in structure, blocking the chain movement of macromolecules, preventing the extension and precipitation of proteins, and maintaining the stability of the three-dimensional structure of protein molecules, thus playing a protective role. Adding sucrose to the SA-PE diluent for preparing the fluorescent reagent can stabilize and protect streptavidin (SA) and phycoerythrin (PE), thereby improving the stability and accuracy of the kit detection.

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

[0013] Furthermore, the kit further includes a reaction buffer, and the reaction buffer contains blockers HBR6 and HBR11.

[0014] HBR is a heterophilic antibody blocking reagent, which is mainly composed of core active components (such as animal-derived IgG, synthetic polypeptides / antibodies) and auxiliary components (buffer, BSA stabilizer, preservative, etc.). Its main function is to preemptively bind to heterophilic antibodies in the sample, occupy their binding sites, and avoid cross-reactions between heterophilic antibodies and detection reagents, thereby improving the accuracy of detection results and reducing the occurrence of false positives. Heterophilic antibodies are endogenous autoantibodies secreted by the human immune system stimulated by known or unknown antigenic substances and can non-specifically bind to animal immunoglobulins. In particular, close contact with animals, ingestion of contaminated food, treatment with monoclonal animal antibody preparations, infection, blood transfusion, etc. may all be the causes of the production of heterophilic antibodies. The sample detected by the kit of the present invention is a human blood sample, and the blood sample contains heterophilic antibodies that will interfere with the detection. The interference principle is that heterophilic antibodies can bind to the fluorescent microspheres conjugated with antibodies. Since heterophilic antibodies can bind to the Fc (crystallizable fragment) and Fab (antigen-binding fragment) epitopes of immunoglobulins, the fluorescent microspheres conjugated with antibodies cannot capture the antigens to be detected in the blood sample, resulting in false positives. The blockers HBR6 and HBR11 used in the present invention are purchased from Scantibodies Company and can prevent heterophilic antibodies in the sample from binding to the fluorescent microspheres conjugated with antibodies and can block sites without biological regulatory functions to reduce non-specific binding. At the same time, during the detection process, after adding the blocker to the reaction buffer, the detection accuracy is significantly improved.

[0015] The present invention screens different commercially available blockers, and finally selects to use a combination of 5% HBR6 and 5% HBR11 as blockers and add them to the reaction buffer, which can effectively eliminate false positives and prevent non-specific binding.

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

[0017] In CN2023111107302, the light incubation time for detecting five proteins, namely Aβ1-42, Aβ1-40, T-Tau, p-Tau-181, and α-synuclein, is as long as 18.5 hours, resulting in a very high time cost for the test samples. The long incubation also increases uncontrollable factors and affects the accuracy of the test results. Moreover, an incubation agent must be added before incubation, and the incubation conditions are shaking incubation at 2-8°C. The preparation raw materials of the incubation agent include many components, such as sorbitol, polyvinylpyrrolidone, Triton X-100, sodium citrate, sodium hyaluronate, and Tris. Subsequent washing may not be complete, which will affect the accuracy and sensitivity of the test results. However, in the present invention, by adding blockers HBR6 and HBR11 to the reaction buffer and adding sucrose to the SA-PE diluent for preparing the fluorescent reagent, there is no longer a need to add an incubation agent, and a 2.5-step room temperature incubation method is achieved. The total incubation time is only 2.5 hours at the shortest. This not only shortens the sample test time, but also has higher accuracy, specificity, and sensitivity in detecting Aβ1-42, Aβ1-40, p-Tau-181, and p-Tau-217.

[0018] Further, the fluorescent reagent also contains SA-PE, and the concentration of SA-PE after dilution with the SA-PE diluent is 0.25-2 μg / mL.

[0019] In some embodiments, the concentration of SA-PE after dilution with the SA-PE diluent is 1 μg / mL.

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

[0021] HEPES is an amphoteric ion buffer that contains both a weakly acidic group (sulfonic acid group) and a weakly basic group (piperazine nitrogen atom). It can buffer the pH fluctuation of the external environment well by accepting and donating protons within a certain pH range. The pH range of HEPES is 7.2-7.5, which can effectively maintain the stability of proteins.

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

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

[0024] In some embodiments, the ratio of the 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 further includes a washing buffer.

[0027] In some embodiments, the components of the washing buffer include KH2PO4, K2HPO4·12H2O, NaCl, KCl, BSA, Proclin300 preservative, and Tween-20.

[0028] Furthermore, the kit further includes a calibrator and a quality control sample.

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

[0030] The detection principle of the kit of the present invention is that the antibody protein conjugated with fluorescent microspheres and the antibody protein conjugated with biotin jointly capture the target protein in the sample to be detected. Then, the antibody protein conjugated with biotin binds to streptavidin conjugated with phycoerythrin, and they jointly form an immune complex. During detection, two beams of excitation light with different wavelengths emitted by the flow cytometer irradiate the immune complex, and the type of detection index is determined by the fluorescence intensity of different fluorescent microspheres, and the content of each detection index is determined by the fluorescence intensity of phycoerythrin.

[0031] On the other hand, the present invention provides a method for detecting Alzheimer's disease-related proteins, which uses the above-mentioned kit for detection and includes the following steps:

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

[0033] (2) Add the sample to be detected to 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 contents of Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217 on a flow cytometer.

