Preserving fluid of p-Tau217 magnetic bead coated antibody and preparation method

Abstract

The invention discloses a preservation solution of a p-Tau217 magnetic bead coated antibody and a preparation method, and belongs to the technical field of immunodetection, the preservation solution is a buffer solution containing 0.05 g / L-0. 2 g / L of a high-molecular polymer, 0.005 g / L-0. 02 g / L of a surfactant, 0.1 g / L-0. 3 g / L of a protein protective agent, 0.2 g / L-4 g / L of a metal chelating agent, 0.1 g / L-0. 5 g / L of a suspending aid and 0.001 g / L-0. 01 g / L of a preservative, the buffer solution is a PBS buffer solution or a Tris-HCl buffer solution, and the pH value is 7.0-7.5; the high-molecular polymer is any one of poly (methacryloyloxyethyl phosphorylcholine), poly (sulphobetaine methacrylate) or polyethylene glycol; the metal chelating agent is sodium hyaluronate or sodium gluconate; the suspending aid is cane sugar or trehalose; according to the invention, the signal-to-noise ratio is increased to gt by constructing a mild metal chelating agent-saccharide suspending agent-high-molecular polymer ternary synergistic protection system; 80, performing repetitive optimization to CVlt; the signal attenuation result after 10 days of acceleration is realized; 5%, and the attenuation lt after low-temperature storage for 20 months; the long-acting stability is 7%.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of immunodetection, and relates to a preservation solution for magnetic bead-coated antibodies, in particular to a preservation solution for p-Tau217 magnetic bead-coated antibodies and a preparation method. BACKGROUND

[0002] Alzheimer's disease (AD) is the main type of neurodegenerative disease, and its early diagnosis relies on high-specificity biomarkers. In recent years, research has found that the phosphorylated Tau protein at threonine 217 (p-Tau217) is significantly increased in the early pathological stage of AD (such as preclinical or mild cognitive impairment period), and can be detected even before the formation of beta-amyloid plaques. Its plasma concentration is highly consistent with the results of cerebrospinal fluid and PET imaging, and the specificity reaches 98.6%. Compared with the traditional marker p-Tau181, p-Tau217 performs better in the differential diagnosis of AD and other dementia, and is listed as an AD-specific biomarker by Nature Medicine. Therefore, developing an ultra-sensitive detection technology (such as chemiluminescence, single-molecule immunization platform) based on p-Tau217 has become a core direction for early screening of AD.

[0003] The immunodetection technology based on magnetic beads can significantly improve the detection sensitivity due to the high specific surface area and easy separation characteristics of magnetic beads, and is especially suitable for low-abundance sample analysis such as plasma. In the detection of p-Tau217, the antibody needs to be covalently coupled to the surface of the carboxylated magnetic bead, and the loading rate needs to be greater than or equal to 90% to ensure the stability of the signal. However, the magnetic bead-antibody complex faces multiple risks of inactivation during storage and detection. The traditional preservation solution relies on a high concentration of bovine serum albumin (BSA) or glycerol as a protein protective agent, but BSA may introduce exogenous interfering antigens, and glycerol increases the viscosity and affects the dispersibility of the magnetic beads. In addition, in order to avoid the catalysis of divalent metal ions (such as Ca²⁺, Mg²⁺) on antibody oxidation, chelating agents such as EDTA or sodium salt need to be added. However, EDTA or sodium salt at high concentrations can destroy the charge stability of the antibody, which may interfere with the antibody-antigen binding reaction. There is another more important problem. In order to ensure that the magnetic beads can be uniformly dispersed in the preservation solution and avoid magnetic bead aggregation, some add protein to make the preservation solution system in a gel state, semi-solid state or solid state at a temperature of 2℃ to 8℃, which causes difficulties in machine operation. The reagent needs to be placed at room temperature before machine operation, but the long time of reagent placement at room temperature will affect the activity of the magnetic bead antibody.

[0004] Based on the above defects, it is necessary to study the preservation solution for magnetic bead-coated antibodies to develop a preservation system with zero exogenous interference, low-temperature liquid anti-aggregation and mild chelation characteristics, and finally to construct a liquid system that can be used immediately after opening the bottle to support the direct detection of p-Tau217 magnetic bead complex without rewarming. SUMMARY

[0005] The purpose of the present application is to overcome the defects of exogenous interference, low-temperature gelation and chelation damage of the existing magnetic bead coated antibody preservation technology, to research a non-EDTA chelation system and an ultra-low concentration stable system, and to provide a new liquid preservation system with zero coagulation risk, no charge disturbance and high dispersity.

[0006] The technical scheme adopted by the present application provides a p-Tau217 magnetic bead coated antibody preservation solution, which is characterized in that the above-mentioned preservation solution is a buffer solution containing 0.05g / L-0.2g / L of a high molecular polymer, 0.005g / L-0.02g / L of a surfactant, 0.1g / L-0.3g / L of a protein protective agent, 0.2g / L-4g / L of a metal chelating agent, 0.1g / L-0.5g / L of a suspending agent and 0.001g / L-0.01g / L of a preservative, the above-mentioned buffer solution is a PBS buffer solution or a Tris-HCl buffer solution, the pH value of the above-mentioned preservation solution is 7.0-7.5, the above-mentioned high molecular polymer is any one of polymethylacryloxyethyl phosphorylcholine, polysulfobetaine methacrylate or polyethylene glycol, the above-mentioned metal chelating agent is sodium hyaluronate or sodium gluconate, and the above-mentioned suspending agent is sucrose or trehalose.

[0007] Specifically, the number average molecular weight of the polymethylacryloxyethyl phosphorylcholine is 9000Da-15000Da, the number average molecular weight of the polysulfobetaine methacrylate is 15000Da-25000Da, and the number average molecular weight of the polyethylene glycol is 6000Da-12000Da.

