A composite immune enhancer for AD marker detection and application thereof

Abstract

The present application relates to the medical immunological in-vitro diagnosis field, provide a kind of for AD marker detection composite immunopotentiator, including 2-methyl acryloyloxy ethyl phosphoryl choline copolymer, polyethylene glycol 20000, polyvinylpyrrolidone-10 and membrane lysing agent.The present application also provides the application of the above-mentioned composite immunopotentiator in the preparation of the kit for detecting Alzheimer's disease biomarker.The present application has the advantages that: the composite immunopotentiator is added to the reagent R1 and reagent R2 of the existing AD marker detection kit, can accelerate the reaction, shorten the immune response time, improve the detection sensitivity and accuracy, and keep stable.

Description

A composite immune enhancer for AD biomarker detection and its application Technical Field

[0001] This invention relates to the field of medical immunodiagnostics, and more particularly to a compound immunostimulant for the detection of AD markers and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system. The pathogenesis of AD is not yet fully understood, and diagnosis is primarily based on clinical manifestations. In 2007, the National Institute of Neurological and Language Disorders Stroke (NINCDS-ADRDA) added biomarkers as a new auxiliary diagnostic indicator to its new diagnostic criteria for Alzheimer's disease. These biomarkers can indicate underlying pathological conditions in AD, thus aiding in its early diagnosis.

[0003] The clinical manifestations of Alzheimer's disease (AD) are highly specific and easy to identify. Magnetic resonance imaging (MRI) is the most widely used auxiliary diagnostic tool, and it has some significance in differentiating AD. However, imaging examinations lack specific imaging features for patients without obvious clinical manifestations and are expensive, hindering early diagnosis. With further research, scholars have discovered pathological changes in the early stages of AD, such as abnormal deposition of extracellular amyloid beta protein (Aβ) and phosphorylation of Tau protein. Therefore, studying changes in these two biomarkers in the blood is of great clinical significance for auxiliary screening of AD.

[0004] Because obtaining cerebrospinal fluid is difficult and causes significant damage, resulting in considerable suffering for patients, studying changes in Alzheimer's markers in the blood is of great clinical significance for assisting in AD screening. However, since the levels of Alzheimer's disease markers (such as Aβ1-42 and P-tau-181) in the blood are lower than those in cerebrospinal fluid, existing methods for detecting Alzheimer's disease markers in the blood are somewhat lacking in sensitivity and accuracy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a composite immunostimulant for the detection of AD biomarkers and its application. When the composite immunostimulant is added to reagents R1 and R2 of an existing AD biomarker detection kit, the reaction can be accelerated, the immune reaction time can be shortened, the detection sensitivity and accuracy can be improved, and the stability can be maintained.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A composite immunostimulant for the detection of AD markers comprises: a copolymer of 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol, polyvinylpyrrolidone, and a membrane lysis agent.

[0008] In this invention, the composite immune enhancer contains polyethylene glycol and PVP, both of which are high molecular weight substances. Polyethylene glycol is a surfactant with excellent solubilizing, suspending, emulsifying, and low-toxicity properties, and can be used as a matrix with good thermal stability. Polyvinylpyrrolidone has good solubility, solubilizing, physiological compatibility, and complexing ability, and can be used as a co-solvent and solubilizer. Both can promote the dissolution of the components in the reagent, thereby accelerating the reaction process.

[0009] The membrane lysing agent in the composite immune enhancer of this invention dissolves the cell membrane by dissolving the lipid components on the cell membrane, allowing antibodies to enter the cell and bind to the antigen. The antibodies fully bind to the corresponding antigen, thereby improving the positive detection rate, enhancing the blocking and sealing effects, and reducing the background value. Furthermore, it has good compatibility with other types of surfactants and strong dissolving properties.