[0037] It has been found through research that adding sucrose to the SA-PE diluent can not only maintain protein stability but also keep the microspheres conjugated with antibodies in a suspended state during incubation, reducing the probability of aggregation and cross-linking of the microspheres conjugated with antibodies, thereby promoting the occurrence of the reaction. On the one hand, sucrose increases the density and viscosity of the solution, helping the fluorescent microspheres to remain stably suspended in the solution. On the other hand, it can wrap the antibodies conjugated on the microspheres to form a carbohydrate vitreous body similar to glass ice in structure, blocking the chain movement of macromolecules and preventing the extension and precipitation of proteins. Therefore, adding sucrose to the SA-PE diluent can allow the reactants to react fully and improve the detection accuracy.

[0038] Further, the incubation conditions in step (2) are light-shielded oscillatory incubation at 4 - 37 °C for 1 - 6 hours; the incubation conditions in step (3) are light-shielded oscillatory incubation at 4 - 37 °C for 1 - 6 hours.

[0039] In some ways, the incubation conditions in step (2) are light-shielded oscillatory incubation at 25 °C for 1 hour; the incubation conditions in step (3) are light-shielded oscillatory incubation at 25 °C for 1 hour.

[0040] Further, the incubation conditions in step (4) are light-shielded oscillatory incubation at 4 - 37 °C for 0.5 hour.

[0041] In the actual application process, the incubation temperature in step (4) within the range of 4 - 37 °C can make the detection results more accurate.

[0042] In some ways, the most preferred incubation temperature in step (4) is 25 °C.

[0043] On the other hand, the present invention also provides the application of the SA-PE diluent and the blocker composition in the preparation of a kit for shortening the incubation time of detecting Alzheimer's disease-related proteins, wherein the SA-PE diluent includes sucrose, and the blocker composition includes HBR6 and HBR11.

[0044] In some ways, it has been experimentally proven that adding sucrose to the SA-PE diluent for preparing the fluorescent reagent and adding the blocker composition HBR6 and HBR11 to the reaction buffer can effectively shorten the incubation time during the detection process, and the total incubation time is only 2.5 h at the shortest, and incubation can be achieved at room temperature (25 °C).

[0045] On the other hand, the present invention provides the use of a blocker composition for preparing a reagent for improving the specific binding ability of detecting Alzheimer's disease-related proteins in human blood, and the blocker composition includes HBR6 and HBR11.

[0046] In some embodiments, six different reaction buffers are prepared by combining the four blockers, namely HBR6, HBR11, HBR9, and HBR2, at a concentration of 5% in pairs. The six different reaction buffers are used to detect solutions that do not contain AD markers but contain interfering substances. The detection results prove that the 5% HBR6 and 5% HBR11 blocker composition can effectively reduce the detection value of the interfering substances. Further, the six different reaction buffers are used to detect AD-related proteins in blood samples, and the detection results are compared with the mass spectrometry detection results, which proves that the 5% HBR6 and 5% HBR11 blocker composition can effectively reduce the influence of interfering substances on the detection of AD markers, thereby improving the detection accuracy.

[0047] The beneficial effects achieved by the present invention are as follows:

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

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

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

[0051] 4. The working concentration of SA-PE, the mixed molar ratio of antibody and biotin, and the incubation conditions are optimized, effectively improving the detection accuracy, specificity, and sensitivity. 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;

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

[0053] Figure 1 It is a schematic diagram of the detection principle of the kit in Example 1;

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

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

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

[0057] Figure 5 Linear evaluation results of p-Tau-217 in Example 1. Detailed implementation manners

[0058] The embodiments of the present invention will be further described in detail below. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention, but do not limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

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

[0060] 1. Preparation of the kit

[0061] (1) Preparation of the fluorescent microsphere solution conjugated with antibodies

[0062] Four kinds of fluorescent microspheres and the antibodies conjugated thereto are shown in Table 1 below. In Table 1, the A1-A4 fluorescent microspheres are 5μm carboxyl fluorescent microspheres with different fluorescence intensities. The Aβ1-40 antibody, Aβ1-42 antibody, and 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 conjugated thereto

[0064]

[0065] Steps for preparing the fluorescent microspheres conjugated with the Aβ1-40 antibody: Take 5×10 of the A1 fluorescent microspheres 6Take 5×10 microspheres of A1, add PBST buffer (phosphate buffer containing 0.05% Tween-20, pH 7.4, purchased from sigma) and wash twice. Add 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) to the washed A1 fluorescent microspheres and let stand for 30 minutes to activate the fluorescent microspheres. Add 100 μg of anti-human Aβ1-40 antibody and react by rotation at room temperature for 5 hours to wash the fluorescent microspheres to remove the excess antibody. After washing the A1 fluorescent microspheres to remove the excess anti-human Aβ1-40 antibody, add 5% skim milk powder by mass fraction to block for 30 minutes. After removing the blocking solution, add Tris buffer with a pH of 7.2 for storage;

