Oxidized low-density lipoprotein detection method based on stabilizer and blocking agent

Abstract

The invention discloses an oxidized low-density lipoprotein detection method based on a stabilizer and a blocking agent, and relates to the field of high-molecular compounds. The method comprises the following steps: copolymerizing a hydrophilic monomer, a hydrophobic monomer and an antioxidant functional group-containing monomer to form a multifunctional polymer stabilizer, representing the structure and performance of the multifunctional polymer stabilizer by adopting a nuclear magnetic resonance spectroscopy, a gel permeation chromatography and a dynamic light scattering method, and simulating and optimizing the monomer composition through molecular dynamics; preparing a sample reaction solution on the basis of a stabilizer; the method comprises the following steps: collecting a serum sample, and preparing a sample for detecting the oxidized low-density lipoprotein through stabilizing treatment of a sample reaction solution; a latex microsphere-labeled oxidized low-density lipoprotein monoclonal antibody is adopted as an antigen trapping agent and is subjected to incubation reaction with a sample, the difference value of absorbance values before and after the reaction is calculated, and the oxidized low-density lipoprotein concentration is calculated according to the standard curve. The stability of the ox-LDL sample can be improved, and the ox-LDL sample can also be used as an efficient blocking agent to reduce non-specific binding.

Description

Technical Field

[0001] The present application relates to the field of polymer compounds, and in particular to a method for detecting oxidized low-density lipoprotein based on stabilizers and blockers. Background Art

[0002] Oxidized low-density lipoprotein (ox-LDL) is an important marker of atherosclerosis, and its detection is of great significance in the prevention, diagnosis and treatment of cardiovascular diseases. In the prior art, ox-LDL detection methods mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA) and radioimmunoassay (RIA). Although these methods have achieved certain results in clinical applications, they still face problems such as poor sample stability, insufficient detection specificity and limited sensitivity.

[0003] Specifically, ox-LDL has a complex molecular structure and is prone to further oxidation or degradation in an in vitro environment, resulting in inaccurate test results. At the same time, due to the similarity of ox-LDL to other lipoprotein molecular structures, existing detection methods often have cross-reactions, affecting the specificity of the test. In addition, traditional blocking agents (such as bovine serum albumin BSA) have limited effects in reducing nonspecific binding and are difficult to meet the needs of high-sensitivity detection.

[0004] Therefore, a technical solution is urgently needed to improve the stability of ox-LDL samples and to reduce nonspecific binding as an efficient blocker. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention provides a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker. The present invention solves the technical problems of the prior art such as poor sample stability and insufficient detection specificity.

[0006] The embodiment of the present application provides an oxidized low-density lipoprotein detection method based on a stabilizer and a blocker, comprising: copolymerizing a hydrophilic monomer, a hydrophobic monomer and a monomer containing an antioxidant functional group to form a multifunctional polymer stabilizer, characterizing its structure and performance by nuclear magnetic resonance spectroscopy, gel permeation chromatography and dynamic light scattering, and optimizing the monomer composition by molecular dynamics simulation; adding a blocker, a surfactant, a flocculant, a metal ion chelator and a preservative to the stabilizer as a basis to prepare a sample reaction solution; collecting a serum sample, and preparing a sample for oxidized low-density lipoprotein detection by stabilizing the sample reaction solution; using a monoclonal antibody labeled with latex microspheres as an antigen capture agent, incubating a reaction with a sample treated with a reaction solution containing a stabilizer and a blocker, calculating the difference in absorbance values ​​before and after the reaction, and calculating the concentration of oxidized low-density lipoprotein according to a standard curve.

[0007] In one possible implementation, a stabilizer is used as a basis, and a blocking agent, a surfactant, a flocculant, a metal ion chelating agent, and a preservative are added to prepare a sample reaction solution, including: The sample reaction solution contains 20mmol / L Tris buffer, 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetic acid, 0.01%-0.05% surfactant, 4g / L-10g / L flocculant, 0.05-0.2 g / L blocking agent, 0.05% proclin300, and the pH value is 4.0-6.5; the blocking agent is an active blocking agent, specifically HBR-X or HBR-ULTRA; the surfactant is TritonX-100 or Tween-20; the flocculant is PEG6000 or PEG8000.

[0008] In one possible implementation, a serum sample is collected, and a sample for oxidized low-density lipoprotein detection is prepared by stabilizing the sample reaction solution, including: collecting a blood sample, separating the serum, and packaging the separated serum into containers for low-temperature storage; mixing and diluting the serum sample with the sample reaction solution to a target concentration range so that it is within the linear range of the detection; and incubating at 37°C for 5 minutes to allow the stabilizer to coat the oxidized low-density lipoprotein particles.

[0009] In a possible implementation, a monoclonal antibody to oxidized low-density lipoprotein labeled with latex microspheres is used as an antigen capture agent, incubated with a sample treated with a reaction solution containing a stabilizer and a blocking agent, the difference in absorbance before and after the reaction is calculated, and the concentration of oxidized low-density lipoprotein is calculated according to a standard curve, including: using a monoclonal antibody to oxidized low-density lipoprotein labeled with double-diameter latex microspheres as an antigen capture agent, adding the sample treated with a sample reaction solution containing a stabilizer, and incubating for 3 minutes; latex microspheres of different particle sizes are cross-linked after the formation of an antigen-antibody complex, recording the absorbance value A0 at the beginning of the reaction and the absorbance value A1 after 3 minutes of incubation, and calculating the difference DA between the absorbance values ​​before and after the reaction; drawing a standard curve based on the concentration of the standard and the difference in absorbance, and calculating the concentration of oxidized low-density lipoprotein in the sample.

[0010] In a possible implementation, a predetermined amount of hydrophilic monomer, hydrophobic monomer and monomer containing antioxidant functional group are mixed, an organic solvent is added, an inert gas is introduced for deoxygenation, a free radical initiator is added, and a free radical copolymerization reaction is carried out at a set temperature for a predetermined time, including: adding a predetermined amount of polyethylene glycol methacrylate monomer, dodecyl methacrylate monomer and vitamin E methacrylate monomer to a reaction container; adding a predetermined amount of tetrahydrofuran solvent to the reaction container, and introducing an inert gas for deoxygenation; adding a predetermined amount of azobisisobutyronitrile initiator to the deoxygenated reaction mixture; placing the reaction container in a constant temperature heating device for polymerization reaction, and maintaining inert gas protection throughout the reaction.

