Stable oxidized low-density lipoprotein cholesterol detection kit and preparation method thereof

Abstract

The invention relates to the technical field of detection reagent preparation, and discloses a stable oxidized low-density lipoprotein cholesterol detection kit and a preparation method thereof.The kit is composed of a component R1 and a component R2, the component R1 at least comprises a buffer solution, a sensitizer, a surfactant, a stabilizer, an anti-interference agent and a preservative, and the component R2 at least comprises a buffer solution, a sensitizer, a surfactant, a stabilizer, an anti-interference agent and a preservative; the component R2 at least comprises an ion buffer solution, a stabilizer, a sealing agent, an antibody-carboxyl microsphere conjugate and a preservative; the antibody-carboxyl microsphere conjugate is a conjugate of an oxidized low-density lipoprotein antibody and carboxyl microspheres. According to the formula of the oxidized low-density lipoprotein reagent composition, the reagent R1 and the reagent R2, the production cost is effectively reduced, the oxidized low-density lipoprotein reagent composition can be used on a full-automatic biochemical analyzer, the automation degree is high, compared with similar products, the oxidized low-density lipoprotein reagent composition has higher accuracy, repeatability and stability, and unexpected technical effects are achieved.

Description

Technical Field

[0001] This invention relates to the field of reagent preparation technology, specifically to a stable oxidized low-density lipoprotein cholesterol detection kit and its preparation method. Background Technology

[0002] Oxidized low-density lipoprotein (oxLDL) is a product of the oxidation and modification of polyunsaturated fatty acids in low-density lipoprotein. In the field of cardiovascular disease risk assessment, oxLDL is a key pathogenic modified lipoprotein, and its accurate detection has significant clinical implications. However, existing detection technologies have many shortcomings, limiting their widespread application and the reliability of results. Traditional enzyme-linked immunosorbent assays (ELISA) are cumbersome and time-consuming, heavily reliant on manual operation, making high-throughput automated detection difficult. Furthermore, reagent stability is poor, with significant batch-to-batch variability, failing to meet the demands of modern clinical laboratories for rapid, efficient, and standardized testing. Although latex-enhanced immunoturbidimetric assays have been developed, achieving preliminary automated analysis, their core performance remains unsatisfactory. Existing methods generally suffer from insufficient reagent stability; antibodies are easily inactivated during storage, and latex microspheres are prone to non-specific aggregation or sedimentation, resulting in short reagent shelf life and signal drift. Simultaneously, these methods have weak anti-interference capabilities; common conditions in patient serum such as hyperlipidemia, hemolysis, and jaundice can easily introduce background interference into turbidity detection, causing false increases or decreases. More critically, existing methods lack optimization in the antibody-microsphere conjugation process, resulting in low conjugation efficiency and insufficient exposure of active sites. This leads to limited detection sensitivity, making it difficult to accurately measure low-concentration samples and achieve a broad linear range between sensitivity and detection upper limit. Furthermore, the core reaction system design of existing kits is inadequate; the buffer environment, polymerization conditions, and blocking scheme fail to create a synergistic effect, making it difficult to simultaneously meet high-end clinical requirements for both precision and accuracy.

[0003] Therefore, developing a highly stable, highly sensitive, highly interference-resistant, and fully automated oxidized low-density lipoprotein detection kit has become an urgent technical challenge. Summary of the Invention

[0004] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a stable oxidized low-density lipoprotein cholesterol detection kit and its preparation method. Technical solution

[0005] To achieve the above objectives, the present invention provides the following technical solution: A stable oxidized low-density lipoprotein cholesterol detection kit, comprising component R1 and component R2, wherein component R1 includes at least buffer, sensitizer, surfactant, stabilizer, anti-interference agent, and preservative, and component R2 includes at least ion buffer, stabilizer, blocking agent, antibody-carboxylic microsphere conjugate, and preservative. The antibody-carboxylic acid microsphere conjugate is an oxidized low-density lipoprotein antibody conjugate with carboxylic acid microspheres.

[0006] Furthermore, in component R1, the buffer solution is at least one of Tris-HCl buffer, glycine-NaOH solution, sodium glutamate solution, borate buffer, and carbonate-bicarbonate buffer.

[0007] More preferably, the buffer solution is a sodium glutamate solution with a concentration of 50-150 mmol / L.