[0066] Steps for preparing Aβ1-42 antibody-conjugated fluorescent microspheres: Take 5×10 microspheres of A2 6 and add PBST buffer to wash twice. Add 100 μg of EDC and 50 μg of NHS to the washed A2 fluorescent microspheres and let stand for 30 minutes to activate the fluorescent microspheres. Add 100 μg of anti-human Aβ1-42 antibody and react by rotation at room temperature for 5 hours to wash the fluorescent microspheres to remove the excess antibody. After washing the A2 fluorescent microspheres to remove the excess anti-human Aβ1-42 antibody, add 5% skim milk powder by mass fraction to block for 30 minutes. After removing the skim milk powder, add Tris buffer with a pH of 7.2 for storage;

[0067] Steps for preparing p-Tau-181 antibody-conjugated fluorescent microspheres: Take 5×10 microspheres of A3 6 and add PBST buffer to wash twice. Add 100 μg of EDC and 50 μg of NHS to the washed A3 fluorescent microspheres and let stand for 30 minutes to activate the fluorescent microspheres. Add 100 μg of anti-human p-Tau-181 antibody and react by rotation at room temperature for 5 hours to wash the fluorescent microspheres to remove the excess antibody. After washing the A3 fluorescent microspheres to remove the excess anti-human p-Tau-181 antibody, add 5% skim milk powder by mass fraction to block for 30 minutes. After removing the skim milk powder, add Tris buffer with a pH of 7.2 for storage;

[0068] Steps for preparing p-Tau-217 antibody-conjugated fluorescent microspheres: Take 5×10 microspheres of A4 6Add, wash twice with PBST buffer. Add 100 μg of EDC and 50 μg of NHS to the washed A4 fluorescent microspheres and let stand for 30 minutes to activate the fluorescent microspheres. Add 100 μg of rabbit anti-human p-Tau-217 antibody and react by rotating at room temperature for 5 hours. Wash the fluorescent microspheres to remove the excess antibody. After washing the A4 fluorescent microspheres to remove the excess rabbit anti-human p-Tau-217 antibody, add 5% skim milk powder to block for 30 minutes. After removing the skim milk powder, add Tris buffer with a pH of 7.2 for storage;

[0069] Take 98 mL of Tris buffer with a pH of 7.2. According to the detection indicators of the kit, add 0.5 mL each of the fluorescent microspheres conjugated with Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217 antibodies to prepare the fluorescent microsphere solution conjugated with the antibody.

[0070] (2)Prepare the biotin-conjugated antibody solution

[0071] Prepare the antibody-biotin complex. The preparation steps are as follows:

[0072] Take 100 μg each 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 described in (1). Add biotin according to the molar ratio of antibody to biotin of 1:40. After incubating at room temperature for 5 hours, obtain the biotin-conjugated anti-human Aβ1-40 antibody, biotin-conjugated anti-human Aβ1-42 antibody, biotin-conjugated anti-human p-Tau-181 antibody, and biotin-conjugated anti-human p-Tau-217 antibody. After removing the unbound biotin, dilute the biotin-conjugated anti-human Aβ1-40 antibody, biotin-conjugated anti-human Aβ1-42 antibody, biotin-conjugated anti-human p-Tau-181 antibody, and biotin-conjugated anti-human p-Tau-217 antibody to 4 μg / mL respectively with 0.01 mol / L PBS solution;

[0073] Take 25 mL each of the above biotin-conjugated anti-human Aβ1-40 antibody, biotin-conjugated anti-human Aβ1-42 antibody, biotin-conjugated anti-human p-Tau-181 antibody, and biotin-conjugated anti-human p-Tau-217 antibody with a concentration of 4 μg / mL and mix them. Then the concentration of each biotin-labeled antibody is 1 μg / mL.

[0074] (3)Prepare the fluorescent reagent

[0075] Prepare SA-PE diluent: Dissolve 9 g of NaCl, 50 g of sucrose, and 6.057 g of Tris in 1000 mL of pure water, add the preservative Proclin300 with a mass concentration of 0.1%, and adjust the pH to 7.4 for standby. SA-PE is purchased from thermofisher, and SA-PE is diluted to 1 μg / mL with SA-PE diluent for standby.

[0076] (4)Prepare reaction buffer

[0077] Take 9 g of NaCl and 6.057 g of Tris and dissolve them in 1000 mL of pure water, add BSA with a mass concentration of 1%, HBR6 with a mass concentration of 5% (purchased from Scantibodies, catalog number 3KC542), HBR11 with a mass concentration of 5% (purchased from Scantibodies, 3KC565), the preservative Proclin300 with a mass concentration of 0.1%, and Tween-20 with a mass concentration of 0.1%, and adjust the pH to 7.4 for standby.

[0078] (5)Prepare washing buffer

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

[0080] (6)Prepare sample diluent

[0081] Dissolve 9 g of NaCl and 2.38 g of HEPES in 1000 mL of pure water, add the preservative Proclin300 with a mass concentration of 0.1%, and adjust the pH to 7.4 for standby.

[0082] (7)Prepare calibrators

[0083] Use the sample diluent to dilute the freeze-dried powders Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217 to different concentrations to establish a calibration curve for detection. The concentration of Aβ1-40 is 1250 pg / mL, the concentration of Aβ1-42 is 625 pg / mL, the concentration of p-Tau-181 is 100 pg / mL, and the concentration of p-Tau-217 is 40 pg / mL.