[0011] In a possible implementation, the reaction solution is poured into an excess of a precipitant to precipitate the target product, the precipitate is collected by centrifugation, repeatedly washed with the precipitant, and dried to constant weight under reduced pressure to obtain a multifunctional polymer stabilizer, including: pouring the reaction solution into an excess of aprotic organic solvent to precipitate the polymer product; collecting the obtained precipitate by centrifugation, and washing it three times with aprotic organic solvent; placing the washed product in a vacuum drying device, and drying it at a relatively low temperature for a preset time to obtain the target multifunctional polymer stabilizer.

[0012] In a possible implementation, the method for calculating the efficiency of the stabilizer coating includes: ,in, represents the stabilizer coating efficiency, The time-dependent reaction rate constant representing the stabilizer concentration ranges from 0.11 to 0.72 min -1 (mL / mg), represents the stabilizer concentration, Indicates the wrapping time, It represents the reaction rate constant related to the molecular weight and hydrophilicity of the stabilizer, and its value range is 10 -5 - 10 -3 mol / g, Indicates the molecular weight of the stabilizer, represents the hydrophilicity influencing factor, Indicates the hydrophilicity index of the stabilizer.

[0013] An embodiment of the present application also provides an oxidized low-density lipoprotein detection device, comprising: a processor, a memory, and a system bus; wherein the processor and the memory are connected via the system bus; the memory is used to store one or more programs, and the one or more programs include instructions, which, when executed by the processor, enable the processor to execute the method described in any one of the above embodiments.

[0014] In the above-provided method and apparatus for detecting oxidized low-density lipoprotein based on stabilizers and blockers, the embodiment of the present application synthesizes an amphiphilic copolymer with multiple functions, which can improve the stability of ox-LDL samples and can also serve as a highly efficient blocker to reduce nonspecific binding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 A schematic diagram of a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker provided in an embodiment of the present application; Figure 2 A schematic diagram of a molecular dynamics simulation trajectory provided in an embodiment of the present application; Figure 3 A schematic diagram of the stabilizer coating efficiency provided in an embodiment of the present application. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present application.

[0018] Those skilled in the art will appreciate that the terms "first", "second" and the like in the embodiments of the present application are only used to distinguish different steps, devices or modules, etc., and do not represent any specific technical meaning, nor do they represent the necessary logical order between them. It should also be understood that in the embodiments of the present application, "multiple" may refer to two or more, and "at least one" may refer to one, two or more. It should also be understood that for any component, data or structure mentioned in the embodiments of the present application, in the absence of explicit limitation or contrary revelation given in the context, it can generally be understood as one or more. In addition, the term "and / or" in the present application is only a kind of association relationship describing the associated objects, indicating that there can be three kinds of relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in the present application generally indicates that the associated objects before and after are an "or" relationship. It should also be understood that the description of each embodiment in the present application emphasizes the differences between the embodiments, and the same or similar parts can refer to each other. For the sake of brevity, they will not be repeated one by one.

[0019] At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present application and its application or use. The techniques, methods and devices known to ordinary technicians in the relevant fields may not be discussed in detail, but where appropriate, the techniques, methods and devices should be considered as part of the specification. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0021] Figure 1 A schematic flow chart of a method for detecting oxidized low-density lipoprotein based on a stabilizer and a blocker provided in an embodiment of the present application. The method aims to solve the technical problems of poor stability and insufficient specificity in existing oxidized low-density lipoprotein (ox-LDL) detection methods. The main innovation of the embodiment of the present application is the development of a new multifunctional polymer stabilizer, which can not only improve the stability of ox-LDL samples, but also act as a blocker to reduce nonspecific binding, thereby improving the accuracy and sensitivity of detection.

[0022] The oxidized low-density lipoprotein refers to low-density lipoprotein that has been oxidatively modified, which plays an important role in the occurrence and development of cardiovascular diseases such as atherosclerosis. The detection of ox-LDL is of great significance for assessing the risk of cardiovascular diseases. However, due to the instability of ox-LDL and the limitations of the detection method, there are some problems with the ox-LDL detection method in the prior art, such as poor sample stability, insufficient detection specificity, etc., which affect the accuracy and reliability of the test results.

[0023] The detection method provided in the embodiment of the present application includes but is not limited to the following key steps: First, a new type of amphiphilic copolymer stabilizer is designed and synthesized. The copolymer is copolymerized by a hydrophilic monomer and a hydrophobic monomer to form a polymer chain with an amphiphilic structure. Among them, the hydrophilic monomer can be but is not limited to polyethylene glycol methacrylate, and the hydrophobic monomer can be but is not limited to dodecyl methacrylate. This amphiphilic structure enables the stabilizer to interact with the lipid and protein parts on the surface of the ox-LDL particles at the same time to form a stable coating layer, thereby improving the stability of the ox-LDL sample.

[0024] Secondly, functional groups with antioxidant properties are introduced into the copolymer molecular chain, such as but not limited to vitamin E derivatives. These antioxidant functional groups can further improve the stability of the ox-LDL sample and prevent it from further oxidation during storage and detection. The introduction of antioxidant functional groups can be achieved by but not limited to the following methods: copolymerizing monomers containing antioxidant groups with hydrophilic monomers and hydrophobic monomers; or grafting antioxidant groups onto the copolymer molecular chain by chemical modification after the copolymer is synthesized.

[0025] Again, an epitope-mimicking structure that can compete with the detection antibody for binding is introduced into the copolymer molecular chain. This allows the stabilizer to also function as a blocker, effectively reducing nonspecific binding and improving the specificity of detection. The epitope-mimicking structure can be, but is not limited to, a mimetic peptide segment of a specific antigenic determinant on the surface of ox-LDL or other structures that can specifically bind to the detection antibody. These structures can be directly introduced into the copolymer molecular chain by chemical synthesis methods, or they can be connected to the copolymer by biocoupling technology.

[0026] In order to obtain the best stability and blocking effect, the present application also uses computational chemistry and molecular dynamics simulation methods to optimize the composition and structure of the copolymer. This includes but is not limited to: simulating the effects of different monomer ratios on the performance of the copolymer; predicting the interaction between the copolymer and ox-LDL; evaluating the optimal distribution of antioxidant functional groups and epitope-mimicking structures in the copolymer, etc. Through these computational simulations, the performance of the copolymer can be predicted and optimized before actual synthesis, thereby improving research and development efficiency.