[0008] In the preferred embodiment described above, the buffer solution is sodium glutamate solution, which maintains the pH value of the reaction system in a stable range of 8.0-9.0. This pH value is conducive to antigen-antibody binding and also helps to maintain the activity of each component.

[0009] Furthermore, in component R1, the sensitizer is at least one of polydextrose sulfate, polyethylene glycol, sodium heparin, dextran sulfate, and polyvinylpyrrolidone.

[0010] More preferably, in component R1, the sensitizer is polydextrose sulfate.

[0011] In the preferred technical solution described above, dextran sulfate is selected as a sensitizer. As a polymer with a large number of negative charges, it can neutralize the negative charge carried by oxLDL itself and reduce some of the negative charge on the surface of antibody microspheres, thereby reducing electrostatic repulsion. As a component in the system, dextran sulfate can synergistically promote the cross-linking and aggregation of immune complexes with surfactants and stabilizers, thereby significantly amplifying the turbidity signal and improving the detection sensitivity of the kit.

[0012] Furthermore, in component R1, the surfactant is at least one of poloxamer 188 (abbreviated as F68), Triton X-100, Tween-80, Tween-20, and sodium deoxycholate.

[0013] More preferably, the surfactant in component R1 is F68.

[0014] In the preferred technical solution described above, F68 is selected as the surfactant to prevent non-specific aggregation and adsorption of proteins and latex particles on the container surface. In the system, in conjunction with a sensitizer, it promotes specific aggregation while inhibiting non-specific aggregation, thereby improving reagent stability and result precision.

[0015] Furthermore, in component R1, the stabilizer is at least one of the following: protein, sugar, polyol, amino acid, metal chelating agent, and antioxidant.

[0016] The protein is at least one of casein, gelatin, protamine, and human serum albumin. The carbohydrate is at least one of trehalose, sucrose, and galactose. The amino acid is at least one of glycine, lysine, and taurine. The metal chelating agent is at least one of EDTA and EGTA. The antioxidant is at least one of ascorbic acid and glutathione.

[0017] More preferably, in component R1, the stabilizer, by mass, is a 10-15 mmol / L NaCl solution containing 0.50%-2.0% sodium octanoate, 0.05%-0.10% fetal bovine serum albumin (hereinafter referred to as BSA), and 0.20%-3.00% galactose.

[0018] In the preferred embodiment described above, the components of the stabilizer form a complex protective system. Sodium caprylate specifically stabilizes the antibody conformation, preventing denaturation during storage. BSA acts as an inert protein filler, reducing non-specific adsorption of other proteins in the sample and protecting the active ingredient. Galactose, as a carbohydrate protectant, stabilizes all protein components through preferential exclusion. The NaCl solution environment provides suitable ionic strength, optimizing the kinetics of antigen-antibody binding.

[0019] Furthermore, the preservative is at least one of antibiotics, isothiazolinones, sodium azide, sodium benzoate, and potassium sorbate.

[0020] Furthermore, in component R1, the antibiotic is at least one of ampicillin, streptomycin, and gentamicin.

[0021] More preferably, in component R1, the preservative is an HPO solution containing gentamicin, and the mass ratio of gentamicin to HPO is (0.01-0.05):(0.01-0.05).

[0022] In the above technical solution, the role of the preservative is to prevent the growth of microorganisms and ensure the long-term stability of the reagent.

[0023] Furthermore, in component R2, the surfactant is poloxamer 188.

[0024] Further, in component R2, the ion buffer is at least one of 1,4-piperazine diethanesulfonic acid (hereinafter referred to as PIPES) buffer, 3-morpholine propanesulfonic acid (hereinafter referred to as MOPS) buffer, 2-morpholine ethanesulfonic acid buffer, and phosphate buffer.

[0025] More preferably, the ion buffer is a PIPES buffer.

[0026] In the above technical solution, the ion buffer selected is PIPES buffer, which serves to provide a mild and stable pH environment for the conjugation of antibodies and microspheres and the long-term storage of the conjugate.

[0027] Furthermore, in component R2, the stabilizer is at least one of galactose, lactoferrin, BSA, casein, trehalose, sucrose, and surfactants.

[0028] More preferably, in component R2, the stabilizer is obtained by mixing galactose and lactoferrin in a mass ratio of (0.1-1):(0.1-0.2).