[0084] (8)Prepare quality control samples

[0085] The calibration solution in (7) above was formulated into low-value quality control product 1 and high-value quality control product 2 with sample diluent to judge the performance of the test results. The concentrations of low-value quality control product 1 and high-value quality control product 2 are shown in Table 2 below.

[0086] Table 2. Concentrations of Low-Value Quality Control Product 1 and High-Value Quality Control Product 2

[0087]

[0088] 2. Detection Procedure

[0089] The detection procedure is as follows:

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

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

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

[0093] (4) Add 25 μL of fluorescent reagent to the sample tube and shake the tube to mix evenly. Incubate in the dark at room temperature with shaking for 0.5 h (shaking amplitude at 600 - 900 r / min). Add 1 mL of 1× washing buffer to the sample tube, resuspend the microspheres by vortexing, centrifuge at 400 g for 5 min, and carefully discard the supernatant;

[0094] (5) According to the sample loading requirement, add 150 μL - 300 μL of 1× 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 1Schematic diagram of the relationship among the specific antibody, microspheres, detection antibody, and phycoerythrin in this embodiment. Among them, 1 is the fluorescent microsphere, 2 is the antibody protein, 3 is the target protein in the sample to be detected, 4 is the antibody protein, 5 is biotin, 6 is streptavidin, and 7 is phycoerythrin. The antibody protein 2 conjugated to the fluorescent microsphere 1 and the antibody protein 4 conjugated to biotin 5 jointly capture the target protein 3. Then, the biotin 5 conjugated to the antibody protein 4 binds to the streptavidin 6 conjugated to phycoerythrin 7, and they jointly form an immune complex. During detection, two beams of excitation light with different wavelengths emitted by the flow cytometer irradiate the immune complex. The type of detection index is determined by the fluorescence intensity of different fluorescent microspheres 1, and the content of each detection index is determined by the fluorescence intensity of phycoerythrin 7.

[0096] 3. Preparation of the calibration curve

[0097] The calibration standards of 1250 pg / mL Aβ1-40, 625 pg / mL Aβ1-42, 100 pg / mL p-Tau-181, and 40 pg / mL p-Tau-217 were respectively diluted at a two-fold dilution ratio into 8 different concentrations (xi). The detection results were obtained according to the detection process described in this embodiment. Each concentration was tested 3 times, and the mean value (yi) of the detection results was calculated respectively to prepare the calibration curve.

[0098] 4. Performance evaluation

[0099] (1) Linear evaluation

[0100] Linear evaluation was performed on the standard curve prepared in 3 above. Taking the dilution concentration (xi) as the independent variable and the mean value (yi) of the detection results as the dependent variable, the linear regression equation was obtained, and 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. 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 This is the linear evaluation result of p-Tau-181 in the embodiment of the present invention. Its linear regression equation is y = 1.0087x - 0.144, R 2 = 0.9999; Figure 5 This is the linear evaluation result of p-Tau-217 in the embodiment of the present invention. Its linear regression equation is y = 0.9454x + 0.1494, R 2 = 0.9999. The R values of the four linear regression equations 2All are greater than 0.99, indicating good linearity.

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

[0103] Detection methods for the blank limit and detection limit: 1 flow cytometer; 2 reagent batches (1 and 2); 3 test days; 5 blank samples and 5 low-concentration level samples; each sample is measured 4 times repetitively. The number of test results for both the blank samples and the low-concentration level samples is 60. The classical method protocol is used to evaluate the blank limit (LoB) and the detection limit (LoD). By default, α = β = 0.05. Analyzed with SPSS software, the test results of the blank samples are non-normally distributed, while those of the low-concentration samples are normally distributed. The blank limit of Aβ1-40 is 0.154 pg / mL, the blank limit of Aβ1-42 is 0.181 pg / mL, the blank limit of p-Tau-181 is 0.169 pg / mL, and the blank limit of p-Tau-217 is 0.049 pg / mL.

[0104] Verification method for the blank limit: Use the zero-concentration calibrator as the sample for detection, detect according to the detection process described in this embodiment, measure 20 times repetitively, obtain the concentration values of the 20 measurement results according to the calibration curve equation of the kit used, calculate their average value (M) and standard deviation (SD), obtain M + 2SD, and compare it with the blank limit. The blank limit verification results are shown in Table 3 below.

[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 values, indicating that the setting of the blank limit of this kit is basically reasonable.

[0108] Verification method for the detection limit: Detect 5 low-value samples with concentrations approximately equal to the detection limit (the approximate detection limit is estimated according to the obtained blank limit value, slightly higher than the blank limit), measure each sample 5 times, sort the detection results by size, and 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 are 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 below.

[0109] Table 4. Detection limit verification results

[0110]

[0111] According to the analysis of Table 4 above, the number of parallel five - time detection results of each sample of Aβ1 - 40, Aβ1 - 42, p - Tau - 181, and p - Tau - 217 lower than the blank limit is less than or equal to 3, indicating that the setting of the detection limit of this kit is basically reasonable.