[0008] Specifically, the above-mentioned surfactant is any one of Tween-20, S24 or Triton X-100, the above-mentioned protein protective agent is bovine serum albumin or glycerol, and the above-mentioned preservative is ProClin300 or sodium azide.

[0009] Preferably, the above-mentioned preservation solution is a PBS buffer solution containing 0.1g / L of a high molecular polymer, 0.01g / L of a surfactant, 0.2g / L of a protein protective agent, 2.0g / L of sodium hyaluronate, 0.2g / L of sucrose and 0.005g / L of a preservative, and the pH value is 7.4; the above-mentioned high molecular polymer is polymethylacryloxyethyl phosphorylcholine with a number average molecular weight of 12000Da or polysulfobetaine methacrylate with a number average molecular weight of 20000Da.

[0010] Further, when the metal chelator is sodium hyaluronate, the concentration of sodium hyaluronate in the preservative solution is 0.4 g / L to 4.0 g / L, and the number average molecular weight of the sodium hyaluronate is 1000 kDa to 1800 kDa; when the metal chelator is sodium gluconate, the concentration of sodium gluconate in the preservative solution is 0.2 g / L to 2.2 g / L.

[0011] Further, when the buffer is a PBS buffer, the concentration of the PBS buffer is 10 mM to 50 mM; when the buffer is a Tris-HCl buffer, the concentration of the Tris-HCl buffer is 20 mM to 50 mM.

[0012] Further, the magnetic beads coated with the p-Tau217 magnetic bead-coated antibody are carboxylated magnetic microspheres, and the antibody is covalently coupled to the magnetic beads, and the concentration of the magnetic beads in the preservative solution is 0.05 g / L to 0.2 g / L.

[0013] It should be noted that the preservative solution is used for single-molecule fluorescent immunoassay as a detection working solution.

[0014] A preparation method of a p-Tau217 magnetic bead-coated antibody preservative solution, for the preservative solution, the key is that the preparation method is specifically: The buffer is selected, and a metal chelator, a suspending agent, a high molecular polymer, a protein protective agent, a surfactant, and a preservative are added, an acid or a base is used to adjust the pH value, the volume is adjusted, and then the mixture is uniformly mixed, filtered through a membrane to obtain the preservative solution.

[0015] Specifically, the acid is a 0.1M hydrochloric acid solution, and the base is a 0.1M sodium hydroxide solution; the membrane filtration uses a 0.22μm PVDF filter membrane.

[0016] Compared with the prior art, the present application has the following beneficial effects: The application realizes a breakthrough in the antibody preservation technology of magnetic beads coating by constructing a ternary synergistic protection system of mild metal chelator-saccharide suspending agent-high molecular polymer. The system significantly improves the performance indicators: signal-to-noise ratio > 80, repeatability CV < 5%. More importantly, it has achieved excellent stability: signal attenuation < 5% after 10 days of acceleration at 37℃, and signal attenuation < 7% after 20 months of storage at 2℃-8℃. This breakthrough is due to the triple synergistic protection mechanism: sodium hyaluronate or sodium gluconate blocks metal ion catalytic oxidation through mild chelation, and the hydrogen bond network of its polysaccharide chain stabilizes the charge conformation of the antibody; sucrose or trehalose forms a glassy solidified antibody structure; and the amphiphilic or similar amphiphilic high molecular polymer constructs a steric hindrance layer on the surface of the magnetic beads, and some can also form a biomimetic hydration layer, synchronously solving the three major problems of metal ion oxidation, low temperature phase transition damage and magnetic bead sedimentation inactivation in traditional technology.

[0017] The prior art usually relies on high concentration of BSA or glycerol as a protective agent, and its protection mechanism is mainly physical masking or increasing viscosity to delay degradation. However, this method has a fundamental defect: high concentration of BSA can non-specifically cover the surface of magnetic beads, but the exogenous protein will competitively occupy the antigen binding site, significantly reducing the signal-to-noise ratio of detection; high viscosity glycerol increases the liquid resistance, leading to difficulty in dispersing magnetic beads, and cannot block the oxidative damage caused by metal ions. The present application uses ultra-low concentration of BSA to form a single-molecule dynamic protection layer on the surface of magnetic beads, breaking through the contradiction between protection concentration and interference threshold; sodium hyaluronate or sodium gluconate is used instead of traditional EDTA, which can also take advantage of its unique chelation-hydration dual function to avoid the strong charge disturbance of EDTA. It is worth noting that sodium hyaluronate has not been applied in this field before, which is due to the neglect of its biocompatibility value in this field, and sodium gluconate has limited performance due to the lack of high molecular hydration protection function.

[0018] In view of the low-temperature solidification and extrusion damage problem in the prior art, the present application introduces a high molecular polymer with optimized molecular weight. Its long chain structure forms a dense barrier on the surface of magnetic beads, which inhibits aggregation through steric hindrance effect; among them, the phosphorylcholine or sulfobetaine group in PMPC or PSBMA can bond water molecules to produce strong repulsive force, so that the magnetic beads remain in a liquid state at 2℃-8℃, realizing direct machine operation without rewarming. The control of molecular weight in the present application has a strict threshold effect: if the molecular weight is too low, the hydrophobic segment is exposed, causing aggregation, and if the molecular weight is too high, the molecular chain is folded, causing steric hindrance failure. PEG is suitable for basic dispersion demand scenarios, but the signal-to-noise ratio is limited.

[0019] Compared with the traditional EDTA+high BSA system, the components of the three-component synergistic system of the application restrict each other, such as EDTA catalytic oxidation, flux limitation of high glycerol system, the three-component synergistic system of the application realizes zero external interference and long-term stability, provides p-Tau217 detection reagent conforming to the standards of open bottle, ultra-sensitivity and low attenuation for Alzheimer's disease super-early diagnosis, promotes the standardization process of AD plasma marker detection technology. DETAILED DESCRIPTION

[0020] The various illustrative embodiments of the application will now be described in detail in connection with the accompanying drawings. This description is not intended to limit the application, but rather to provide an exemplification of certain aspects and features thereof. Thus, those skilled in the art will recognize that modifications can be made to the preferred embodiments without departing from the scope of the present application.