[0010] The copolymer of 2-methacryloyloxyethyl phosphorylcholine in the composite immune enhancer of this invention has excellent stability and reproducibility. It can significantly enhance the signal intensity in the sandwich method, enhance sensitivity, improve antibody stability, reduce batch-to-batch differences in nonspecific binding, and inhibit nonspecific binding.

[0011] In the buffer solution containing the aforementioned composite immune enhancer, the copolymer of 2-methacryloyloxyethyl phosphorylcholine has a mass-volume percentage of 0.01% to 1%, for example, it can be 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, preferably 0.1% to 0.5%.

[0012] In the buffer solution containing the aforementioned composite immune enhancer, the polyethylene glycol has a mass-volume percentage of 0.01% to 0.5%, for example, it can be 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%, preferably 0.05% to 0.3%.

[0013] In the buffer solution containing the aforementioned composite immune enhancer, the mass-volume percentage of the polyvinylpyrrolidone is 0.01% to 1%, for example, it can be 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, preferably 0.05% to 0.5%.

[0014] In the buffer solution containing the composite immune enhancer, the volume percentage of the membrane lysis agent is 0.05% to 2%, for example, it can be 0.05%, 0.08%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, or 2%, preferably 0.1% to 1%.

[0015] As one of the preferred embodiments of the present invention, the copolymer of 2-methacryloyloxyethylphosphorylcholine is selected from... 205. 103 or Any one or at least two of 405.

[0016] The polyethylene glycol is selected from polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000 or polyethylene glycol 20000.

[0017] The polyvinylpyrrolidone is selected from PVP-10 or PVP-40.

[0018] The membrane lysis agent is selected from Triton X-100, Triton X-114, Triton X-405 or Triton X-450.

[0019] As one of the preferred embodiments of the present invention, a composite immune enhancer for the detection of AD biomarkers comprises: 205. Polyethylene glycol 20000 (PEG20000), PVP-10 (polyvinylpyrrolidone-10) and Triton X-100.

[0020] In this invention, 205 is a polymer with excellent stability and reproducibility; and it has been verified that, compared to other biolipidures, it exhibits superior performance. 103. 405 and similar materials have high hydrophilicity and are generally used in latex aggregation, sideflow testing, and metal surface modification. The material selected in this invention... The relative hydrophobicity of 205 can significantly enhance the signal intensity in the sandwich method, improve sensitivity, enhance antibody stability, reduce batch-to-batch differences in nonspecific binding, and inhibit nonspecific binding.

[0021] PEG20000 possesses excellent properties such as solubilization, suspending, emulsification, and low toxicity. Adding PEG20000 as a component of the composite immune enhancer in this invention can improve solution stability. PVP-10 exhibits good solubility, susceptibility, physiological compatibility, and complexing ability, and can be used as a co-solvent and solubilizer. Both PEG20000 and PVP-10 can promote the dissolution of components in the reagent and accelerate the reaction process.

[0022] Triton X-100 is a nonionic surfactant with strong dissolving properties. It can dissolve the cell membrane in immunocytochemistry by dissolving the lipid components on the cell membrane, allowing antibodies to enter the cell and bind to the antigen. The antibodies fully bind to the corresponding antigen, thereby improving the positive detection rate of cells. It can also enhance the blocking and sealing effects and reduce background influence.

[0023] The composite immune enhancer is dispersed in a buffer solution, wherein the copolymer of 2-methacryloyloxyethylphosphorylcholine has a mass-volume percentage of 0.1%–0.5% (w / v), polyethylene glycol 20000 (PEG20000) has a mass-volume percentage of 0.05%–0.3% (w / v), polyvinylpyrrolidone-10 (PVP-10) has a mass-volume percentage of 0.05%–0.5% (w / v), and Triton X-100 has a volume percentage of 0.1%–1% (v / v).

[0024] Preferably, the copolymer of 2-methacryloyloxyethylphosphorylcholine has a mass-volume percentage of 0.1% to 0.5%, for example, it can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%, and is preferably 0.2%.