[0112] (3)Repeatability evaluation

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

[0114] Table 5. Detection results of repeatability experiment

[0115]

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

[0117] (4)Inter - batch difference evaluation

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

[0119] Table 6. Detection results of inter - batch difference

[0120]

[0121] According to the result analysis of Table 6, the inter - batch difference CVs of Aβ1 - 40, Aβ1 - 42, p - Tau - 181, and p - Tau - 217 for 3 batches are all within 10%, indicating that the inter - batch difference of this kit is very small.

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

[0123] Example 2. Influence of different sugars on detection

[0124] The difference between this embodiment and Embodiment 1 lies in the types and concentrations of sugars in the SA-PE diluent. The different sugars and their concentrations are 3% sucrose, 5% sucrose, 10% sucrose, 5% trehalose, 5% glucose, and 5% fructose. Their detection stabilities after long-term storage at 2-8°C were tested. The SA-PE diluents containing different sugars and concentrations were placed at 2-8°C for 1 month, 3 months, 6 months, 9 months, 12 months, and 18 months respectively, and then formulated into fluorescence reagents for detection. The detection sample was Quality Control Product 1. Each quality control product was detected three times and the average value was taken to calculate the relative deviation from the theoretical value. The results are shown in Table 7 below.

[0125] Table 7. Detection Results of Stability Experiments of Different Sugars and Different Concentrations of Sugars

[0126]

[0127]

[0128] According to the results analysis in Table 7, by comparing the effects of SA-PE diluents prepared with 3%, 5%, and 10% sucrose on detection after being placed at 2-8°C for different times, the detection results of 3% sucrose deviated significantly from the theoretical value, while the detection results of 5% and 10% sucrose were close to the theoretical value, and their relative deviation values were close, both controlled within ±5%. Then, by comparing the effects of SA-PE diluents prepared with 5% sucrose, 5% trehalose, 5% glucose, and 5% fructose on detection after being placed at 2-8°C for different times, the relative deviation of the detection results of the SA-PE diluent prepared with 5% sucrose was always controlled within ±5% as the storage time prolonged, while the relative deviation of the detection results of the SA-PE diluents prepared in the other groups became larger and larger as the storage time prolonged. This shows that 5% sucrose can significantly improve the stability of the SA-PE diluent and thus improve the accuracy of detection. Considering the detection performance and cost comprehensively, adding 5% sucrose to the SA-PE diluent can enable the SA-PE diluent to be stored at 2-8°C for more than 18 months.

[0129] Example 3. Influence of Blocking Agent in Reaction Buffer on Detection

[0130] When no blocker is added, there will be relatively serious non-specific binding problems. In order to reduce false positive or false negative test results caused by non-specific binding in samples and improve the accuracy and specificity of detection. It is necessary to add blockers to the reaction buffer. In this example, different blockers HBR2 (purchased from Scantibodies, catalog number 3KC535), HBR6 (purchased from Scantibodies, catalog number 3KC542), HBR9 (purchased from Scantibodies, 3KC564), and HBR11 (purchased from Scantibodies, 3KC565) are combined as shown in Table 8 below. In this example, the kit provided in Example 1 was used to add interfering substances (rheumatoid factor 1500 IU / mL, HAMA 1000 ng / mL) to samples close to the blank limit (PBS buffer without AD-related markers), and at the same time, four markers, namely Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217, were detected. Different blocker compositions shown in Table 8 below were added to the reaction buffer respectively to investigate the influence of different blocker compositions on the blank limit test results. Each group was repeated three times, and the average value was calculated. The experimental results are shown in Table 8 below.

[0131] Table 8. Comparison of interference removal detection with different blocker compositions

[0132]

[0133] According to the result analysis in Table 8, by comparing the interference removal detection results of different blocker compositions, the combination of 5% HBR6 and 5% HBR11 minimizes the occurrence of interference, thereby reducing the appearance of false positives, improving specific binding, and further improving the accuracy of detection.

[0134] Furthermore, the different blocker compositions in Table 8 above were used to detect four AD markers, namely Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217, in non-specific samples (blood samples). The influence of different blockers on the test results was investigated. Each group was repeated three times, and the average value was calculated. The test results were compared with the mass spectrometry test results to calculate the relative deviation. The test results are shown in Table 9 below.

[0135] Table 9. Test results of detecting non-specific samples with different blockers

[0136]

[0137] According to the result analysis in Table 9, by comparing the relative deviations of the test results of different blocker compositions from the mass spectrometry test values, only the test results of the 5% HBR6 + 5% HBR11 blocker composition have a relative deviation controlled within ±5%, and the detection accuracy is higher.

[0138] Example 4, Influence of Adding Blocking Agent to Reaction Buffer and Adding Sucrose to SA-PE Diluent on Detection

[0139] 1. Two-step Incubation Method

[0140] The incubation method described in CN2023111107302 is a two-step incubation method, specifically: Add 25 μL of reaction buffer and 25 μL of microsphere solution conjugated with antibody into the sample tube, mix well, and shake with a shaker for more than 30 seconds; Add 75 μL of sample into the sample tube; Add 25 μL of biotin-conjugated antibody and 25 μL of incubator into the sample tube, and the incubator contains sorbitol, polyvinylpyrrolidone, Triton X-100, sodium citrate, sodium hyaluronate, and tris(hydroxymethyl)aminomethane; Incubate the sample tube at 2-8 °C in the dark with shaking for 18 hours, and the shaking frequency is 500 r / min; Add fluorescent reagent into the sample tube, and incubate at room temperature in the dark with shaking for 0.5 hour; Add 1000 μL of 1× washing buffer into the sample tube, resuspend the microspheres by vortexing, mix well, and shake with a shaker for more than 30 seconds, then centrifuge at 300 g for 5 minutes and discard the supernatant; Add 150 μL - 300 μL of 1× washing buffer into the sample tube, resuspend the microspheres by vortexing, mix well, and shake with a shaker for more than 30 seconds, and detect the fluorescence type and fluorescence signal intensity on a flow cytometer.