[0021] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each individual intermediate value and each smaller range that falls within the range of values. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.

[0023] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from consideration of the description and practice of the application disclosed herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0024] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0025] The specific conditions not specified in the examples can be carried out according to the conventional conditions; the reagents or instruments not specified by the manufacturer are all conventional products that can be purchased on the market. Among them, the number average molecular weight of the commercially available sodium hyaluronate is usually expressed in the form of a range, and the number average molecular weight (expressed by Mn) of the sodium hyaluronate used in the application is 1000 kDa-1800 kDa.

[0026] Example One In this example, the preparation of the p-Tau217 magnetic bead coated antibody preservative solution was carried out, and different concentrations of magnetic bead reagents were prepared using the preservative solution, and the specific process was as follows: S1, select p-Tau217 magnetic bead coated antibody: The magnetic beads used were carboxylated magnetic microspheres with a particle size of 2.7 μm, and the p-Tau217 antibody was covalently coupled to the magnetic beads, with an antibody loading rate of 92.3%.

[0027] S2, preparation of preservative solution: To 800 mL of 20 mM PBS buffer, add 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 0.1 g of polymethyl methacryloyl oxyethyl phosphocholine (abbreviated as PMPC), 0.2 g of bovine serum albumin (abbreviated as BSA), 0.01 g of S24 and 0.005 g of ProClin300, adjust the pH value to 7.4 using 0.1 M sodium hydroxide solution, and then use 20 mM PBS buffer to make up to 1 L, mix well, and filter through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances that affect the stability of the magnetic beads, to obtain the p-Tau217 magnetic bead coated antibody preservative solution, which is denoted as preservative solution sample 1. In this example, sodium hyaluronate is used as a metal chelating agent, which is pre-dissolved in 20 mM PBS buffer before use; sucrose is used as a suspending agent; PMPC is an amphiphilic polymer, and the Mn of PMPC in this example is 12000 Da; BSA is used as a protein protective agent.

[0028] S3, preparation of magnetic bead reagent: To the above preservative solution, add the p-Tau217 magnetic bead coated antibody selected in step S1, to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, respectively, denoted as magnetic bead reagent sample 1-1, magnetic bead reagent sample 1-2 and magnetic bead reagent sample 1-3.

[0029] Example Two In this example, the preparation of the p-Tau217 magnetic bead coated antibody preservative solution was carried out, and different concentrations of magnetic bead reagents were prepared using the preservative solution, and the specific process was as follows: S1, select p-Tau217 magnetic bead coated antibody: same as step S1 of Example One.

[0030] S2, preparation of preservative solution: To 850 mL of 10 mM PBS buffer, 2.2 g of sodium gluconate, 0.5 g of trehalose, 0.05 g of PMPC, 0.3 g of glycerol, 0.005 g of Triton X-100 and 0.01 g of sodium azide were added, the pH value was adjusted to 7.0 using 0.1 M sodium hydroxide solution, and after being diluted to 1 L with 10 mM PBS buffer, it was mixed, filtered through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of the magnetic beads, to obtain a p-Tau217 magnetic bead coated antibody storage solution, denoted as storage solution sample 2. In this embodiment, sodium gluconate is used as a metal chelating agent; trehalose is used as a suspending agent; the Mn of PMPC in this embodiment is 9000 Da; glycerol is used as a protein protective agent.

[0031] S3, preparation of magnetic bead reagent: To the above storage solution, the p-Tau217 magnetic bead coated antibody selected in step S1 was added to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, denoted as magnetic bead reagent sample 2-1, magnetic bead reagent sample 2-2 and magnetic bead reagent sample 2-3, respectively.

[0032] Example Three In this embodiment, the preparation of a p-Tau217 magnetic bead coated antibody storage solution was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as step S1 of Example One.

[0033] S2, preparation of storage solution: To 820 mL of 30 mM Tris-HCl buffer, 0.4 g of sodium hyaluronate, 0.1 g of sucrose, 0.2 g of PMPC, 0.2 g of BSA, 0.02 g of Tween-20 and 0.008 g of ProClin300 were added, the pH value was adjusted to 7.5 using 0.1 M hydrochloric acid solution, and after being diluted to 1 L with 30 mM Tris-HCl buffer, it was mixed, filtered through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of the magnetic beads, to obtain a p-Tau217 magnetic bead coated antibody storage solution, denoted as storage solution sample 3. In this embodiment, sodium hyaluronate is used as a metal chelating agent, which is pre-dissolved in 30 mM Tris-HCl buffer before use; sucrose is used as a suspending agent; the Mn of PMPC in this embodiment is 15000 Da; BSA is used as a protein protective agent.

[0034] S3, preparation of magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in S1 was added to the above storage solution to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, respectively, which were recorded as magnetic bead reagent sample 3-1, magnetic bead reagent sample 3-2 and magnetic bead reagent sample 3-3.

[0035] Example Four In this example, the preparation of the storage solution of the p-Tau217 magnetic bead coated antibody was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as S1 step of example one.

[0036] S2, preparation of storage solution: To 800 mL of 20 mM PBS buffer, 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 0.1 g of polysulfobetaine methacrylate (abbreviated as PSBMA), 0.2 g of BSA, 0.01 g of S24 and 0.005 g of ProClin300 were added, the pH value was adjusted to 7.4 using 0.1 M sodium hydroxide solution, and the volume was adjusted to 1 L using 20 mM PBS buffer, then mixed, filtered through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of the magnetic beads, to obtain the storage solution of the p-Tau217 magnetic bead coated antibody, recorded as storage solution sample 4. In this example, sodium hyaluronate is used as a metal chelating agent, which is used after pre-dissolving in 20 mM PBS buffer; sucrose is used as a suspending agent; PSBMA is an amphiphilic polymer, and the Mn of PSBMA in this example is 20000 Da; BSA is used as a protein protective agent.