[0025] Preferably, the mass-volume percentage of the polyethylene glycol 20000 is 0.05% to 0.3%, for example, it can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25% or 0.3%, etc., and is preferably 0.1%.

[0026] Preferably, the mass-volume percentage of the polyvinylpyrrolidone-10 is 0.05% to 0.5%, for example, it can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%, and is preferably 0.1%.

[0027] Preferably, the volume percentage of the Triton X-100 is 0.1% to 1%, for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, etc., preferably 0.2%.

[0028] This invention also provides the application of a composite immune enhancer for the detection of AD biomarkers in the preparation of a kit for detecting Alzheimer's disease biomarkers.

[0029] The AD markers include p-Tau-181, p-Tau-217, or p-Tau-231.

[0030] As one of the preferred embodiments of the present invention, the composite immune enhancer is added to reagents R1 and / or reagents R2 of the kit for detecting Alzheimer's disease biomarkers, respectively.

[0031] The composite immunostimulant provided by this invention can be added as a universal reagent to kits for detecting Alzheimer's disease biomarkers, or to magnetic particle chemiluminescence detection kits. Adding the composite immunostimulant shortens the immune reaction time, improves detection sensitivity and accuracy, and maintains stability.

[0032] A p-Tau-181 magnetic microparticle chemiluminescence detection kit includes reagent R1, reagent R2, magnetic separation reagent M, calibrator and / or quality control, luminescent substrate solution A, and luminescent substrate solution B; the above-mentioned composite immunostimulant is added to reagent R1 and reagent R2 respectively.

[0033] As one of the preferred embodiments of the present invention, the reagent R1 comprises a biotin-labeled p-Tau-181 antibody, a Tris buffer, and the composite immunostimulant, wherein the buffer is preferably a Tris buffer.

[0034] The reagent R2 comprises an acridinium-labeled p-Tau-181 antibody, a buffer solution, and the composite immunostimulant, wherein the buffer solution is preferably PBS buffer.

[0035] The magnetic particles in the magnetic separation reagent M are streptavidin magnetic beads.

[0036] As one of the preferred embodiments of the present invention, the preparation method of the p-Tau-181 magnetic particle chemiluminescence detection kit is as follows:

[0037] (1) The copolymer of 2-methacryloxyethylphosphorylcholine, polyethylene glycol, polyvinylpyrrolidone and membrane lysis agent were dispersed in a buffer to prepare R1 buffer containing a composite immunostimulant; Biotin and p-Tau-181 antibody were mixed and reacted, purified and diluted to working concentration with the R1 buffer to obtain reagent R1;

[0038] (2) The copolymer of 2-methacryloyloxyethylphosphorylcholine, polyethylene glycol, polyvinylpyrrolidone and membrane lysis agent were dispersed in a buffer to prepare R2 buffer containing a composite immunostimulant; acridinium ester and p-Tau-181 antibody were mixed and reacted, purified and diluted to working concentration with the R2 buffer to obtain reagent R2;

[0039] (3) Prepare magnetic separation reagent M, calibrator, luminescent substrate A and luminescent substrate B solutions respectively to obtain the p-Tau-181 magnetic microparticle chemiluminescence detection kit.

[0040] Specifically, the preparation method includes the following steps:

[0041] Preparation of reagent R1: Commercially available biotin was dissolved in DMSO to prepare a 10 mM solution. p-Tau-181 antibody was added, and the mixture was reacted with shaking at room temperature for 4 h. After purification using a molecular sieve and filtration through a 0.22 μm membrane, the solution was diluted with R1 buffer to a working concentration (0.5 μg / mL) to obtain reagent R1. The R1 buffer consisted of Tris buffer and the aforementioned composite immunostimulant.