[0027] Specific implementation steps, such as Figure 1As shown, at step S101, a hydrophilic monomer, a hydrophobic monomer and a monomer containing an antioxidant functional group are copolymerized to form a multifunctional polymer stabilizer, and its structure and performance are characterized by nuclear magnetic resonance spectroscopy, gel permeation chromatography and dynamic light scattering, and the monomer composition is optimized by molecular dynamics simulation. This step can be carried out by methods such as free radical polymerization, controlled / active free radical polymerization, ionic polymerization, etc. The polymerization reaction can be carried out in an appropriate solvent, such as water, an organic solvent or a mixture thereof. The conditions of the polymerization reaction, such as temperature, time, initiator type, etc., need to be optimized according to the specific monomer combination. The synthesized multifunctional polymer stabilizer needs to be characterized to confirm its structure and performance. The embodiment of the present application adopts but is not limited to the following methods for characterization: nuclear magnetic resonance spectroscopy (NMR) is used to determine the chemical structure and composition of the copolymer; gel permeation chromatography (GPC) is used to determine the molecular weight and distribution of the copolymer; dynamic light scattering (DLS) is used to determine the particle size and dispersibility of the copolymer in solution. In addition, other characterization methods, such as Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-Vis), etc., can be used to obtain more comprehensive structural information.

[0028] Specifically, the method comprises: mixing a predetermined amount of a hydrophilic monomer, a hydrophobic monomer and a monomer containing an antioxidant functional group, adding an organic solvent, introducing an inert gas to remove oxygen, adding a free radical initiator, and performing a free radical copolymerization reaction at a set temperature for a predetermined time; pouring the reaction solution into an excess of a precipitant to precipitate a target product, collecting the precipitate by centrifugation, repeatedly washing with a precipitant, and drying to a constant weight under reduced pressure to obtain a multifunctional polymer stabilizer; determining the composition of the copolymer by nuclear magnetic resonance spectroscopy, determining the molecular weight and its distribution by gel permeation chromatography, and characterizing the particle size distribution of the copolymer in an aqueous solution by dynamic light scattering; using a molecular dynamics simulation method to simulate the interaction between the copolymer and oxidized low-density lipoprotein under physiological conditions, and optimizing the molar ratio of the monomers by analyzing the simulation trajectory (e.g. Figure 2 The optimal copolymer composition was determined.

[0029] The method comprises mixing a predetermined amount of a hydrophilic monomer, a hydrophobic monomer and a monomer containing an antioxidant functional group, adding an organic solvent, introducing an inert gas for deoxygenation, adding a free radical initiator, and performing a free radical copolymerization reaction at a set temperature for a predetermined time, including: adding a predetermined amount of a polyethylene glycol methacrylate monomer, a dodecyl methacrylate monomer and a vitamin E methacrylate monomer into a reaction container; adding a predetermined amount of a tetrahydrofuran solvent into the reaction container, and introducing an inert gas for deoxygenation; adding a predetermined amount of azobisisobutyronitrile initiator into the reaction mixture after deoxygenation; placing the reaction container in a constant temperature heating device for polymerization reaction, and maintaining inert gas protection throughout the reaction.

[0030] The method comprises the steps of: pouring the reaction liquid into an excess of a precipitant to precipitate a target product, collecting the precipitate by centrifugal separation, repeatedly washing with a precipitant, and drying to a constant weight under reduced pressure to obtain a multifunctional polymer stabilizer, including: pouring the reaction liquid into an excess of aprotic organic solvent to precipitate the polymer product; collecting the obtained precipitate by centrifugal separation, and washing it three times with aprotic organic solvent; placing the washed product in a vacuum drying device, and drying it at a relatively low temperature for a preset time to obtain the target multifunctional polymer stabilizer.

[0031] In one embodiment, first, raw materials are prepared, hydrophilic monomer: polyethylene glycol methacrylate (PEGMA, Mn = 500 g / mol); hydrophobic monomer: dodecyl methacrylate (DDMA); antioxidant functional group: α-tocopherol methacrylate (VE-MA); initiator: azobisisobutyronitrile (AIBN); solvent: tetrahydrofuran (THF).

[0032] Synthesis steps: PEGMA (5g, 10mmol), DDMA (2.54g, 10mmol) and VE-MA (0.5g, 1mmol) were added to a 100mL round-bottom flask. 50mL THF was added as a solvent, and nitrogen was introduced for deoxygenation for 15 minutes. AIBN (0.033g, 0.2mmol) was added as an initiator. The reaction was carried out in a 65°C oil bath for 24 hours, with nitrogen protection being maintained during the reaction. After the reaction was completed, the reaction solution was poured into an excess of ice ether to precipitate the polymer. The precipitate was collected by centrifugation and washed 3 times with ether. The product was dried in a 40°C vacuum drying oven for 24 hours to obtain a final multifunctional polymer stabilizer.

[0033] For characterization, nuclear magnetic resonance hydrogen spectroscopy (1H NMR) can be used: a Bruker AVANCE III 400 MHz NMR instrument is used to determine the composition of the copolymer. Gel permeation chromatography (GPC): a Waters Alliance e2695 system is used to determine the molecular weight and molecular weight distribution. Dynamic light scattering (DLS): a Malvern Zetasizer Nano ZS is used to determine the particle size distribution of the copolymer in aqueous solution.

[0034] Next, computational chemistry and molecular dynamics simulation methods were used to optimize the composition and structure of the copolymer. Molecular dynamics simulations were performed using the GROMACS software package, and the simulation conditions were as follows: force field: OPLS-AA; temperature: 310 K (physiological temperature); pressure: 1 atm; simulation time: 100 ns; time step: 2 fs. By analyzing the simulation trajectory, the interaction energy between the copolymer and the simulated ox-LDL surface was calculated to optimize the composition ratio of the copolymer. For example, according to the optimization results, the optimal monomer molar ratio can be obtained as PEGMA:DDMA:VE-MA = 10:10:1.