[0037] S3, preparation of magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in S1 was added to the above storage solution to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, respectively, which were recorded as magnetic bead reagent sample 4-1, magnetic bead reagent sample 4-2 and magnetic bead reagent sample 4-3.

[0038] Example Five In this example, the preparation of the storage solution of the p-Tau217 magnetic bead coated antibody was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as S1 step of example one.

[0039] S2, preparation of storage solution: To 830 mL of 50 mM PBS buffer, 1.2 g of sodium gluconate, 0.3 g of sucrose, 0.3 g of PSBMA, 0.1 g of BSA, 0.02 g of Triton X-100 and 0.008 g of ProClin300 were added, the pH value was adjusted to 7.5 using 0.1 M sodium hydroxide solution, and after constant volume to 1 L with 50 mM PBS buffer, it was mixed, filtered through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of magnetic beads, to obtain a p-Tau217 magnetic bead coated antibody storage solution, denoted as storage solution sample 5; In this embodiment, sodium gluconate is used as a metal chelating agent; sucrose is used as a suspending agent; the Mn of PSBMA in this embodiment is 15000 Da; BSA is used as a protein protective agent.

[0040] S3, preparation of magnetic bead reagent: To the above storage solution, the p-Tau217 magnetic bead coated antibody selected in step S1 was added to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, denoted as magnetic bead reagent sample 5-1, magnetic bead reagent sample 5-2 and magnetic bead reagent sample 5-3, respectively.

[0041] Example Six In this embodiment, the preparation of a p-Tau217 magnetic bead coated antibody storage solution was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as step S1 of example one.

[0042] S2, preparation of storage solution: To 800 mL of 40 mM Tris-HCl buffer, 0.2 g of sodium gluconate, 0.4 g of trehalose, 0.2 g of PSBMA, 0.2 g of glycerol, 0.005 g of Tween-20 and 0.008 g of ProClin300 were added, the pH value was adjusted to 7.1 using 0.1 M hydrochloric acid solution, and after constant volume to 1 L with 40 mM Tris-HCl buffer, it was mixed, filtered through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of magnetic beads, to obtain a p-Tau217 magnetic bead coated antibody storage solution, denoted as storage solution sample 6; In this embodiment, sodium gluconate is used as a metal chelating agent; trehalose is used as a suspending agent; the Mn of PSBMA in this embodiment is 25000 Da; glycerol is used as a protein protective agent.

[0043] S3, preparation of magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in S1 was added to the above storage solution to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, respectively, which were recorded as magnetic bead reagent sample 6-1, magnetic bead reagent sample 6-2 and magnetic bead reagent sample 6-3.

[0044] Example Seven In this example, the preparation of the p-Tau217 magnetic bead coated antibody storage solution was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as S1 step of example one.

[0045] S2, preparation of storage solution: To 850 mL of 20 mM PBS buffer, 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 0.1 g of PEG8000, 0.2 g of BSA, 0.01 g of Tween-20 and 0.005 g of ProClin300 were added, and the pH value was adjusted to 7.4 using 0.1 M sodium hydroxide solution. After constant volume to 1 L with 20 mM PBS buffer, mix well, filter through 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of magnetic beads, to obtain the p-Tau217 magnetic bead coated antibody storage solution, recorded as storage solution sample 7. In this example, sodium hyaluronate is used as a metal chelating agent, which is used after pre-dissolving in 20 mM PBS buffer; sucrose is used as a suspending agent; PEG8000 is a high molecular polymer, polyethylene glycol as a hydrophilic polymer, which plays a spatial stabilization function similar to amphiphilic polymer through molecular chain hydration, but the molecular structure does not contain hydrophobic block; BSA is used as a protein protective agent.

[0046] S3, preparation of magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in S1 was added to the above storage solution to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, respectively, which were recorded as magnetic bead reagent sample 7-1, magnetic bead reagent sample 7-2 and magnetic bead reagent sample 7-3.

[0047] Example Eight In this example, the preparation of the p-Tau217 magnetic bead coated antibody storage solution was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as S1 step of example one.

[0048] S2, preparation of storage solution: To 800 mL of 20 mM Tris-HCl buffer, add 1.2 g of sodium gluconate, 0.5 g of sucrose, 0.05 g of PEG6000, 0.3 g of BSA, 0.01 g of S24 and 0.01 g of ProClin300, adjust the pH value to 7.2 using 0.1 M hydrochloric acid solution, and then use 20 mM Tris-HCl buffer to make up to 1 L, mix, filter through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of magnetic beads, and obtain a p-Tau217 magnetic bead coated antibody storage solution, which is recorded as storage solution sample 8. In this embodiment, sodium gluconate is used as a metal chelating agent; sucrose is used as a suspending agent.

[0049] S3, preparation of magnetic bead reagent: To the above storage solution, add the p-Tau217 magnetic bead coated antibody selected in step S1, and prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, which are recorded as magnetic bead reagent sample 8-1, magnetic bead reagent sample 8-2 and magnetic bead reagent sample 8-3, respectively.

[0050] Example Nine In this embodiment, the preparation of a p-Tau217 magnetic bead coated antibody storage solution is carried out, and different concentrations of magnetic bead reagents are prepared using the storage solution, and the specific process is as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as step S1 of Example One.