[0042] Preparation of magnetic separation reagent M: Take commercially available streptavidin magnetic beads, add PBS buffer containing 5% bovine serum albumin and dilute to the working concentration (1.0 mg / mL) to obtain magnetic separation reagent M;

[0043] Preparation of reagent R2: Commercially available acridil ester NSP-DMAE-NHS was dissolved in DMF to prepare a 1.5 mM solution. p-Tau-181 antibody was added, and the mixture was reacted with shaking at room temperature in the dark for 2 h. Blocking agent was added, and the mixture was reacted with shaking at room temperature in the dark for 1 h. The mixture was purified using a molecular sieve, filtered through a 0.22 μm membrane, and diluted with R2 buffer to a working concentration (1.0 μg / mL) to obtain reagent R2. The R2 buffer consisted of PBS buffer and the aforementioned composite immunostimulant.

[0044] Preparation of calibration / quality control samples: Weigh the required amounts of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, bovine serum albumin, Tween 20, and preservative Proclin 300 into a container and stir to dissolve evenly; finally, add purified water to make up to the target volume; adjust the pH value to 7.20±0.10; after 0.22μm filtration, add p-Tau-181 antigen to prepare two concentrations (40pg / mL and 80pg / mL);

[0045] Preparation method of substrate A solution: Weigh hydrogen peroxide and concentrated nitric acid and add them to a container. Stir and dissolve evenly, then filter using a 0.22μm filter membrane.

[0046] Preparation method of substrate B solution: Weigh sodium hydroxide and surfactant Tween 20 and add them to a container. Stir and dissolve evenly, then filter using a 0.22μm filter membrane.

[0047] A detection method for the above-mentioned p-Tau-181 magnetic particle chemiluminescence detection kit includes the following steps:

[0048] (1) Add the sample to be tested, reagent R1 and reagent R2 together into the reaction vessel and incubate at 37°C;

[0049] (2) Add magnetic separation reagent M to the sample after incubation in step (1), and continue incubation at 37°C;

[0050] (3) Washing to remove unbound substances;

[0051] (4) Add luminescent substrate A and luminescent substrate B to the reaction complex to excite light and detect the light intensity;

[0052] (5) Using a four-parameter fitting method, a calibration curve is established with the luminescence value of the calibrator as the Y-axis and the corresponding concentration as the X-axis; the corresponding concentration value of p-Tau-181 is calculated back based on the luminescence value of the sample to be tested.

[0053] Detection Principle: A sandwich immunoassay based on direct chemiluminescence technology; First, p-Tau-181 in the sample reacts with reagent R1 (biotin-labeled p-Tau-181 antibody) to form an antigen-antibody complex. Next, reagent R2 (acridoid-labeled p-Tau-181 antibody) binds to the complex from the first step, forming an antibody-antigen-antibody complex; simultaneously, reagent M (streptavidin-coated magnetic microparticles) binds to the immune complex; then, unbound substances are washed away with instrument cleaning solution, and luminescent substrate solutions A and B are added to the reaction complex using the instrument, and the relative luminescence intensity (RLU) is measured.

[0054] The advantages of this invention compared to the prior art are: it provides a composite immunostimulant and kit for AD biomarker detection. Adding this composite immunostimulant to reagents R1 and R2 in existing AD biomarker detection kits can accelerate the reaction, shorten the immune reaction time, improve detection sensitivity and accuracy, and maintain stability. Attached Figure Description

[0055] Figure 1 is a comparison of the test results of the experimental group and the control group in the performance test of SO solution;

[0056] Figure 2 is a comparison of the test results of the experimental group and the control group for S1 solution in the performance test;

[0057] Figure 3 is a comparison of the test results of the experimental group and the control group for S2 solution in the performance test;

[0058] Figure 4 is a comparison of the test results of the experimental group and the control group for S3 solution in the performance test;

[0059] Figure 5 is a comparison of the test results of the experimental group and the control group for S4 solution in the performance test;

[0060] Figure 6 is a comparison of the test results of the experimental group and the control group for S5 solution in the performance test;

[0061] Figure 7 is a correlation curve between the kit of the present invention and commercially available test kits in the performance test. Detailed Implementation