[0141] The above incubation method requires the addition of an incubation agent, and the composition of the incubation agent is complex, including sorbitol, polyvinylpyrrolidone, Triton X-100, sodium citrate, sodium hyaluronate, tris(hydroxymethyl)aminomethane (Tris). Incomplete removal during subsequent washing may affect the detection results. The incubation conditions of the incubation agent are 2-8°C because low temperature can reduce non-specific binding, stabilize fluorescent labels, and maintain the activity of biomolecules. However, low-temperature incubation means that relatively strict conditions need to be controlled. The research team of this invention thought that the blocker has a similar effect to the incubation agent. In addition, the blocker helps to provide a relatively stable chemical environment for biomolecules at room temperature, reducing problems such as 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 blockers are added to the reaction buffer to replace the incubation agent. In addition, the research team of this invention thought that in addition to maintaining protein stability, sucrose can also keep the microspheres conjugated with antibodies in a suspended state during incubation, reducing the probability of aggregation and cross-linking of the microspheres conjugated with antibodies, and thus achieving the effect of promoting the reaction. Therefore, sucrose is added to the SA-PE diluent for preparing the fluorescent reagent. To verify whether adding HBR6 and HBR11 blockers to the reaction buffer can replace the incubation agent to achieve room-temperature incubation and whether adding sucrose to the SA-PE diluent for preparing the fluorescent reagent can improve the detection stability and thus improve the accuracy, the following five groups are set:

[0142] Group 1: According to the incubation method described in the above CN2023111107302;

[0143] Group 2: Change the incubation temperature of 2-8°C in the incubation method described in CN2023111107302 to room temperature (25°C);

[0144] Group 3: Replace the incubation agent in the incubation method described in CN2023111107302 with 5% HBR6 and 5% HBR11 blockers, and change the incubation temperature of 2-8°C to room temperature (25°C);

[0145] Group 4: Add 5% sucrose to the SA-PE diluent for preparing the fluorescent reagent in the incubation method described in CN2023111107302;

[0146] Group 5: Replace the incubation agent in the incubation method described in CN2023111107302 with 5% HBR6 and 5% HBR11 blockers, change the incubation temperature of 2-8°C to room temperature (25°C), and then add 5% sucrose to the SA-PE diluent for preparing the fluorescent reagent.

[0147] Using the above five groups of incubation methods, the quality control products 1 of Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217 were detected respectively. Each quality control product was detected three times and the average value was taken. The detection results are shown in Table 10 below.

[0148] Table 10. Influence of replacing the incubation agent with a blocker on detection

[0149]

[0150] According to the data analysis in Table 10, comparing the data of the first and second groups, it shows that directly changing the low-temperature incubation temperature from 2-8°C to room temperature (25°C) according to the incubation method described in the above CN2023111107302 will lead to inaccurate detection results; however, comparing the data of the first, second, and third groups, it shows that replacing the incubation agent with 5% HBR6 and 5% HBR11 blockers can achieve changing the low-temperature incubation temperature from 2-8°C to room temperature (25°C), and the detection results are not affected; comparing the data of the first and fourth groups, it shows that adding 5% sucrose to the SA-PE diluent for preparing the fluorescent reagent 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, indicating that using 5% HBR6 and 5% HBR11 blockers to replace the incubation agent realizes room temperature (25°C) incubation, and adding 5% sucrose to the SA-PE diluent for preparing the fluorescent reagent further improves the detection accuracy.

[0151] Therefore, only by replacing the incubation agent described in CN2023111107302 with 5% HBR6 and 5% HBR11 blockers and adding 5% sucrose to the SA-PE diluent for preparing the fluorescent reagent can the low-temperature incubation condition of 2-8°C be changed to room temperature (25°C) 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. However, the microsphere reagent conjugated with the antibody and the biotin-conjugated antibody reagent are still co-incubated. In order to make the fluorescent microspheres conjugated with the antibody and the biotin-conjugated antibody bind more fully with the sample to be detected, the optimized two-step incubation method in the above CN2023111107302 is split into a 2.5-step incubation method. The specific steps of the 2.5-step incubation method are as follows:

[0154] (1)Step 1 Incubation: Add 25 μL of reaction buffer (containing 5% HBR6 and 5% HBR11 blocker) and 25 μL of microsphere solution conjugated with antibodies into the sample tube, mix well, and shake on a shaker for more than 30 seconds; add 75 μL of the sample into the sample tube, mix well by shaking, and incubate in the dark at room temperature (25 °C) with shaking for 9 h at a shaking amplitude of 500 r / min;