[0051] S2, preparation of storage solution: To 800 mL of 50 mM Tris-HCl buffer, add 4.0 g of sodium hyaluronate, 0.1 g of trehalose, 0.1 g of PEG12000, 0.1 g of glycerol, 0.01 g of Triton X-100 and 0.008 g of sodium azide, adjust the pH value to 7.5 using 0.1 M hydrochloric acid solution, and then use 50 mM Tris-HCl buffer to make up to 1 L, mix, filter through a 0.22 μm PVDF filter membrane to remove bacteria and insoluble substances affecting the stability of magnetic beads, and obtain a p-Tau217 magnetic bead coated antibody storage solution, which is recorded as storage solution sample 9. In this embodiment, sodium hyaluronate is used as a metal chelating agent, which is pre-dissolved in 50 mM Tris-HCl buffer before use; trehalose is used as a suspending agent.

[0052] S3, preparation of magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in step S1 was added to the above storage solution to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, which were recorded as magnetic bead reagent sample 9-1, magnetic bead reagent sample 9-2 and magnetic bead reagent sample 9-3, respectively.

[0053] Comparative Example One In this comparative example, the preparation of the storage solution of the p-Tau217 magnetic bead coated antibody was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as Example One.

[0054] S2, preparation of the storage solution: To 800 mL of 20 mM PBS buffer, 2.0 g of EDTA, 0.2 g of sucrose, 0.1 g of PMPC, 0.2 g of BSA, 0.01 g of S24 and 0.005 g of ProClin300 were added, and the pH value was adjusted to 7.4 using 0.1 M sodium hydroxide solution. After being diluted to 1 L with 20 mM PBS buffer and mixed, it was filtered through a 0.22 μm PVDF filter membrane to obtain the storage solution of the p-Tau217 magnetic bead coated antibody, which was recorded as storage solution control 1. In this comparative example, EDTA was used as a metal chelating agent; sucrose was used as a suspending agent; the Mn of PMPC in this comparative example was 12000 Da; and BSA was used as a protein protective agent.

[0055] S3, preparation of the magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in step S1 was added to the above storage solution to prepare magnetic bead reagents with concentrations of 0.05 g / L, 0.1 g / L and 0.2 g / L, which were recorded as magnetic bead reagent sample 9-1, magnetic bead reagent sample 9-2 and magnetic bead reagent sample 9-3, respectively.

[0056] Comparative Example Two In this comparative example, the preparation of the storage solution of the p-Tau217 magnetic bead coated antibody was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as Example One.

[0057] S2, preparation of the storage solution: To 800 mL of 20 mM PBS buffer, 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 5 g of proteose peptone, 0.2 g of BSA, 0.01 g of S24 and 0.005 g of ProClin300 were added, the pH value was adjusted to 7.4 using 0.1 M sodium hydroxide solution, and after constant volume to 1 L using 20 mM PBS buffer, it was mixed, filtered through a 0.22 μm PVDF filter membrane, and a p-Tau217 magnetic bead coated antibody storage solution was obtained, which was recorded as storage solution control 2. In the present comparative example, sodium hyaluronate is used as a metal chelating agent, which is pre-dissolved in 20 mM PBS buffer before use; sucrose is used as a suspending agent; and BSA is used as a protein protective agent.

[0058] S3, preparation of magnetic bead reagent: To the above storage solution, the p-Tau217 magnetic bead coated antibody selected in step S1 was added, and a magnetic bead reagent with a concentration of 0.05 g / L and 0.1 g / L was prepared, which was recorded as magnetic bead reagent control 2-1 and magnetic bead reagent control 2-2, respectively.

[0059] Comparative Example Three In the present comparative example, the preparation of the p-Tau217 magnetic bead coated antibody storage solution was carried out, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as Example One.

[0060] S2, preparation of storage solution: To 800 mL of 20 mM PBS buffer, 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 0.1 g of PMPC, 5 g of BSA, 0.01 g of S24 and 0.005 g of ProClin300 were added, the pH value was adjusted to 7.4 using 0.1 M sodium hydroxide solution, and after constant volume to 1 L using 20 mM PBS buffer, it was mixed, filtered through a 0.22 μm PVDF filter membrane, and a p-Tau217 magnetic bead coated antibody storage solution was obtained, which was recorded as storage solution control 3. In the present comparative example, sodium hyaluronate is used as a metal chelating agent, which is pre-dissolved in 20 mM PBS buffer before use; sucrose is used as a suspending agent; and BSA is used as a protein protective agent.

[0061] S3, preparation of magnetic bead reagent: To the above storage solution, the p-Tau217 magnetic bead coated antibody selected in step S1 was added, and a magnetic bead reagent with a concentration of 0.05 g / L and 0.1 g / L was prepared, which was recorded as magnetic bead reagent control 2-1 and magnetic bead reagent control 2-2, respectively.

[0062] Comparative Example Four The comparative example is to prepare the storage solution of p-Tau217 magnetic bead coated antibody, and different concentrations of magnetic bead reagents are prepared by using the storage solution, and the specific process is as follows: S1, selecting p-Tau217 magnetic bead coated antibody: same as example one.

[0063] S2, preparation of storage solution: Into 800 mL of 20 mM PBS buffer, 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 0.1 g of PMPC, 0.2 g of BSA, 0.01 g of S24 and 0.005 g of ProClin300 are added, 0.1 M sodium hydroxide solution is used to adjust the pH value to 7.4, and then 20 mM PBS buffer is used to constant volume to 1 L after mixing, and then filtered through 0.22 μm PVDF filter membrane to obtain the storage solution of p-Tau217 magnetic bead coated antibody, which is recorded as storage solution control 4. In the comparative example, sodium hyaluronate is used as a metal chelating agent, which is used after pre-dissolving in 20 mM PBS buffer; sucrose is used as a suspending agent; the Mn of PMPC in the example is 20000 Da; BSA is used as a protein protective agent.