[0062] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0063] Example 1

[0064] This embodiment provides a composite immune enhancer for AD biomarker detection, comprising: 2050.2% (w / v), PEG-20000 0.1% (w / v), PVP-10 0.1% (w / v), Triton X-100 0.2% (v / v), balance: purified water;

[0065] The reagent kit buffer was prepared using the aforementioned composite immune enhancer, following these steps:

[0066] Add 1g of PEG20000 and 1g of PVP10 to 1L of buffer solution, dissolve completely, then add 2g of PVP10. Dissolve 205 and 2 mL of Triton X-100 to prepare the kit buffer.

[0067] Example 2

[0068] This embodiment provides a composite immune enhancer for AD biomarker detection, comprising: 2050.5% (w / v), PEG20000 0.3% (w / v), PVP-10 0.5% (w / v), Triton X-100 1% (v / v).

[0069] Example 3

[0070] This embodiment provides a composite immune enhancer for AD biomarker detection, comprising: 2050.1% (w / v), PEG-20000 0.05% (w / v), PVP-10 0.05% (w / v), Triton X-100 0.1% (v / v).

[0071] Example 4

[0072] This embodiment provides a composite immune enhancer for AD biomarker detection, comprising: 2050.01% (w / v), PEG-20000 0.5% (w / v), PVP-10 0.01% (w / v), Triton X-100 2% (v / v).

[0073] Example 5

[0074] This embodiment provides a composite immune enhancer for AD biomarker detection, comprising: 2051% (w / v), PEG-20000 0.01% (w / v), PVP-10 1% (w / v), Triton X-1000.05% (v / v).

[0075] The specific components and their contents are shown in Table 1 below:

[0076] Table 1

[0077]

[0078] Example 6

[0079] The difference from Example 1 is that the components of the compound immune enhancer are: 103, PEG-6000, PVP-10 and Triton X-114, the content and preparation method are consistent with those in Example 1.

[0080] Example 7

[0081] The difference from Example 1 is that the components of the compound immune enhancer are: 405, PEG-6000, PVP-40 and Triton X-100, with the same content and preparation method as in Example 1.

[0082] Example 8

[0083] The difference from Example 1 is that the components of the compound immune enhancer are: 205, PEG-8000, PVP-10 and Triton X-405, with the same content and preparation method as in Example 1.

[0084] Example 9

[0085] The difference from Example 1 is that the components of the compound immune enhancer are: 103, PEG-20000, PVP-40 and Triton X-100, with the same content and preparation method as in Example 1.

[0086] The specific components and their contents are shown in Table 2 below:

[0087] Table 2

[0088]

[0089] Comparative Example 1

[0090] The difference from Example 1 is that the compound immune enhancer does not contain PVP-10, and its specific component is 0.25%. 205, 0.1% PEG-20000 and 0.25% Triton X-100.

[0091] Comparative Example 2

[0092] The difference from Example 1 is that the compound immune enhancer does not contain... 205, specifically composed of 0.15% PEG-20000, 0.15% PVP-10 and 0.25% Triton X-100.

[0093] Comparative Example 3

[0094] The difference from Example 1 is that the composite immune enhancer does not contain PEG-20000, and its specific component is 0.25%. 205, 0.1% PVP-10 and 0.25% Triton X-100.

[0095] The specific components and their contents are shown in Table 3 below:

[0096] Table 3

[0097]

[0098] Application examples

[0099] This embodiment of a p-Tau-181 (a biomarker for detecting Alzheimer's disease) magnetic microparticle chemiluminescence detection kit includes reagent R1, reagent R2, magnetic separation reagent M, calibrator, quality control, luminescent substrate solution A, and luminescent substrate solution B; reagent R1 and reagent R2 are respectively supplemented with the composite immunostimulant provided in Example 1.