[0029] In the preferred embodiment described above, the stabilizer comprises galactose and lactoferrin. Lactoferrin, as a basic protein, acts as a high-level blocking agent, effectively blocking incompletely blocked sites on the microspheres and reducing non-specific binding to negatively charged substances such as DNA and acidic proteins in the sample. In addition to its protective effect on antibody activity as described above, galactose, combined with lactoferrin, further promotes antibody activity during long-term storage and strongly inhibits non-specific reactions, thereby improving the signal-to-noise ratio.

[0030] Furthermore, in component R2, the blocking agent is at least one of small molecule blocking agents, protein blocking agents, and charged polymer blocking agents.

[0031] Among them, the small molecule blocking agent is at least one of ethanolamine, glycine, and lysine; the protein blocking agent is at least one of casein and skim milk powder; and the charged polymer blocking agent is at least one of polyethyleneimine, protamine sulfate, and glucan sulfate.

[0032] More preferably, in component R2, the blocking agent is a 1-3 wt% glucan sulfate solution.

[0033] In the preferred embodiment described above, the blocking agent is used in the coupling step to covalently block the remaining carboxyl groups on the microspheres and convert them into positively charged groups, which helps to reduce non-specific adsorption and may assist the initial reaction through charge interaction.

[0034] Furthermore, in component R2, the preservative is an HPO solution containing kanamycin, and the mass ratio of kanamycin to HPO is 0.05:(0.05-0.1).

[0035] Furthermore, in component R2, the antibody-carboxylic acid microsphere conjugate is specifically prepared by the following steps: A1. The carboxyl-modified polystyrene microsphere suspension was centrifuged and washed with MOPS buffer and then resuspended to obtain a microsphere suspension; anti-human oxLDL monoclonal antibody was taken and diluted with MOPS buffer to obtain an antibody solution; the microsphere suspension and antibody solution were mixed in a centrifuge tube and the mixture was incubated at room temperature. A2. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as EDC) and N-hydroxysuccinimide (hereinafter referred to as NHS) solution to the mixture in A1, and react at room temperature in the dark for 1-2 hours. A3. After the reaction is complete, add BSA solution directly to the reaction system and incubate the system for 1-2 hours. A4. Transfer the blocked reaction solution from A3 to a centrifuge tube, add the coupling washing buffer and centrifuge. Discard the supernatant, repeat the steps of adding coupling washing buffer and centrifugation, and wash 2-3 times to obtain the antibody-carboxylic microsphere conjugate.

[0036] Furthermore, in component R2, the antibody-carboxylic acid microsphere conjugate is specifically prepared by the following steps: A1. Wash the 10% (w / v) carboxyl-modified polystyrene microsphere suspension twice with 20 mmol / L MOPS buffer at pH 7.0 by centrifugation at 12000 rpm for 20 minutes to remove the additives in the original storage solution; resuspend the washed microsphere precipitate in 20 mmol / L MOPS buffer at pH 7.0 to prepare a 2% (w / v) microsphere suspension; Take anti-human oxLDL monoclonal antibody and dilute it with the same MOPS buffer to a concentration between 0.5-2.0 mg / mL to obtain antibody solution; mix 1 volume of 2% microsphere suspension with 1-2 volumes of antibody solution in a centrifuge tube, place the mixture at room temperature (25°C), gently mix on a rotary mixer, and incubate for 15-30 minutes. A2. Prepare 0.4 M EDC and 0.1 M NHS solutions using MOPS buffer, and adjust the pH of the system to neutral. Store in an ice bath away from light. Add the freshly prepared EDC and NHS solutions to the mixture in A1 at a final EDC concentration of 10-20 mmol / L and a final NHS concentration of 2-5 mmol / L. Immediately transfer the reaction system to a rotary mixer at room temperature (25°C) and react in the dark for 1-2 hours. A3. After the reaction is complete, add 10% BSA solution directly to the reaction system to make its final concentration in the system reach 1-2 (v / v)%; place the system in a shaker at 37℃ and 200 rpm for 1-2 hours. A4. Transfer the blocked reaction solution from A3 to a centrifuge tube, add sufficient conjugation wash buffer (20 mmol / L MOPS buffer containing 0.1 wt% BSA and 0.05 wt% Tween-20), and mix thoroughly. Centrifuge at 4°C and 15000-20000 ×g for 20-30 minutes, discard the supernatant, and repeat the conjugation wash buffer and centrifugation steps 2-3 times to obtain the antibody-carboxylic acid microsphere conjugate.