[0064] S3, preparation of magnetic bead reagent: Into the above storage solution, the p-Tau217 magnetic bead coated antibody selected in step S1 is added to prepare magnetic bead reagents with concentrations of 0.1 g / L and 0.2 g / L, which are recorded as magnetic bead reagent control 4-1 and magnetic bead reagent control 4-2, respectively.

[0065] Comparative Example Five The comparative example is to prepare the storage solution of p-Tau217 magnetic bead coated antibody, and different concentrations of magnetic bead reagents are prepared by using the storage solution, and the specific process is as follows: S1, selecting p-Tau217 magnetic bead coated antibody: same as example one.

[0066] S2, preparation of storage solution: Into 800 mL of 20 mM PBS buffer, 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 0.1 g of PMPC, 0.2 g of BSA, 0.01 g of S24 and 0.005 g of ProClin300 are added, 0.1 M sodium hydroxide solution is used to adjust the pH value to 7.4, and then 20 mM PBS buffer is used to constant volume to 1 L after mixing, and then filtered through 0.22 μm PVDF filter membrane to obtain the storage solution of p-Tau217 magnetic bead coated antibody, which is recorded as storage solution control 4. The sodium hyaluronate in the present comparative example is used as a metal chelating agent, and is used after being pre-dissolved in 20 mM PBS buffer; the sucrose is used as a suspending agent; the PMPC in the present example has an Mn of 6000 Da; and the BSA is used as a protein protective agent.

[0067] S3, preparation of magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in the step S1 was added to the above-mentioned storage solution to prepare a magnetic bead reagent with a concentration of 0.1 g / L, which was recorded as magnetic bead reagent control 5-1.

[0068] Comparative example six The present comparative example was prepared by preparing a storage solution for the p-Tau217 magnetic bead coated antibody, and different concentrations of magnetic bead reagents were prepared using the storage solution, and the specific process was as follows: S1, selection of p-Tau217 magnetic bead coated antibody: same as example one.

[0069] S2, preparation of storage solution: In 800 mL of 20 mM PBS buffer, 2.0 g of sodium hyaluronate, 0.2 g of sucrose, 0.2 g of BSA, 5.0 g of sodium caseinate, 0.01 g of S24 and 0.005 g of ProClin300 were added, the pH value was adjusted to 7.4 using 0.1 M sodium hydroxide solution, and the volume was adjusted to 1 L using 20 mM PBS buffer, then mixed, filtered through a 0.22 μm PVDF filter membrane, to obtain a storage solution for the p-Tau217 magnetic bead coated antibody, which was recorded as storage solution control 6. The sodium hyaluronate in the present comparative example is used as a metal chelating agent, and is used after being pre-dissolved in 20 mM PBS buffer; the sucrose is used as a suspending agent; the PMPC in the present example has an Mn of 6000 Da; and the BSA is used as a protein protective agent.

[0070] S3, preparation of magnetic bead reagent: The p-Tau217 magnetic bead coated antibody selected in the step S1 was added to the above-mentioned storage solution to prepare a magnetic bead reagent with a concentration of 0.1 g / L, which was recorded as magnetic bead reagent control 5-1.

[0071] Analysis and test Each magnetic bead reagent sample prepared by the present application was in a liquid state at 2℃-8℃, without solid-liquid separation or gel mass, and showed a flowing continuity when poured. The control prepared in comparative example two contained proteose peptone and was in a semi-solid state at 2℃-8℃; the controls prepared in comparative examples one, three, four, five and six were also in a liquid state at 2℃-8℃.

[0072] I. Signal-to-noise ratio experiment Experimental method: The AEB signals of blank reagent and low concentration sample 1 (p-Tau217 concentration of 0.26 pg / mL) were determined by using a full-automatic single molecule fluorescence immunoassay analyzer for the magnetic bead reagent samples obtained from examples 1-9 and the magnetic bead reagent control samples obtained from comparative examples 1-6, and the signal-to-noise ratio S / N was calculated, and the results are shown in Table 1.

[0073] The formula for calculating the signal-to-noise ratio S / N is formula 1: S / N = sample signal intensity / blank signal intensity Formula 1.

[0074] Table 1: Results of signal-to-noise ratio experiment

[0075] In the field, the signal-to-noise ratio of AD diagnostic reagents is required to be not less than 40.

[0076] As can be seen from the results in Table 1, the signal-to-noise ratios of magnetic bead reagent samples 1-2, 1-3, 4-2 and 4-3 are higher than 80, meeting the requirements of ultra-sensitive detection, among which the magnetic bead reagent sample 4-3 is a high-concentration magnetic bead reagent, still maintaining excellent signal-to-noise ratio, proving that the anti-agglomeration advantage of PSBMA is more significant under high magnetic bead concentration. This may be because sodium hyaluronate can chelate metal ions gently, avoiding the charge disturbance of EDTA; PMPC or PSBMA with appropriate molecular weight can provide the best steric hindrance, preventing magnetic bead agglomeration and maintaining uniform dispersion; BSA at an ultra-low concentration (such as 0.2 g / L) forms a dynamic protective layer at a trace level, blocking non-specific adsorption on the surface of magnetic beads and avoiding high-concentration BSA from masking p-Tau217 antigen epitopes. Sodium gluconate is also a mild chelating agent, which can chelate metal ions, but lacks the hydration protection effect of high molecular chains, and has limited effect on improving charge stability, resulting in a lower signal-to-noise ratio than the sodium hyaluronate group. PEG can be used as a high molecular polymer, which can provide basic dispersion, but lacks biomimetic groups such as phosphatidylcholine / sulfobetaine, and has weak anti-protein adsorption capacity, resulting in a significantly lower S / N than the PSBMA / PMPC group.