[0155] (2)Step 2 Incubation: Add 1 mL of 1× wash buffer into the sample tube, resuspend the microspheres by vortexing, centrifuge at 400 g for 5 min, and carefully remove the supernatant; add 25 μL of detection antibody reagent into the sample tube, mix well by shaking, and incubate in the dark at room temperature (25 °C) with shaking for 9 h at a shaking amplitude of 500 r / min;

[0156] (3)Step 2.5 Incubation: Add 25 μL of fluorescent reagent (containing 5% sucrose) into the sample tube, mix well by shaking the tube, incubate at room temperature in the dark with shaking for 0.5 h (shaking amplitude is 600 - 900 r / min), add 1 mL of 1× wash buffer into the sample tube, resuspend the microspheres by vortexing, centrifuge at 400 g for 5 min, and carefully remove the supernatant; according to the sample loading requirement, add 150 μL - 300 μL of 1× wash buffer into the tube, resuspend the microspheres by vortexing, mix well, and shake on a shaker for more than 30 seconds, then detect the fluorescence type and fluorescence signal intensity on a flow cytometer.

[0157] Furthermore, to verify whether the 2.5-step incubation method can further improve the detection accuracy, the following experiment was conducted.

[0158] Set the following four groups:

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

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

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

[0162] The fourth group: Detect according to the 2.5-step incubation method above, but replace 5% HBR6 and 5% HBR11 blocker with the incubation agent in CN2023111107302.

[0163] The quality control product 1 of Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217 was detected using the above four groups of detection methods respectively. Each quality control product was detected three times and the average value was taken. The detection results are shown in Table 11 below.

[0164] Table 11. Influence of different 2.5-step incubation conditions on detection

[0165]

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

[0167] In summary, this embodiment improves the two-step incubation method described in CN2023111107302, uses the blockers 5% HBR6 and 5% HBR11 to replace the incubator, adds 5% sucrose to the SA-PE diluent for preparing the fluorescent reagent, and also provides a new 2.5-step incubation method. Under the combined action of the above three changes, the incubation temperature changes from 2-8°C to room temperature (25°C), and the detection accuracy is further improved.

[0168] Example 5. Screening of conditions for 2.5-step room temperature shaking incubation

[0169] It has been proven in Example 4 that adding the blocker 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 it is possible to achieve incubation at room temperature (25°C) through the 2.5-step incubation method, but the total incubation time still needs 18.5 h, which cannot meet the detection requirements of some emergency samples.

[0170] Since increasing the incubation temperature can shorten the incubation time, and the above 25°C is already the result of increasing the incubation temperature from the original low-temperature incubation (2-8°C), it is thus conjectured whether the incubation time has reached saturation and whether the incubation time can be shortened. To confirm the above conjecture and explore the room temperature incubation temperature range, time, and the optimal sample loading amount, the following experiment was set up:

[0171] The incubation temperatures were 4°C, 25°C, and 37°C, the total incubation times were 2.5 h, 4.5 h, 6.5 h, and 18.5 h, and the sample loading volumes were 25 μL, 75 μL, and 100 μL respectively. The total incubation time was the sum of the incubation times in the 2.5-step incubation method in Example 4. The settings of the total incubation time are specifically shown in Table 12 below:

[0172] Table 12. Total incubation time

[0173]

[0174] Four experimental groups with total incubation times of 2.5 h, 4.5 h, 6.5 h, and 18.5 h were set at 4°C, while three experimental groups with total incubation times of 2.5 h, 4.5 h, and 6.5 h were set at each of 25°C and 37°C.

[0175] The quality control product 2 was named calibrator C1 and serially diluted 2-fold to calibrators C2 - C8. C0 was the sample diluent for blank control. The C1 concentration of Aβ1-40 was 1250 pg / mL, the C1 concentration of Aβ1-42 was 625 pg / mL, the C1 concentration of p-Tau-181 was 100 pg / mL, and the C1 concentration of p-Tau-217 was 40 pg / mL. The fluorescence signal values of Aβ1-40 and Aβ1-42 in C1, C2, C4, C7, and C8 were detected, and the fluorescence signal values of p-Tau-181 and p-Tau-217 in C1, C3, C5, C7, and C8 were detected. Each sample was detected three times and the average value was taken. At the same time, the sample loading volume was screened. In addition, 4 samples with known concentrations were selected for detection. The Aβ1-40 concentration of sample 1 was 150.12 pg / mL, the Aβ1-42 concentration of sample 2 was 75.06 pg / mL, the p-Tau-181 concentration of sample 3 was 25 pg / mL, and the p-Tau-217 concentration of sample 4 was 10 pg / mL. The detection results are shown in Tables 13 - 16 below.

[0176] Table 13. Results of detecting Aβ1-40 at different incubation temperatures and times

[0177]

[0178] Table 14. Results of detecting Aβ1-42 at different incubation temperatures and times

[0179]

[0180] Table 15. Results of detecting p-Tau-181 at different incubation temperatures and times

[0181]

[0182] Table 16. Results of detecting p-Tau-217 at different incubation temperatures and incubation times

[0183]

[0184] According to the result analysis of Tables 13 - 16, convert all the above fluorescence signal values into concentrations. Regardless of which incubation condition is used, when the sample loading volume for detection is 75 μL, the detected value is closest to the theoretical value. Therefore, the sample loading volume is selected as 75 μL.