[0037] In step A1 of the above technical solution, the antibody and microspheres are thoroughly mixed and equilibrated in a mild buffer system before coupling, preparing for subsequent covalent binding. In step A2, the carboxyl groups on the surface of the microspheres are activated using the cross-linking agent EDC to form an active O-acylisourea intermediate. This intermediate can spontaneously react with the primary amino groups on the antibody molecule to form stable amide bonds, thereby achieving covalent coupling. After the coupling reaction, a large number of activated but unreacted ester bonds remain on the microspheres. By adding a large amount of primary amino blocking agent (BSA) to the system, these active sites can be rapidly quenched, preventing them from non-specifically binding to irrelevant proteins or sample components in subsequent steps. Finally, in step A4, uncoupled free antibody, excess coupling agent, blocking agent, and reaction byproducts are thoroughly removed by centrifugation and washing to obtain a pure antibody-microsphere conjugate precipitate.

[0038] A method for preparing a stable oxidized low-density lipoprotein cholesterol detection kit, specifically including the following steps: S1. In deionized water, add buffer, stabilizer and sensitizer in sequence, stir until dissolved, then add surfactant and preservative, make up to volume with deionized water, adjust the pH of the system to 8.5 with 1 mol / L NaOH solution and 1 mol / L HCl solution, filter sterilize with 0.22 μm filter membrane, and store in the dark at 0-4℃ to obtain component R1. S2. Prepare component R2 according to the formula, then filter the system through a 0.22μm filter membrane for sterilization. After pre-cooling at 0-4℃, take 10-20 mL of the above R2 basic stock solution, add the antibody-carboxylic acid microsphere conjugate, and use a probe-type sonicator to sonicate three times under ice bath and low power conditions, each time for 10 seconds, with a 10-second interval, until the precipitate is completely resuspended and the solution is a homogeneous, semi-transparent emulsion. Transfer all the resuspended solution to a clean container and dilute it with R2 basic stock solution so that the concentration of antibody-microsphere conjugate in the final R2 reagent is 0.10%-0.30%, thus obtaining component R2. S3. Dispense components R1 and R2 into a double reagent bottle according to the preset volume ratio to obtain a stable oxidized low-density lipoprotein cholesterol detection kit.

[0039] In step S2 of the above preparation, the purified conjugate is resuspended in a storage buffer with optimized composition. PIPES provides a stable pH; galactose and lactoferrin synergistically stabilize antibody activity and enhance blocking; F68 prevents microsphere aggregation; and sonication ensures monodispersity.

[0040] Furthermore, the preset volume ratio is R1 component: R2 component = (2-4): 1.

[0041] Beneficial technical effects In the technical solution of this invention, the detection kit employs a composite stabilizer system. Specifically, sodium caprylate and galactose in reagent R1 work synergistically to effectively stabilize antibody activity and maintain the native conformation of proteins. Lactoferrin in reagent R2, as a high-performance blocking agent, strongly blocks non-specific binding sites on the microsphere surface. Combined with a dual buffer system composed of monosodium glutamate and PIPES, this ensures extremely low performance degradation of the reagents during storage and strong resistance to interfering substances such as lipids, bilirubin, hemoglobin, and other miscellaneous proteins commonly found in serum samples, thereby guaranteeing high specificity and accuracy of the detection results.

[0042] In the technical solution of this invention, by optimizing the molecular weight and concentration of the sulfated dextran sensitizer and using a strictly parameter-controlled EDC-NHS coupling process to prepare high-density, highly active antibody carboxyl microspheres, the formation efficiency of immune complexes is significantly improved, the signal amplification effect is obvious, and low concentrations of oxidized low-density lipoprotein in clinical samples can be accurately detected.

[0043] In the technical solution of this invention, the reagent kit is easy to operate and has good compatibility. Its dual-liquid reagent form and ready-to-use characteristics, combined with a clearly defined mixing ratio of R1 and R2, make it perfectly compatible with fully automated biochemical analyzers, realizing automation and high throughput of the detection process, greatly improving work efficiency, and reducing human error.