[0077] Comparative Example 1 uses EDTA instead of sodium hyaluronate or sodium gluconate to chelate metal ions, but the strong negative charge of EDTA can disturb the antibody conformation and destroy the specific binding ability with p-Tau217; Comparative Example 2 uses proteose peptone instead of high molecular polymer, proteose peptone causes the low-temperature gelation of the preservation system, and the magnetic bead aggregation leads to a sharp decrease in the effective binding area; Comparative Example 3 increases the amount of BSA, and high-concentration BSA non-specifically occupies the antigen binding site, forming steric hindrance; the molecular weight of PMPC in Comparative Example 4 and Comparative Example 5 is not suitable, and the molecular weight of PMPC is too high, resulting in high steric hindrance, and too low, making the hydrophobic segment exposed, causing magnetic bead aggregation, which all have adverse effects on the signal-to-noise ratio; Comparative Example 6 adds casein as a blocking agent, which will non-specifically adsorb p-Tau217, significantly increasing the background noise.

[0078] II. Reproducibility Experiment Experimental method: The magnetic bead reagent samples obtained in Examples 1-9 and the magnetic bead reagent control samples obtained in Comparative Examples 1-6 were repeatedly determined for low-concentration sample 2 (p-Tau217 concentration of 0.6 pg / mL) and high-concentration sample 1 (p-Tau217 concentration of 6.0 pg / mL) using a full-automatic single-molecule fluorescence immunoassay instrument, and the coefficient of variation, i.e., the CV value, was calculated. During the experiment, the first determination was made in the order of the magnetic bead reagent samples of Examples 1-9 and the magnetic bead reagent control samples of Comparative Examples 1-6, and the second determination was made in the order, and so on. By sequentially determining all the samples of Examples and Comparative Examples (each sample was determined at a certain interval), the stability of the magnetic bead reagent in the suspended state can also be fully evaluated. In this experiment, from the addition of the first magnetic bead reagent to the completion of the determination of the last reagent, the total time was controlled within 2 hours. The results are shown in Table 2.

[0079] Table 2: Results of reproducibility experiment

[0080] As can be seen from Table 2, the CV values of low concentration samples of magnetic bead reagent samples 1-2 and 4-2 are as low as 4.108% and 3.877% respectively, and the CV values of high concentration samples are all <4.5%, far exceeding the industry standard (CV≤10%). Among them, the 0.1 g / L magnetic bead concentration performs best in both groups, while the high concentration of 0.2 g / L magnetic beads needs to be matched with high molecular weight polymers (such as PSBMA, Mn=20 kDa) to maintain dispersibility; if the molecular weight of the polymer is insufficient (such as PEG, Mn=6000), the CV will rise to more than 9.0%, for example, the CV of magnetic bead reagent sample 8-1 is 9.154%. This is because: PMPC or PSBMA forms a dense hydration layer on the surface of the magnetic beads, which inhibits the collision and aggregation of the magnetic beads through steric hindrance effect, especially in high concentration, which ensures the uniformity of the reaction; the threshold of 0.1 g / L magnetic bead concentration balances the signal strength and dispersibility requirements, avoiding the fluid dynamic collision caused by too high concentration; the buffer protection effect of sodium hyaluronate maintains the stability of the system charge, reducing the influence of antibody conformation fluctuation on binding efficiency. The repeatability of the PSBMA group is slightly better than that of the PMPC group, because the sulfobetaine group can provide stronger hydrophilicity and anti-protein adsorption capacity; although the PEG group has basic dispersibility, it lacks biomimetic polar groups, and the magnetic beads gradually settle in long-term detection, resulting in an increase in CV during the experiment.

[0081] Comparative Example 1 uses EDTA to chelate metal ions, which has strong negative charge and causes antibody conformation drift, resulting in signal fluctuation, with CV value around 10%; Comparative Example 2 uses protein peptone instead of polymer, and low-temperature gelation causes magnetic bead aggregation and sedimentation, with CV value rising to 12.6%~13.1%; the high concentration of BSA in Comparative Example 3 causes uneven viscosity of the system, which intensifies signal dispersion; the PMPC molecular weight in Comparative Example Four and Comparative Example Five is improper, i.e. too high to cause molecular chain entanglement failure, and too low to expose the hydrophobic segment and cause magnetic bead aggregation; after removing the polymer in Comparative Example Six, the magnetic beads quickly settle due to the lack of anti-settling mechanism.

[0082] III. Thermal stability experiment Experimental method: after accelerating the magnetic bead reagent samples obtained from Examples 1-9 and the magnetic bead reagent control samples obtained from Comparative Examples 1-6 at 37℃ for 10 days, the signal deviation (%) of low concentration sample 2 (p-Tau217 concentration of 0.6 pg / mL) was measured using a fully automatic single molecule fluorescence immunoassay analyzer, repeated 3 times, and the average value was taken as the signal intensity after 10 days of acceleration. The initial signal intensity is the average value in the repeatability experiment, and the results are shown in Table 3.

[0083] Among them, the calculation formula of signal deviation after 10 days of acceleration is formula 2: Relative deviation (%) = (signal intensity after 10 days of acceleration - initial signal intensity) / initial signal intensity x 100% Formula 2.

[0084] IV. Long-term stability experiment Experimental method: The magnetic bead reagent samples of Example One and Example Four (i.e. magnetic bead reagent samples 1-1, 1-2, 1-3, 4-1, 4-2, 4-3) were stored at 2-8℃ for 20 months, and the signal deviation (%) of low concentration sample 2 (p-Tau217 concentration of 0.6 pg / mL) was measured at 3 months, 6 months, 12 months and 20 months using a full-automatic single molecule fluorescence immunoassay analyzer (based on low concentration sample 2), repeated 3 times, and the average value was taken as the measured signal intensity. The corresponding initial signal intensity was the average value in the repeatability experiment. The results are shown in Table 3.