[0100] Preparation method:

[0101] (1) Preparation of reagent R1: Take commercially available biotin, dissolve it in DMSO to prepare a 10mM solution, add p-Tau-181 antibody, shake at room temperature for 4h, purify using molecular sieve, filter through a 0.22μm membrane, and dilute to 0.5μg / mL with R1 buffer (composed of 100mM Tris buffer and the aforementioned composite immunostimulant) to obtain reagent R1.

[0102] (2) Preparation of magnetic separation reagent M: Take commercially available streptavidin magnetic beads, add PBS buffer containing 5% bovine serum albumin and dilute to 1.0 mg / mL to obtain magnetic separation reagent M.

[0103] (3) Preparation of reagent R2: Take commercially available acridinium ester NSP-DMAE-NHS, dissolve it in DMF to prepare a 1.5 mM solution, add p-Tau-181 antibody, and react with shaking at room temperature in the dark for 2 h. Add blocking agent and react with shaking at room temperature in the dark for 1 h. Purify using molecular sieve, filter through a 0.22 μm membrane, and dilute to 1.0 μg / mL with R2 buffer (composed of 50 mM PBS buffer and the aforementioned composite immunostimulant) to obtain reagent R2.

[0104] (4) Preparation of calibration / quality control materials: Weigh the required amounts of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, bovine serum albumin, Tween 20, and preservative Proclin 300 into a container and stir to dissolve evenly; finally, add purified water to make up to the target volume; adjust the pH value to 7.20±0.10; filter at 0.22μm and add p-Tau-181 antigen to prepare two concentrations of 40pg / mL and 80pg / mL.

[0105] (5) Preparation method of luminescent substrate A solution: Weigh hydrogen peroxide and concentrated nitric acid and add them to a container. Stir and dissolve evenly, then filter with a 0.22μm filter membrane.

[0106] (6) Preparation method of luminescent substrate B solution: Weigh sodium hydroxide and surfactant Tween 20 and add them to a container. Stir and dissolve evenly, then filter with a 0.22μm filter membrane.

[0107] Regarding the preparation of R1 buffer (taking the compound immune enhancer formulation of Example 1 as an example): Add 1g of PEG20000 and 1g of PVP10 to 1L of Tris buffer, dissolve completely, and then add 2g of... Dissolve 205 in 2 mL of Triton X-100 to prepare R1 buffer for reagent R1.

[0108] Regarding the preparation of R2 buffer (taking the compound immunostimulant formulation of Example 1 as an example): Add 1g of PEG20000 and 1g of PVP10 to 1L of PBS buffer, dissolve completely, and then add 2g of [unclear text - possibly a continuation of the previous sentence] Dissolve 205 in 2 mL of Triton X-100 to prepare R2 buffer for reagent R2.

[0109] Detection method:

[0110] (1) Add 50 μL of the sample to be tested, 50 μL of reagent R1 and 50 μL of reagent R2 into the reaction vessel, and incubate at 37°C for 10 min.

[0111] (2) Add 30 μL of magnetic separation reagent M to the sample after incubation in step (1), and then continue incubation at 37°C for 5 min;

[0112] (3) Washing to remove unbound substances;

[0113] (4) Add 100 μL of luminescent substrate A solution and luminescent substrate B solution to the reaction complex for light excitation and detect the light intensity RLU;

[0114] (5) Using a four-parameter fitting method, a calibration curve is established with the luminescence value of the calibrator as the Y-axis and the corresponding concentration as the X-axis; the corresponding concentration value of p-Tau-181 is calculated back based on the luminescence value of the sample to be tested.

[0115] The p-Tau-181 biomarker for detecting Alzheimer's disease, prepared using the above method, was used sequentially with the composite immunostimulants prepared in Examples 1-9 and Comparative Examples 1-3 to obtain detection kits 1-12 (wherein, kits 1-9 use the composite immunostimulants of Examples 1-9, and kits 10-12 use the composite immunostimulants of Comparative Examples 1-3).