[0035] In step S102, a blocker, a surfactant, a flocculant, a metal ion chelating agent, and a preservative are added to the stabilizer to prepare a sample reaction solution. The following steps may be used but are not limited to: The sample reaction solution contained 20mmol / L Tris buffer, 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetate, 0.01%-0.05% surfactant, 4g / L-10g / L flocculant, 0.05-0.2 g / L blocking agent, 0.05% proclin300, and the pH value was 4.0-6.5; The blocker is an active blocker, and specifically HBR-X or HBR-ULTRA can be selected; in this embodiment, 0.05 g / L HBR-ULTRA is preferred; The surfactant may be TritonX-100 or Tween-20; in this embodiment, 0.02% TritonX-100 is preferred; The flocculant can be PEG6000 or PEG8000; in this embodiment, 10 g / L PEG6000 is preferred; Among them, the stabilizer can be quickly adsorbed on the surface of the antigen, reducing further oxidation of the OxLDL antigen, exposing the antibody recognition epitope, and facilitating the rapid reaction of the latex microsphere-labeled antibody with the OxLDL antigen.

[0036] In step S103, a serum sample is collected, and a sample for oxidized low-density lipoprotein detection is prepared by stabilizing the sample reaction solution; In this step S103, it specifically includes: collecting blood samples, separating serum, and packaging the separated serum into containers for low-temperature storage; mixing the serum sample with the sample reaction solution and diluting it to the target concentration range so that it is within the linear range of detection; incubating at 37°C for 5 minutes to allow the stabilizer to coat the oxidized low-density lipoprotein particles.

[0037] The following steps may be adopted but are not limited to: first, a whole blood sample is collected from the subject's vein and serum is obtained by centrifugation. Then, the serum sample is diluted with a buffer solution according to a predetermined ratio, and the dilution ratio can be adjusted according to the expected ox-LDL concentration range. Next, an appropriate amount of a multifunctional polymer stabilizer is added to the diluted serum sample and mixed thoroughly so that the stabilizer and the ox-LDL in the sample fully interact to form a stable complex. Specifically, it includes: collecting a blood sample, separating serum, and packaging the separated serum into containers for low-temperature storage; mixing and diluting the serum sample with a buffer solution to a target concentration range so that it is within the linear range of the test; mixing the diluted serum sample with a multifunctional polymer stabilizer solution at a target volume ratio, and incubating at room temperature so that the stabilizer coats the oxidized low-density lipoprotein particles.

[0038] Wherein, the calculation method of the efficiency of the stabilizer coating (such as Figure 3 shown), including: ,in, represents the stabilizer coating efficiency, The time-dependent reaction rate constant representing the stabilizer concentration ranges from 0.11 to 0.72 min -1 (mL / mg), represents the stabilizer concentration, Indicates the wrapping time, It represents the reaction rate constant related to the molecular weight and hydrophilicity of the stabilizer, and its value range is 10 -5 - 10 - 3 mol / g, Indicates the molecular weight of the stabilizer, represents the hydrophilicity influencing factor, Indicates the hydrophilicity index of the stabilizer.

[0039] In one implementation scenario, sample pretreatment first requires serum sample collection: 5 mL of fasting venous blood from the subject is collected using a vacuum blood collection tube. After standing at room temperature for 30 minutes, the serum is separated by centrifugation at 3000 rpm for 15 minutes. The separated serum is aliquoted into 1.5 mL centrifuge tubes and stored at -80°C for later use.

[0040] In step S104, a monoclonal antibody against oxidized low-density lipoprotein labeled with latex microspheres is used as an antigen capture agent to incubate with a sample treated with a reaction solution containing a stabilizer and a blocking agent, the difference in absorbance before and after the reaction is calculated, and the concentration of oxidized low-density lipoprotein is calculated based on a standard curve.

[0041] The step S104 specifically includes: using a monoclonal antibody of oxidized low-density lipoprotein labeled with double-diameter latex microspheres as an antigen capture agent, adding a sample treated with a sample reaction solution containing a stabilizer, and incubating the sample for 3 minutes; Latex microspheres of different particle sizes are cross-linked together after the formation of antigen-antibody complexes. The absorbance value A0 at the beginning of the reaction and the absorbance value A1 after 3 minutes of incubation are recorded, and the absorbance difference DA before and after the reaction is calculated. A standard curve is drawn based on the standard concentration and the absorbance difference, and the concentration of oxidized low-density lipoprotein in the sample is calculated.

[0042] Example 1: As an example of the sample reaction solution described in the present invention, the sample reaction solution components of this example include the following: 20mmol / L Tris buffer (pH6.0), 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetate, 0.02% TritonX-100, 10g / L polyethylene glycol 6000, 0.05% proclin300.

[0043] Example 2: As an example of the sample reaction solution described in the present invention, the sample reaction solution components of this example include the following: 20mmol / L Tris buffer (pH6.0), 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetic acid, 0.02% TritonX-100, 10g / L polyethylene glycol 6000, 0.05% proclin300.

[0044] Example 3: As an example of the sample reaction solution described in the present invention, the sample reaction solution components of this example include the following: 20mmol / L Tris buffer (pH6.0), 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetic acid, 0.02% TritonX-100, 10g / L polyethylene glycol 6000, 0.05g / L HBR-ULTRA, 0.05% proclin300.

[0045] HBR-ULTRA was purchased from Scantibodies Laboratory, Inc, catalog number: 3KC102.

[0046] Example 4: As an example of the sample reaction solution described in the present invention, the sample reaction solution components of this example include the following: 20mmol / L Tris buffer (pH6.0), 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetic acid, 0.02% TritonX-100, 10g / L polyethylene glycol 6000, 0.05g / L HBR-X, 0.05% proclin300.

[0047] HBR-X was purchased from Scantibodies Laboratory, Inc, with the catalog number 3KC029.

[0048] Example 5: As an example of the sample reaction solution described in the present invention, the sample reaction solution components of this example include the following: 20mmol / L Tris buffer (pH6.0), 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetic acid, 0.02% Tween-20, 10g / L polyethylene glycol 6000, 0.05g / L HBR-X, 0.05% proclin300.

[0049] Example 6: As an example of the sample reaction solution described in the present invention, the sample reaction solution components of this example include the following: 20mmol / L Tris buffer (pH6.0), 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetic acid, 0.02% TritonX-100, 10g / L polyethylene glycol 8000, 0.05g / L HBR-X, 0.05% proclin300.

[0050] Example 7: As an example of the sample reaction solution described in the present invention, the sample reaction solution components of this example include the following: 20mmol / L Tris buffer (pH4.2), 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetic acid, 0.02% TritonX-100, 10g / L polyethylene glycol 8000, 0.05g / L HBR-X, 0.05% proclin300.