[0044] In the technical solution of this invention, the entire preparation process parameters are clearly defined and highly reproducible. From the ultrasonic resuspension conditions of microsphere coupling to the final product calibration, each step has quantitative standards, effectively ensuring the high uniformity of reagent kit performance between different production batches, laying a solid foundation for reliable comparison and long-term monitoring of clinical test results. Attached Figure Description

[0045] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is a graph showing the linear correlation results obtained after testing the sample using the reagent kit in Example 4 of this invention.

[0047] Figure 2 This is a graph showing the linear correlation results obtained after testing the sample in the reagent kit in Example 5 of this invention.

[0048] Figure 3 This is a graph showing the linear correlation results obtained after testing the sample in the reagent kit in Example 5 of this invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] The raw materials used in the embodiments of this invention are shown below, and all reagents used are analytical grade.

[0051] The carboxyl-modified polystyrene microspheres, with a particle size of 100-300 nm, were purchased from Suzhou Weidu Co., Ltd.; oxidized low-density lipoprotein antigen and antibody were purchased from Beijing Dacheng Biotechnology Co., Ltd.; F68 was purchased from Shanghai Xibao Biotechnology Co., Ltd.; sodium caprylate, galactose, and PIPES were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.; lactoferrin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; MOPS were purchased from Nanjing Xuanguang Technology Co., Ltd.; EDC and NHS were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; HPO: preservative, was purchased from Beijing Dacheng Biotechnology Co., Ltd.; the automated biochemical analyzer was a Hitachi 7180 automated biochemical analyzer.

[0052] The following Examples 1-3 provide a method for preparing antibody-carboxylic acid microsphere conjugates, as well as the products obtained by this method. The general steps are as follows: The antibody-carboxylic acid microsphere conjugate is prepared by the following steps: A1. Wash the 10% (w / v) carboxyl-modified polystyrene microsphere suspension twice with 20 mmol / L MOPS buffer at pH 7.0 by centrifugation at 12000 rpm for 20 minutes to remove the additives in the original storage solution; resuspend the washed microsphere precipitate in 20 mmol / L MOPS buffer at pH 7.0 to prepare a 2% (w / v) microsphere suspension; Take anti-human oxLDL monoclonal antibody and dilute it with the same MOPS buffer to a concentration of 1.0 mg / mL to obtain antibody solution; mix 1 volume of microsphere suspension with 1-2 volumes of antibody solution in a centrifuge tube, place the mixture at room temperature (25°C) and mix on a rotary mixer for 30 minutes; A2. Prepare 0.4 M EDC and 0.1 M NHS solutions using MOPS buffer, and adjust the pH of the system to neutral. Store in an ice bath away from light. Add the freshly prepared EDC and NHS solutions to the mixture in A1 at a final EDC concentration of 20 mmol / L and a final NHS concentration of 5 mmol / L. Immediately transfer the reaction system to a rotary mixer at room temperature (25°C) and react for 2 hours away from light. A3. After the reaction is complete, add 10% BSA solution directly to the reaction system to make its final concentration in the system reach 2 (v / v)%; place the system in a shaker at 37°C and 200 rpm for 2 hours. A4. Transfer the blocked reaction solution from A3 to a centrifuge tube, add sufficient conjugation wash buffer (20 mmol / L MOPS buffer, pH 7, containing 0.1 wt% BSA and 0.05 wt% Tween-20), and mix thoroughly. Centrifuge at 20000 ×g for 20 minutes at 4°C, discard the supernatant, and repeat the conjugation wash buffer and centrifugation steps three times to obtain the antibody-carboxylic acid microsphere conjugate.

[0053] Example 1

[0054] This embodiment provides a method for preparing an antibody-carboxylated microsphere conjugate and the product obtained by the method. Using the aforementioned general method, in step A1, the volume ratio of the microsphere suspension to the antibody solution is 1:1.

[0055] Example 2

[0056] This embodiment provides a method for preparing an antibody-carboxylated microsphere conjugate and the product obtained by the method. Using the aforementioned general method, in step A1, the volume ratio of the microsphere suspension to the antibody solution is 1:1.5.

[0057] Example 3

[0058] This embodiment provides a method for preparing an antibody-carboxylated microsphere conjugate and the product obtained by the method. Using the aforementioned general method, in step A1, the volume ratio of the microsphere suspension to the antibody solution is 1:2.