[0116] Furthermore, the specific embodiments of this invention also include a detection kit, numbered Detection Kit 13-14, which uses the composite immune enhancer provided in Example 1, with the difference being:

[0117] Test kit 13 contains only a compound immunostimulant in reagent R1;

[0118] Test kit 14 contains only a compound immune enhancer in reagent R2.

[0119] Performance testing

[0120] I. Accelerated Stability Test

[0121] Experimental method: The above p-Tau-181 magnetic microparticle chemiluminescence detection kit (i.e., detection kit 1-12) was placed in 4℃ and 37℃ for 7 days, and the concentration (pg / mL) of each kit was determined by quality control sample, with 3 tests for each kit.

[0122] Experimental results: The specific test results of test kit 1 are shown in Table 4; the overall test results of test kits 1-12 are shown in Table 5.

[0123] Table 4 shows the accelerated stability results of test kit 1.

[0124]

[0125] The results in Table 4 show that the reagent degradation rate is ≤ ±10%, indicating that the reagent of this invention has good stability.

[0126] Table 5

[0127]

[0128]

[0129] Based on Table 5, a comparison of test kit 1 and test kits 10-12 (i.e., Example 1 and Comparative Examples 1-3) shows that all four components of the compound immune enhancer are indispensable. Even with the same total usage, the instability is poor and the variation is quite large, far exceeding the acceptable range, if any one component is missing.

[0130] A comparison of test kit 1 and test kits 6-9 (i.e., Examples 1 and 6-9) shows that, in the composite immune enhancer, The optimal combination is 205, PEG-20000, PVP-10, and Triton X-100; other combinations may be used... 103, PEG-6000, and PVP-40, among other commonly used surfactants, also failed to achieve the stability shown in Example 1; therefore, it is evident that in this invention... 205, PEG-20000, PVP-10 and Triton X-100 work synergistically in the kit to improve its stability;

[0131] A comparison of test kit 1 and test kits 2-5 (i.e., Examples 1 and 2-5) shows that, in the composite immune enhancer, The preferred amounts of 205 are 0.1% to 0.5%, PEG-20000 are 0.05% to 0.3%, PVP-10 are 0.05% to 0.5%, and Triton X-100 are 0.1% to 1%. Buffers obtained within these ranges and used to prepare the kit can further improve the stability of the kit.

[0132] Furthermore, the descriptions of test kits 1 and 13-14 indicate that adding a compound immunostimulant to reagents R1 and R2 in the test kits does not change the reagent value by more than ±10%, but adding it to both reagents R1 and R2 simultaneously will result in better results.

[0133] In summary, the composite immune enhancer described in this invention can improve the stability of the reagent kit.

[0134] II. Reaction Time Test

[0135] Experimental methods:

[0136] First, an experimental group and a control group were set up; (1) The experimental group was: detection kit 1, which added the compound immune enhancer of Example 1, wherein the compound immune enhancer was added to both reagent R1 and reagent R2; (2) The control group was: the above p-Tau-181 magnetic particle chemiluminescence detection kit without the addition of the compound immune enhancer.

[0137] Next, p-Tau-181 standards were prepared into p-Tau-181 standard solutions of different concentrations using calibration buffer, with concentrations of 0 pg / mL, 1 pg / mL, 5 pg / mL, 25 pg / mL, 100 pg / mL and 200 pg / mL, respectively, and denoted as S0, S1, S2, S3, S4 and S5.

[0138] Finally, S0, S1, S2, S3, S4, and S5 were tested using the experimental and control groups, and the full reaction time of each group was observed.

[0139] Experimental results: The reaction times were set to 10 min, 15 min, 30 min and 60 min respectively. A line graph was constructed with the reaction time (min) as the x-axis and the luminescence value (RLU) as the y-axis.

[0140] The final results are shown in Figures 1-6:

[0141] Figure 1 shows a comparison of the detection results of the experimental group and the control group in detecting the S0 solution.