[0051] Example 8: Surfactant A commonly used surfactant is selected, and the surfactant can increase the dispersibility of OxLDL. In this example, two surfactants, Tween-20 and TritonX-100, are used to prepare R1 reaction solutions with contents of 0.01%, 0.05%, 0.1%, and 0.2%, respectively. Then, different concentrations of calibrants (blank, 5U / L, 10U / L, 20U / L, 40U / L, 80U / L, 120U / L) are used for testing. The test results of each group are shown in Table 1 and Table 2: Table 1 Test results of Tween-20 calibrators at different concentrations

[0052] Table 2 Test results of TritonX-100 calibrators at different concentrations

[0053] From the results of surfactant optimization test, it can be seen that as the amount of Tween-20 and TritonX-100 added increases, the reactivity value shows a significant downward trend, indicating that as the concentration of the surfactant increases, its dispersion effect on the antigen weakens, which in turn affects the occurrence of antigen-antibody, resulting in a decreasing reactivity trend; from the perspective of the discrimination of each point, as the concentration of the surfactant increases, the discrimination of each point increases significantly, but after increasing to more than 0.1%, the discrimination of the low value becomes worse, affecting the sensitivity of the product. From the comparison of the two surfactants, the effect of TritonX-100 on the signal value is slightly smaller than that of Tween-20, and it can be preferred. Therefore, both Tween-20 and TritonX-100 can be used in the R1 reaction solution, and it is relatively advantageous to use it in the concentration range of 0.01%-0.05% (i.e. 0.1g / L-0.5g / L).

[0054] Example 9: According to the influence of PEG6000 and PEG8000 flocculants on the test results, the dosage of the two flocculants was optimized. The dosage of flocculants was set at 0.2%, 0.4%, 0.8%, 1%, and 1.5%, and the R1 reaction solution was prepared according to the set dosage. After the preparation was completed, the calibration test was carried out with calibration products of various concentrations (blank, 5U / L, 10U / L, 20U / L, 40U / L, 80U / L, 120U / L), and the reaction degree of each concentration was recorded and the discrimination of each concentration point was calculated. The test results are recorded as shown in Tables 3 and 4 below: Table 3 PEG6000 test results

[0055] Table 4 PEG8000 test results

[0056] From the test results of different dosages of EPG6000, it can be seen that when the dosage is below 1% PEG6000, the reactivity decreases to a certain extent, and the differentiation of each point can meet the requirements; the reactivity increases significantly at 1.5%, and the differentiation decreases. When the dosage of 0.1% PEG6000 is used, the reactivity is the lowest, and the differentiation decreases to a certain extent, indicating that the flocculant effect is not good. The reactivity and differentiation of each point of the flocculant PEG6000 at a content of 0.4%-1% can meet the test requirements. Therefore, the dosage of PEG6000 should be in the range of 0.4%~1.0%, that is, 4g / L-10g / L.

[0057] From the test results of different dosages of PEG8000, it can be seen that the reactivity of the calibration test increases with the dosage. When the dosage is 1.5%, the high value area discrimination decreases; when the dosage is 0.1%, the high value discrimination also decreases; in the dosage range of 0.4-1.0%, the reactivity and discrimination can meet the test requirements. Therefore, the appropriate dosage of PEG8000 is in the range of 0.4-1.0%, that is, 4g / L-10g / L.

[0058] Example 10: The blockers initially screened were HBR-X and HBR-ULTRA, and their usage was optimized in this experiment. The optimized dosages of the two blockers were set to 0.02 mg / ml, 0.05 mg / ml, 0.1 mg / ml, and 0.2 mg / ml. R1 reaction solutions were prepared with the above-mentioned different dosages of blockers. After preparation, a calibration test was first performed using a calibrator with a fixed value result, and the calibration reactivity and the discrimination of each point were recorded. Then 10 clinical samples (which had been assigned values ​​using the Mercodia oxidized LDLELISA Kit) were selected for testing, and the test results were analyzed for relative deviations from the results of ELISA. The data are recorded in Tables 5 and 6 below, and the results of the clinical sample tests with the two blockers and the deviation analysis are shown in Tables 7 and 8. Table 5 Calibration data of each group of HBX-X

[0059] Table 6 HBR-ULTRA calibration data for each group

[0060] Table 7 Results of clinical samples tested with two blockers

[0061] Table 8 Bias analysis of clinical samples tested with two blockers

[0062] The calibration test results of the two blockers showed that different amounts of blockers had relatively little effect on the reactivity of each concentration of calibrant, and the discrimination of each point did not change significantly. From the test results of clinical samples, it can be seen that when using 0.02mg / ml of blocker, the test results of some clinical samples were high. When using 0.05-0.2mg / ml of blocker, the results of clinical samples were close to the results of ELISA control group assignment, and the relative deviation met the requirements for clinical use. Therefore, the dosage of blockers HBR-X and HBR-ULTRA should be 0.05-0.2mg / ml. Based on cost factors, the test preferably uses 0.05mg / ml.

[0063] Example 11: Optimization test of the pH value of the R1 reaction solution. In the experiment, hydrochloric acid was used to adjust the pH values ​​to 8.4, 7.4, 6.5, 5.0, and 4.0 to obtain R1 reaction solutions with different pH values. After preparation, the concentrations of calibration products (blank, 5U / L, 10U / L, 20U / L, 40U / L, 80U / L, and 120U / L) were tested. The test results of each group are shown in Table 9 below: Table 9 Test results of each group under different pH conditions

[0064] From the test results of different pH values, we can see that: within the pH range of 4.0-8.4, the reaction value decreases to a certain extent with the pH value, and the decrease is relatively small; from the perspective of the discrimination of each point, after the pH value increases to above 6.5, the discrimination of each high-value point gradually decreases and the discrimination becomes worse. Therefore, the more suitable range of pH value reaction is 4.0-6.5.

[0065] Example 12: This example is a method for detecting oxidized low-density lipoprotein, including the following contents: Human serum samples were collected and centrifuged at 3000 rpm for 10 minutes to separate serum. Serum samples were concentrated using ultrafiltration centrifuge tubes (100 kDa molecular weight cutoff). Samples were diluted to appropriate concentrations using PBST buffer (pH 7.4). A series of ox-LDL standard solutions (0-150 U / L) were prepared.