[0185] When the sample loading volume is 75 μL, compare the detection results of incubating for a total duration of 2.5 h, 4.5 h, 6.5 h, and 18.5 h at a low temperature of 4°C respectively. Convert the fluorescence signal values into concentration values. The deviation between the detected concentration and the theoretical value is the smallest when incubating for 18.5 h, indicating that at a low temperature, the total incubation duration required is 18.5 h. Compare the detection results of incubating for a total duration of 2.5 h, 4.5 h, and 6.5 h at 25°C and 37°C respectively. Convert the fluorescence signal values into concentration values. The detection results under these two temperatures do not differ much, and the relative deviation values of the detections are all controlled within 10%. However, the relative deviation of the detection during incubation at 25°C is smaller, and there is no significant difference when the total incubation duration is 2.5 h, 4.5 h, or 6.5 h. The relative deviation values of the detection results are all controlled within 5%. To save detection time, it is preferably incubated at 25°C for 2.5 h. Then compare the detection results of incubating for a total duration of 18.5 h at a low temperature of 4°C and incubating for a total duration of 2.5 h at room temperature of 25°C. Convert the fluorescence signal values into concentration values. The detection result of incubating for a total duration of 2.5 h at room temperature of 25°C has a smaller deviation and is more accurate. Therefore, when the sample loading volume is 75 μL, by increasing the incubation temperature of the 2.5-step incubation method, the total incubation duration can be shortened without affecting the accuracy of the detection.

[0186] Based on the above comprehensive analysis, the preferred sample loading volume for the 2.5-step incubation method is 75 μL, the preferred incubation temperature is 25°C, and the preferred total incubation duration is 2.5 h.

[0187] Example 6. Optimization of the mixed molar ratio of antibody and biotin and the concentration of SA-PE in the fluorescent reagent

[0188] The difference between this embodiment and Embodiment 1 lies in changing the molar ratio of the antibody to biotin and the concentration of SA-PE in the fluorescent reagent. The molar ratios of the antibody to biotin are 1:10, 1:20, 1:30, 1:40, 1:50, and 1:60 respectively, and the concentrations of SA-PE in the fluorescent reagent are 0.25 μg / mL, 0.50 μg / mL, 1.00 μg / mL, and 2.00 μg / mL respectively. Different molar ratios of the antibody to biotin are combined with different concentrations of SA-PE in the fluorescent reagent to detect the concentration of the AD biomarker in the sample. The sample to be detected is Quality Control Product 1 in Embodiment 1. Each quality control product is detected 3 times and the average value is taken, 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 antibody to biotin and different combinations of SA-PE concentrations

[0190]

[0191] According to the result analysis 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 deviations of the detection results of the four AD biomarkers can be controlled within ±5%.

[0192] Although the present invention is disclosed as above, the present invention 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 invention. Therefore, the protection scope of the present invention 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 kit includes a microsphere solution conjugated with an antibody, a biotin-conjugated antibody solution, and a fluorescent reagent; the fluorescent reagent contains an SA-PE diluent, and the SA-PE diluent contains sucrose; the antibody is one or more of an Aβ1-40 antibody, an Aβ1-42 antibody, a p-Tau-181 antibody, and a p-Tau-217 antibody.

2. The kit according to claim 1, wherein It further includes a reaction buffer, and the reaction buffer contains blockers HBR6 and HBR11.

3. The kit according to claim 1, characterized in that, The fluorescent reagent further contains SA-PE, and the concentration of SA-PE after dilution with the SA-PE diluent is 0.25 - 2 μg / mL.

4. The kit according to claim 1, characterized in that, The molar ratio of the antibody to biotin added in the biotin-conjugated antibody solution is 1:(10 - 60).

5. The kit according to claim 1, characterized in that, 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.

6. A detection method for Alzheimer's disease-related proteins, characterized in that, Using the kit according to any one of claims 1 - 5 for detection includes the following steps: (1) Add the reaction buffer and the microsphere solution conjugated with an antibody to a sample tube and mix well. (2) Add the test sample to the sample tube, mix well and incubate. (3) After washing, add the biotin-conjugated antibody solution to the sample tube, mix well and incubate. (4) Add the fluorescent reagent to the sample tube, mix well and incubate. (5) Detect the contents of Aβ1-40, Aβ1-42, p-Tau-181, and p-Tau-217 on a flow cytometer.

7. The detection method according to claim 6, wherein, The incubation condition in step (2) is to incubate under light avoidance and oscillation at 4 - 37°C for 1 - 6 hours; the incubation condition in step (3) is to incubate under light avoidance and oscillation at 4 - 37°C for 1 - 6 hours.

8. The detection method according to claim 6, characterized in that The incubation condition in step (4) is to incubate under light avoidance and oscillation at 4 - 37°C for 0.5 hour.

9. Use of the SA-PE diluent and blocker composition in the preparation of a kit for shortening the incubation time of detecting Alzheimer's disease-related proteins, characterized in that, The SA-PE diluent includes sucrose, and the blocker composition includes HBR6 and HBR11.

10. Use of a blocker composition for preparing a reagent for enhancing the specific binding ability of Alzheimer's disease-related proteins in a test sample, characterized in that, The blocker composition includes HBR6 and HBR11.

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