[0142] Figure 2 is a comparison of the detection results of the experimental group and the control group for S1 solution;

[0143] Figure 3 is a comparison of the detection results of S2 solution in the experimental group and the control group;

[0144] Figure 4 is a comparison of the detection results of the experimental group and the control group for S3 solution;

[0145] Figure 5 is a comparison of the detection results of the experimental group and the control group for S4 solution;

[0146] Figure 6 is a comparison of the detection results of the experimental group and the control group for S5 solution.

[0147] As shown in Figures 1-6, the experimental group showed no further fluctuations at a reaction time of 15 minutes, indicating a sufficient reaction; while the control group showed the best reaction at a reaction time of 60 minutes. This demonstrates that the compound immune enhancer of the present invention can significantly shorten the reaction time and improve sensitivity.

[0148] III. Accuracy Test

[0149] Experimental method: The p-Tau-181 magnetic microparticle chemiluminescence detection kit (detection kit 1) of the present invention and a commercially available p-Tau-181 detection kit were used to detect 52 clinically determined samples of Anqun reagent to verify the accuracy of the kit of the present invention.

[0150] Experimental results: The results are shown in Figure 7.

[0151] As shown in Figure 7, the clinical correlation between the test results of the kit of the present invention and the commercially available test kit is >0.95, which indicates that the test results of the method of the present invention have high accuracy.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composite immune enhancer for the detection of AD biomarkers, characterized in that, The compound immune enhancer consists of: 0.2% Biolipidure® 205, 0.1% PEG-20000, 0.1% PVP-10, and 0.2% Triton X-100.

2. The use of the composite immune enhancer for detecting AD biomarkers as described in claim 1 in the preparation of a kit for detecting Alzheimer's disease biomarkers.

3. The application of the composite immune enhancer according to claim 2 in the preparation of a reagent kit for detecting Alzheimer's disease biomarkers, characterized in that, The composite immune enhancer is added to reagent R1 and / or reagent R2 of the kit for detecting Alzheimer's disease biomarkers.

4. A p-Tau-181 magnetic microparticle chemiluminescence detection kit, characterized in that, It includes reagent R1, reagent R2, magnetic separation reagent M, calibrator and / or quality control sample, luminescent substrate solution A and luminescent substrate solution B; the composite immune enhancer as described in claim 1 is added to reagent R1 and / or reagent R2.

5. The p-Tau-181 magnetic microparticle chemiluminescence detection kit according to claim 4, characterized in that, The reagent R1 comprises a biotin-labeled p-Tau-181 antibody, a buffer solution, and the composite immunostimulant; the reagent R2 comprises an acridinium-labeled p-Tau-181 antibody, a buffer solution, and the composite immunostimulant; the magnetic particles in the magnetic separation reagent M are streptavidin magnetic beads.

6. A method for preparing the p-Tau-181 magnetic microparticle chemiluminescence detection kit as described in claim 4 or 5, characterized in that, The preparation method is as follows: (1) The copolymer of 2-methacryloxyethyl phosphorylcholine, polyethylene glycol, polyvinylpyrrolidone and membrane lysis agent are dispersed in a buffer to prepare R1 buffer containing a composite immunostimulant; Biotin and p-Tau-181 antibody are mixed and reacted, purified and diluted to the working concentration using the R1 buffer to obtain reagent R1; (2) The copolymer of 2-methacryloxyethyl phosphorylcholine, polyethylene glycol, polyvinylpyrrolidone and membrane lysis agent are dispersed in a buffer to prepare R2 buffer containing a composite immunostimulant; Acridinium ester and p-Tau-181 antibody are mixed and reacted, purified and diluted to the working concentration using the R2 buffer to obtain reagent R2; (3) Magnetic separation reagent M, calibrator, luminescent substrate A solution and luminescent substrate B solution are prepared respectively to obtain the p-Tau-181 magnetic particle chemiluminescence detection kit.

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