[0066] Take 3 μL of standard solution or sample, add it to 180 μL of sample reaction solution, mix well, incubate at 37°C for 5 minutes to obtain a stabilized sample. Then immediately add 60 μL of double-particle latex microsphere marker mixture and continue to incubate at 37°C for 3 minutes. Use an automatic biochemical analyzer (such as Hitachi 7180) to track and measure the absorbance at 570 nm during the entire incubation reaction. Then take the absorbance value A0 recorded immediately after adding the modified double-particle latex microspheres and the absorbance value A1 after the 3-minute incubation reaction, and calculate the absorbance difference (DA) of each concentration standard solution as DA=A1-A0.

[0067] Data processing can draw a standard curve: use the concentration of ox-LDL standard as the horizontal axis and the absorbance difference (DA) as the vertical axis to draw a five-parameter logistic curve. Sample concentration calculation Calculate the concentration of ox-LDL in the sample according to the standard curve equation.

[0068] Three concentrations of quality control products, high, medium and low, are set for each test. The intra-batch coefficient of variation (CV) should be less than 10%, and the inter-batch CV should be less than 15%. Detection sensitivity and linear range: Detection sensitivity: 5U / L. Linear range: 5-150U / L. In addition, all reagents should be fully mixed and returned to room temperature before use. Strictly control the temperature and time of each step to ensure the accuracy and repeatability of the test.

[0069] Implementation Case 9: 20 clinical samples were selected and tested using the sample reaction solutions prepared in Examples 1-7 and latex microsphere-labeled oxidized low-density lipoprotein according to the test method of Example 8. At the same time, the control reagent Oxidized LDL ELISA KIT (Cat. No. 10-1143-01) from Mercodia AB, Sweden, was used for testing. The relative deviations of each example and the control reagent were calculated, and the results are shown in the following table: Unit U / L

[0070] The relative deviation results of each embodiment and the control group are shown in the following table:

[0071] For samples No. 1-15, which are normal samples, after adding stabilizers, the deviation of test results in Examples 2-7 was significantly improved compared with Example 1; for samples No. 16-20, which are interference samples, after adding blockers, the deviation of test results in Examples 3-7 was significantly improved compared with Example 2. Examples 3, 5-7 show that surfactants, flocculants and pH values ​​have little effect on test results.

[0072] In summary, the multifunctional polymer stabilizer described in the examples of the present application can not only improve the stability of ox-LDL samples, but also act as a blocker to reduce nonspecific binding. This is because the epitope mimicking structure introduced on the stabilizer molecular chain can compete with the detection antibody for binding, thereby reducing the binding of the detection antibody to nonspecific sites. This dual-functional design significantly improves the accuracy and sensitivity of the detection.

[0073] Figure 2 A schematic diagram of a molecular dynamics simulation trajectory provided in an embodiment of the present application. The embodiment of the present application relates to a method for detecting oxidized low-density lipoprotein (ox-LDL) based on a stabilizer and a blocker, wherein one of the key steps is the synthesis and optimization of a novel multifunctional polymer stabilizer. The figure shows a molecular dynamics simulation trajectory of the interaction between the polymer stabilizer and the ox-LDL surface, which is of great significance for understanding and optimizing the performance of the stabilizer.

[0074] Overall, the graph shows how the particles start to move at different initial heights and gradually aggregate toward the surface. As can be seen from the figure, the simulation system contains three different types of monomer units, represented by blue, red and green respectively. These three colors correspond to the hydrophilic monomer polyethylene glycol methacrylate (PEGMA), the hydrophobic monomer dodecyl methacrylate (DDMA) and α-tocopherol methacrylate (VE-MA) with antioxidant function. The selection of these three monomers reflects the design concept of the multifunctional polymer stabilizer in the embodiment of the present application, that is, to achieve efficient stabilization and protection of ox-LDL by rationally combining monomers with different functions.

[0075] The simulation trajectory shows the movement of these monomer units in three-dimensional space, and each colored line represents the movement trajectory of a monomer unit during the simulation. From the distribution of the trajectory, it can be observed that different types of monomer units show different movement characteristics and spatial distribution. This difference reflects the different roles of each monomer unit in the polymer chain: PEGMA provides good water solubility and biocompatibility, DDMA gives the polymer a certain hydrophobicity to enhance the interaction with ox-LDL, and VE-MA plays an antioxidant protection role in the entire structure.

[0076] The gray plane at the bottom of the figure represents the simulated ox-LDL surface. It can be observed that many polymer chain trajectories tend to be close to this surface, indicating that the polymer stabilizer can indeed interact effectively with the ox-LDL surface. This interaction is key to achieving ox-LDL stabilization because it can prevent further oxidation and aggregation of ox-LDL, thereby maintaining its structural integrity and function.

[0077] The simulation system uses periodic boundary conditions, which can be seen from the continuity of the trajectory in the x and y directions. This treatment method can effectively simulate the behavior of large systems while reducing the consumption of computing resources. The time scale and spatial scale of the simulation have also been optimized to strike a balance between computational efficiency and simulation accuracy.

[0078] From the density and distribution of the trajectories, it can be inferred that the polymer chains exhibit a certain degree of flexibility and dynamics in solution. This property is crucial for the function of the stabilizer because it allows the polymer chains to adapt to different configurations of the ox-LDL surface, thereby achieving more effective coverage and protection. At the same time, the diversity of the trajectories also reflects the thermodynamic equilibrium state of the system, indicating that the simulation has fully sampled the configuration space.

[0079] By analyzing the spatial distribution and motion characteristics of different types of monomer units, it is possible to evaluate whether the current monomer ratio is optimal. For example, if excessive aggregation of PEGMA units or uneven distribution of VE-MA units is observed, the molar ratio of the monomers may need to be adjusted. This optimization process based on simulation results is an important part of the design of polymer stabilizers in the embodiments of the present application.

[0080] The simulation results can also be used to calculate the interaction energy between the polymer chain and the ox-LDL surface. By analyzing the particle density and trajectory characteristics in different regions, the main binding sites between the polymer chain and the surface can be identified. This information can guide further structural optimization, such as adjusting the chain length of the hydrophobic monomer or introducing new functional groups.

[0081] It is worth noting that the simulation uses a simplified Lennard-Jones potential function to describe the interaction between particles. Although this approach makes a trade-off between computational efficiency and physical accuracy, it can still capture the key features of the system. In practical applications, more complex force field models, such as the OPLS-AA force field, can be introduced as needed to obtain more accurate simulation results.