[0085] The calculation formula of the signal deviation measured in the long-term stability process is formula 3: Relative deviation (%) = (measured signal intensity - initial signal intensity) / initial signal intensity x 100% Formula 3.

[0086] Table 3: Results of thermal stability experiment and long-term stability experiment

[0087] As can be seen from the results in Table 3, the signal deviation of magnetic bead reagent samples 1-2 and 4-2 was only -4.7% and -4.5% after 10 days of acceleration at 37℃, and the deviation was still stable at -6.0% and -5.5% after 20 months of storage at 2-8℃, which was much better than the industry standard (20 months deviation ≤20%), and during the long-term stability experiment, each magnetic bead reagent sample was a stable liquid without precipitation, stratification, and no aggregation phenomenon. This is due to the synergistic effect of the ternary protection system of the present application, i.e. sodium hyaluronate efficiently chelates metal ions, blocking the oxidative degradation pathway; sucrose and trehalose can form a glassy network to lock the antibody active conformation; PMPC and PSBMA can maintain low-temperature liquid flowability to avoid freeze-thaw damage.

[0088] Among them, PSBMA has stronger hydration layer stability due to its sulfobetaine group, which inhibits protein denaturation at high temperature, and has a significant long-term advantage; the long-term stability of PMPC is slightly weaker due to its phosphorylcholine-dependent hydrogen bond.

[0089] The comparative example one uses EDTA chelating agent, which causes irreversible inactivation of the antibody due to catalytic oxidation, and the 20-month deviation reaches -32.9%; the protein peptone in the comparative example two causes gel shrinkage and extrusion of the magnetic beads at low temperature, accelerates the antibody shedding, and the 20-month deviation reaches -21.5%; the high concentration of BSA in the comparative example three introduces the risk of exogenous protein polymerization, accelerates the degradation of the complex, and the 20-month deviation reaches -37.1%; the PMPC molecular weight in the comparative example four and the comparative example five is inaccurate, and too high causes a loose hydration layer, and too low causes intensified hydrophobic aggregation and thermal instability; the magnetic beads are completely inactivated due to gravity settlement in long-term storage after the removal of the polymer in the comparative example six.

[0090] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope defined by the claims of the present application.

Claims

1. A preservation solution for p-Tau217 magnetic bead-coated antibodies, characterized in that, The preservative solution is a buffer solution containing 0.05 g / L-0.2 g / L of a high molecular polymer, 0.005 g / L-0.02 g / L of a surfactant, 0.1 g / L-0.3 g / L of a protein protective agent, 0.2 g / L-4 g / L of a metal chelating agent, 0.1 g / L-0.5 g / L of a suspending agent, and 0.001 g / L-0.01 g / L of a preservative, wherein the buffer solution is a PBS buffer solution or a Tris-HCl buffer solution, the pH value of the preservative solution is 7.0-7.5, the high molecular polymer is any one of polymethylacryloyloxyethyl phosphorylcholine, polysulfobetaine methacrylate or polyethylene glycol, and the metal chelating agent is sodium hyaluronate or sodium gluconate.

2. The preservative solution according to claim 1, wherein The number average molecular weight of the polymethylacryloyloxyethyl phosphorylcholine is 9000 Da-15000 Da, the number average molecular weight of the polysulfobetaine methacrylate is 15000 Da-25000 Da, and the number average molecular weight of the polyethylene glycol is 6000 Da-12000 Da.

3. The preservative solution of claim 1, wherein The surfactant is any one of Tween-20, S24 or Triton X-100, the protein protective agent is bovine serum albumin or glycerol, and the preservative is ProClin300 or sodium azide.

4. The preservative solution of claim 1, wherein The preservative solution is a PBS buffer solution containing 0.1 g / L of a high molecular polymer, 0.01 g / L of a surfactant, 0.2 g / L of a protein protective agent, 2.0 g / L of sodium hyaluronate, 0.2 g / L of sucrose and 0.005 g / L of a preservative, and the pH value is 7.4, wherein the high molecular polymer is polymethylacryloyloxyethyl phosphorylcholine with a number average molecular weight of 12000 Da or polysulfobetaine methacrylate with a number average molecular weight of 20000 Da.

5. The preservative solution of claim 1, wherein When the metal chelating agent is sodium hyaluronate, the concentration of sodium hyaluronate in the preservative solution is 0.4 g / L-4.0 g / L, and the number average molecular weight of the sodium hyaluronate is 1000 kDa-1800 kDa; when the metal chelating agent is sodium gluconate, the concentration of sodium gluconate in the preservative solution is 0.2 g / L-2.2 g / L.

6. The preservative solution of claim 1, wherein When the buffer solution is a PBS buffer solution, the concentration of the PBS buffer solution is 10 mM-50 mM; when the buffer solution is a Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 20 mM-50 mM.

7. The preservative solution of claim 1, wherein The magnetic beads for coating the p-Tau217 magnetic bead antibody are carboxylated magnetic microspheres, and the antibody is covalently coupled to the magnetic beads, and the concentration of the magnetic beads in the preservative solution is 0.05 g / L-0.2 g / L.

8. The preservative solution of claim 1, wherein The preservative solution is used for single molecule fluorescent immunoassay as a detection working solution.

9. A method for preparing a preservation solution for p-Tau217 magnetic bead-coated antibodies, for preparing the preservation solution according to claim 1, characterized in that, The preparation method is specifically as follows: A buffer solution is selected, and a metal chelating agent, a suspending agent, a high molecular polymer, a protein protective agent, a surfactant and a preservative are added, an acid or a base is used to adjust the pH value, the solution is uniformly mixed and filtered through a membrane to obtain the preservative solution.

10. The method of claim 9, wherein, The acid is 0.1 M hydrochloric acid solution, the base is 0.1 M sodium hydroxide solution; the membrane filtration uses 0.22 μm PVDF filter membrane.

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