[0059] Examples 4-6 below provide a stable oxidized low-density lipoprotein cholesterol detection kit and its preparation method. The preparation method specifically adopts the following general steps: A method for preparing a stable oxidized low-density lipoprotein cholesterol detection kit, specifically including the following steps: S1. In deionized water, add buffer, stabilizer and sensitizer in sequence, stir until dissolved, then add surfactant and preservative, make up to volume with deionized water, adjust the pH of the system to 8.5 with 1 mol / L NaOH solution and 1 mol / L HCl solution, filter sterilize with 0.22 μm filter membrane, and store at 0℃ in the dark to obtain component R1. S2. Prepare component R2 according to the formula, then filter the system through a 0.22μm filter membrane for sterilization. After pre-cooling at 0℃ for 30 minutes, take 10-20 mL of the above R2 basic stock solution, add the antibody-carboxylic acid microsphere conjugate, and use a probe-type sonicator to sonicate three times at 150W power in an ice bath for 10 seconds each time, with a 10-second interval, until the precipitate is completely resuspended and the solution is a homogeneous, semi-transparent emulsion. Transfer all the resuspended solution to a clean container and dilute it with R2 basic stock solution so that the concentration of the antibody-microsphere conjugate in the final R2 reagent is 0.10%-0.30%, thus obtaining component R2. S3. Dispense components R1 and R2 in a 3:1 ratio into a double reagent bottle to obtain a stable oxidized low-density lipoprotein cholesterol detection kit.

[0060] Example 4

[0061] This embodiment provides a stable oxidized low-density lipoprotein cholesterol (LDL-C) assay kit, prepared using a general preparation method, with the pH of component R1 adjusted to 8.5 and component R2 adjusted to 7.5. The final content of each component in the kit product (calculated separately for components R1 and R2) is shown in Table 1 below.

[0062] Table 1

[0063] Example 5 This embodiment provides a stable oxidized low-density lipoprotein cholesterol (LDL-C) assay kit, prepared using a general preparation method, with the pH of component R1 adjusted to 8.5 and component R2 adjusted to 7.4. The final content of each component in the kit product (calculated separately for components R1 and R2) is shown in Table 2 below.

[0064] Table 2

[0065] Example 6 This embodiment provides a stable oxidized low-density lipoprotein cholesterol (LDL-C) assay kit, prepared using a general preparation method, with the pH of component R1 adjusted to 8.5 and component R2 adjusted to 7.4. The final content of each component in the kit product (calculated separately for components R1 and R2) is shown in Table 3 below.

[0066] Table 3

[0067] Control reagent: Xi'an Jinci Bio-oxidized low-density lipoprotein kit.

[0068] The performance of the kits used in Examples 4-6 and the control reagents was then tested. The specific test methods are as follows: Forty samples were randomly tested using kits obtained from different groups, and test values ​​and control values ​​for Examples 4-6 were obtained respectively. The units of test values ​​and control values ​​are μg / dl. The specific test results are shown in Table 4 below.

[0069] Table 4

[0070] As shown in Table 4, the linear correlation results corresponding to Examples 1-3 are as follows: Figure 1 As shown, the linear correlation curve for Example 4 is y = 1.0217x + 1.0004, and the correlation coefficient r is... 2 =0.9871; The linear correlation curve of Example 5 is y=1.0645x+0.1423, and the correlation coefficient r is 0.9871. 2 =0.9880; The linear correlation curve for Example 6 is y = 1.0287x + 0.3713, and the correlation coefficient r is 0.9880. 2 =0.9815; This indicates that the results obtained from the comparison tests of Examples 1-3 and the control reagent are all correlated.

[0071] The intra-batch precision of the samples in Examples 4-6 was then tested. Serum samples from different groups were tested at three concentration levels, with each serum sample tested 10 times. The mean and standard deviation (SD) were calculated. Parallel experiments were performed using control reagents. The results are shown in Table 5 below.

[0072] Table 5

[0073] As can be seen from the table above, the intra-assay precision of serum detection in Examples 1-3 of the present invention does not exceed 4%, while the intra-assay precision of serum detection by the control reagent cannot be guaranteed to be within the range of 5%.