[0082] In general, this molecular dynamics simulation provides an important theoretical basis for the design and optimization of the novel multifunctional polymer stabilizer in the examples of this application. By visualizing the behavior of the polymer chain and its interaction with the surface of ox-LDL, we can deeply understand the working mechanism of the stabilizer and make targeted improvements. This computationally assisted design method can not only accelerate the development process, but also reduce experimental costs and improve the performance of the final product.

[0083] Figure 3A schematic diagram of the coating efficiency of a stabilizer provided in an embodiment of the present application. The embodiment of the present application provides a sample pretreatment method for oxidized low-density lipoprotein (ox-LDL) detection, wherein the key step includes stabilizing the serum sample using a multifunctional polymer stabilizer. Figure 3 The curve showing the change of the coating efficiency of the stabilizer over time is one of the core technical points of the embodiment of the present application. Figure 3 It can be seen that the stabilizer coating efficiency (SE) shows a nonlinear growth trend over time. It grows rapidly in the initial stage, and then the growth rate gradually slows down and tends to be stable.

[0084] Figure 3 Three curves are shown, corresponding to different stabilizer concentrations. It can be observed that as As the concentration of stabilizer increases, the curve moves upward as a whole, indicating that a higher stabilizer concentration can achieve a higher coating efficiency. This phenomenon is consistent with the theoretical model of the embodiment of the present application, that is, the stabilizer concentration is one of the key factors affecting the coating efficiency. However, it should be noted that too high a stabilizer concentration may increase nonspecific binding or interfere with subsequent detection steps, so in practical applications, it is necessary to balance the stabilization effect and detection sensitivity. As shown in the figure, =0.1 mg / mL, a higher coating efficiency can be achieved within a reasonable time, which is consistent with the stabilizer concentration recommended in the examples of the present application.

[0085] On the timeline, Figure 3 The 3-minute position is specially marked. The selection of this time point is of great significance because at this time all three curves are close to their respective stable levels. This shows that an incubation time of 3 minutes is sufficient for the stabilizer to fully exert its effect and achieve effective coating of ox-LDL particles, and the coating rate reaches a basically saturated state within 5 minutes. Although extending the incubation time can slightly improve the coating efficiency, the gain effect is not significant, and it may increase the complexity and time cost of sample processing. Therefore, the embodiment of the present application selects 3 minutes as the optimal incubation time, which not only ensures a sufficient stabilization effect, but also takes into account the convenience and efficiency of operation.

[0086] The embodiments of the present application optimize these molecular parameters to design a multifunctional polymer stabilizer that can not only effectively stabilize the sample but also has the function of blocking nonspecific binding, thereby simplifying the sample pretreatment process.

[0087] It is worth noting that the curves in the figure all show a high final coating efficiency, which can reach up to more than 80%. This feature highlights the high efficiency of the stabilizer used in the embodiments of the present application, and a good stabilization effect can be achieved even at lower concentrations. This not only helps to reduce reagent costs, but also minimizes the potential interference of the stabilizer with subsequent detection steps. At the same time, the efficient coating process also means that the ox-LDL particles in the sample can be quickly and comprehensively protected, effectively preventing degradation or denaturation during storage and detection, thereby improving the accuracy and reliability of the test results.

[0088] The method for calculating the coating efficiency of the stabilizer of the embodiment of the present application not only takes into account the factors of time and concentration, but also introduces the influence of molecular weight and hydrophilicity. This multi-parameter model can more comprehensively describe the kinetic characteristics of the coating process and provide theoretical guidance for the design and optimization of stabilizers. By adjusting these parameters, the most suitable stabilization strategy can be customized for different types of biomarkers. For example, for easily degradable protein markers, the molecular weight and hydrophilicity of the stabilizer can be increased to provide a stronger protective effect; for small molecule markers, these parameters can be appropriately reduced to reduce the impact on the properties of the analyte itself.

[0089] Furthermore, an embodiment of the present application also provides an oxidized low-density lipoprotein detection device, comprising: a processor, a memory, and a system bus; the processor and the memory are connected via the system bus; the memory is used to store one or more programs, and the one or more programs include instructions, which, when executed by the processor, enable the processor to execute any of the above methods.

[0090] Furthermore, an embodiment of the present application also provides a computer program product, which, when executed on a terminal device, enables the terminal device to execute any of the above methods.

[0091] It can be known from the description of the above implementation mode that those skilled in the art can clearly understand that all or part of the steps in the above-mentioned embodiment method can be implemented by means of software plus a necessary general hardware platform. Based on such an understanding, the technical solution of the present application can be essentially or partly embodied in the form of a software product that contributes to the prior art. The computer software product can be stored in a storage medium such as ROM / RAM, a disk, an optical disk, etc., including several instructions for enabling a computer device (which can be a personal computer, a server, or a network communication device such as a media gateway, etc.) to execute the methods described in the various embodiments of the present application or certain parts of the embodiments.

[0092] It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the various embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part description.

[0093] It should also be noted that, in the embodiments of the present application, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also includes other elements that are not clearly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of more restrictions, the elements limited by the statement "comprise one..." do not exclude the presence of other identical elements in the process, method, article or equipment including the elements.

[0094] The above description of the disclosed embodiments enables professionals and technicians in the field to implement or use the present application. Various modifications to these embodiments will be apparent to professionals and technicians in the field, and the general principles defined in the embodiments of the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the embodiments of the present application, but will conform to the widest range consistent with the principles and novel features disclosed in the embodiments of the present application.

Claims

1. A method for detecting oxidized low-density lipoprotein based on stabilizers and blockers, characterized in that: include: A multifunctional polymer stabilizer was formed by copolymerizing a hydrophilic monomer, a hydrophobic monomer and a monomer containing an antioxidant functional group. Its structure and properties were characterized by nuclear magnetic resonance spectroscopy, gel permeation chromatography and dynamic light scattering, and the monomer composition was optimized by molecular dynamics simulation. Based on the stabilizer, a blocking agent, a surfactant, a flocculant, a metal ion chelating agent, and a preservative are added to prepare a sample reaction solution; collecting serum samples, and preparing samples for oxidized low-density lipoprotein detection by stabilizing the sample reaction solution; The monoclonal antibody against oxidized low-density lipoprotein labeled with latex microspheres was used as an antigen capture agent to incubate with the sample treated with a reaction solution containing a stabilizer and a blocking agent. The difference in absorbance before and after the reaction was calculated, and the concentration of oxidized low-density lipoprotein was calculated according to the standard curve.

2. The method for detecting oxidized low-density lipoprotein according to claim 1, characterized in that: in, The hydrophilic monomer, the hydrophobic monomer and the monomer containing the antioxidant functional group are copolymerized to form a multifunctional polymer stabilizer. Its structure and properties are characterized by nuclear magnetic resonance spectroscopy, gel permeation chromatography and dynamic light scattering, and the monomer composition is optimized by molecular dynamics simulation, including: A predetermined amount of hydrophilic monomer, hydrophobic monomer and monomer containing antioxidant functional group are mixed, an organic solvent is added, an inert gas is introduced to remove oxygen, a free radical initiator is added, and a free radical copolymerization reaction is carried out at a set temperature for a predetermined time; The reaction solution is poured into an excess of a precipitant to precipitate the target product, the precipitate is collected by centrifugation, repeatedly washed with the precipitant, and dried under reduced pressure to a constant weight to obtain a multifunctional polymer stabilizer; The composition of the copolymers was determined by nuclear magnetic resonance spectroscopy, the molecular weight and its distribution were determined by gel permeation chromatography, and the particle size distribution of the copolymers in aqueous solution was characterized by dynamic light scattering; Molecular dynamics simulation was used to simulate the interaction between the copolymer and oxidized low-density lipoprotein under physiological conditions. The molar ratio of the monomers was optimized by analyzing the simulation trajectory to determine the optimal copolymer composition.

3. The method for detecting oxidized low-density lipoprotein according to claim 1, characterized in that: in, Based on the stabilizer, a blocking agent, a surfactant, a flocculant, a metal ion chelating agent, and a preservative are added to prepare a sample reaction solution, including: The sample reaction solution contained 20mmol / L Tris buffer, 0.5g / L stabilizer, 10g / L sodium chloride, 20mmol / L disodium ethylenediaminetetraacetate, 0.01%-0.05% surfactant, 4g / L-10g / L flocculant, 0.05-0.2 g / L blocking agent, 0.05% proclin300, and the pH value was 4.0-6.5; The blocker is an active blocker, specifically HBR-X or HBR-ULTRA; The surfactant is TritonX-100 or Tween-20; The flocculant is PEG6000 or PEG8000.

4. The method for detecting oxidized low-density lipoprotein according to claim 1, characterized in that: in, Collect serum samples, and prepare samples for oxidized low-density lipoprotein detection by stabilizing the sample reaction solution, including: Collect blood samples, separate serum, and store the separated serum in containers at low temperatures; The serum sample and the sample reaction solution are mixed and diluted to the target concentration range so that it is within the linear range of the test; The stabilizer was coated on the oxidized low-density lipoprotein particles by incubating at 37°C for 5 minutes.

5. The method for detecting oxidized low-density lipoprotein according to claim 1, characterized in that: in, The monoclonal antibody of oxidized low-density lipoprotein labeled with latex microspheres is used as an antigen capture agent, and is incubated with the sample treated with a reaction solution containing a stabilizer and a blocking agent. The difference in absorbance before and after the reaction is calculated, and the concentration of oxidized low-density lipoprotein is calculated according to the standard curve, including: The monoclonal antibody against oxidized low-density lipoprotein labeled with double-size latex microspheres was used as an antigen capture agent, and the sample treated with the sample reaction solution containing a stabilizer was added and incubated for 3 minutes; Latex microspheres of different particle sizes are cross-linked together after the formation of antigen-antibody complexes. The absorbance value A0 at the beginning of the reaction and the absorbance value A1 after 3 minutes of incubation are recorded, and the absorbance difference DA before and after the reaction is calculated. A standard curve is drawn based on the standard concentration and the absorbance difference, and the concentration of oxidized low-density lipoprotein in the sample is calculated.

6. The method for detecting oxidized low-density lipoprotein according to claim 2, characterized in that: in, A predetermined amount of hydrophilic monomer, hydrophobic monomer and monomer containing antioxidant functional group are mixed, an organic solvent is added, an inert gas is introduced to remove oxygen, a free radical initiator is added, and a free radical copolymerization reaction is carried out at a set temperature for a predetermined time, including: Adding predetermined amounts of polyethylene glycol methacrylate monomer, dodecyl methacrylate monomer, and vitamin E methacrylate monomer into a reaction vessel; Adding a predetermined amount of tetrahydrofuran solvent into the reaction container, and introducing an inert gas to perform deoxygenation treatment; adding a predetermined amount of azobisisobutyronitrile initiator to the deoxygenated reaction mixture; The reaction container is placed in a constant temperature heating device to carry out polymerization reaction, and inert gas protection is maintained throughout the reaction.

7. The method for detecting oxidized low-density lipoprotein according to claim 2, characterized in that: in, The reaction solution is poured into an excess of a precipitant to precipitate the target product, the precipitate is collected by centrifugation, repeatedly washed with the precipitant, and dried to a constant weight under reduced pressure to obtain a multifunctional polymer stabilizer, including: The reaction solution is poured into an excess of aprotic organic solvent to precipitate the polymer product; The obtained precipitate is collected by centrifugation and washed three times with an aprotic organic solvent; The washed product is placed in a vacuum drying device and dried at a relatively low temperature for a preset time to obtain a target multifunctional polymer stabilizer.

8. The method for detecting oxidized low-density lipoprotein according to claim 4, characterized in that: in, The method for calculating the efficiency of the stabilizer coating comprises: ， in, represents the stabilizer coating efficiency, represents the time-dependent reaction rate constant of the stabilizer concentration, ranging from 0.11 – 0.72 min^(-1)·(mL / mg), represents the stabilizer concentration, Indicates the wrapping time, It represents the reaction rate constant related to the molecular weight and hydrophilicity of the stabilizer, and its value range is 10^(-5) - 10^(-3) mol / g. Indicates the molecular weight of the stabilizer, represents the hydrophilicity influencing factor, Indicates the hydrophilicity index of the stabilizer.

9. An oxidized low-density lipoprotein detection device, characterized in that: include: A processor, a memory, and a system bus; wherein the processor and the memory are connected via the system bus; The memory is used to store one or more programs, wherein the one or more programs include instructions, and when the instructions are executed by the processor, the processor executes the method according to any one of claims 1 to 8.

Images