[0074] The products obtained in Examples 4-6 are now subjected to stability testing. The specific testing methods are as follows: The products prepared in different groups were placed in a 37℃ water bath for 1, 3, 7, 14, and 28 days, respectively, and the serum samples were tested three times. Parallel comparisons were performed using a control reagent. The test results after accelerated testing at 37℃ are shown in Table 6.

[0075] Table 6

[0076] As shown in Table 6, compared with the control reagent, the CV of the reagent kit samples prepared in Examples 4-6 of this invention is less than 2.5% after being stored at 37°C for 28 days, indicating that the reagent kit samples prepared in Examples 4-6 can still maintain stability after being stored at 37°C for 28 days.

[0077] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0078] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0079] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments.

Claims

1. A stable oxidized low-density lipoprotein cholesterol detection kit, comprising component R1 and component R2, characterized in that, Component R1 includes at least a buffer, a sensitizer, a surfactant, a stabilizer, an anti-interference agent, and a preservative; component R2 includes at least an ion buffer, a stabilizer, a blocking agent, an antibody-carboxylic microsphere conjugate, and a preservative. The antibody-carboxylic acid microsphere conjugate is an oxidized low-density lipoprotein antibody conjugate with carboxylic acid microspheres.

2. The stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R1, the buffer solution is at least one of Tris-HCl buffer, glycine-NaOH solution, sodium glutamate solution, borate buffer, and carbonate-bicarbonate buffer.

3. The stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R1, the sensitizer is at least one of polydextrose sulfate, polyethylene glycol, sodium heparin, dextran sulfate, and polyvinylpyrrolidone.

4. The stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R1, the surfactant is at least one of poloxamer 188, Triton X-100, Tween-80, Tween-20, and sodium deoxycholate.

5. A stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R1, the stabilizer, by mass, is a 10-15 mmol / L NaCl solution containing 0.50%-2.0% sodium octanoate, 0.05%-0.10% fetal bovine serum albumin, and 0.20%-3.00% galactose.

6. The stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R1, the preservative is an HPO solution containing gentamicin, and the mass ratio of gentamicin to HPO is (0.01-0.05):(0.01-0.05).

7. A stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R2, the stabilizer is prepared by galactose and lactoferrin in a mass ratio of (0.1-1):(0.1-0.2).

8. A stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R2, the preservative is an HPO solution containing kanamycin, and the mass ratio of kanamycin to HPO is 0.05:(0.05-0.1).

9. A stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, characterized in that, In component R2, the antibody-carboxylic acid microsphere conjugate is specifically prepared by the following steps: A1. Wash and resuspend the carboxyl-modified polystyrene microsphere suspension to obtain a microsphere suspension; take the anti-human oxLDL monoclonal antibody, dilute it with buffer to obtain an antibody solution; mix the microsphere suspension and the antibody solution and incubate the mixture at room temperature. A2. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide solution to the mixture in A1, and react at room temperature in the dark for 1-2 hours. A3. After the reaction is complete, add fetal bovine serum albumin solution directly to the reaction system and incubate the system for 1-2 hours; A4. Transfer the reaction solution from A3 to a centrifuge tube, add the coupling washing buffer, and centrifuge. Discard the supernatant, and repeat the steps of adding coupling washing buffer and centrifugation. Wash 2-3 times to obtain the antibody-carboxylic microsphere conjugate.

10. A method for preparing a stable oxidized low-density lipoprotein cholesterol detection kit according to any one of claims 1-9, characterized in that, Includes the following steps: S1. In deionized water, add buffer, stabilizer and sensitizer in sequence, stir until dissolved, then add surfactant and preservative, bring the volume to a final volume with deionized water, adjust the pH of the system to 8-9, filter to sterilize, store in the dark, and obtain component R1. S2. Prepare component R2 according to the formula, then filter and sterilize the system. After pre-cooling at 0-4℃, obtain the basic R2 stock solution. Take 10-20 mL and add the antibody-carboxy microsphere conjugate to it. Sonicate in an ice bath to obtain a resuspension. Dilute the resuspension with the basic R2 stock solution so that the concentration of the antibody-microsphere conjugate in the final R2 reagent is 0.10%-0.30%, thus obtaining component R2. S3. Dispense components R1 and R2 into a double reagent bottle according to the preset volume ratio to obtain a stable oxidized low-density lipoprotein cholesterol detection kit.

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