Atherosclerosis risk assessment kit and assessment method

Abstract

The invention discloses an atherosclerosis risk assessment kit and an assessment method, the kit takes a proinflammatory-proregression oxidized lipid combination as a detection target, and comprises a core detection assembly, a sample pretreatment assembly and an auxiliary assembly; a specific monoclonal antibody in the core detection assembly has high specificity and high sensitivity, and can accurately and quantitatively detect the concentration of four key oxidized lipids in plasma; the sample pretreatment assembly can effectively remove impurities such as protein in plasma, and the detection accuracy is improved. According to the risk assessment method, early risk early warning of atherosclerosis is realized by calculating a proinflammatory / proregression lipid ratio and combining with each index concentration threshold value. According to the present invention, the proinflammatory-proregression oxidized lipid combination is adopted as the detection target, and the optimized ELISA detection method is combined, such that the early-stage, accurate and rapid risk evaluation can be performed on the atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a kit and method for assessing the risk of atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is a chronic vascular disease characterized by lipid deposition in the arterial intima, fibrosis, and inflammation. It is the main pathological basis for serious cardiovascular and cerebrovascular events such as coronary heart disease and cerebral infarction. Statistics show that more than 50% of cardiovascular and cerebrovascular deaths worldwide are related to atherosclerosis.

[0003] Early identification of atherosclerosis risk and timely intervention are key to reducing the incidence of cardiovascular and cerebrovascular events.

[0004] Currently, the diagnosis and risk assessment of atherosclerosis mainly rely on imaging examinations (such as carotid ultrasound and coronary CT angiography) and traditional biochemical indicators (such as blood lipids and C-reactive protein).

[0005] However, imaging examinations have a significant lag, and can only detect existing arterial plaques, failing to identify the early stages of vascular endothelial dysfunction.

[0006] Traditional biochemical indicators have low specificity and are easily affected by various factors such as diet, exercise, and liver disease, making it difficult to accurately reflect the early pathological process of atherosclerosis.

[0007] Therefore, developing detection technologies that can accurately assess the risk of atherosclerosis at an early stage has become an urgent clinical problem to be solved.

[0008] Imbalanced polyunsaturated fatty acid oxidation metabolism mediated by lipoxygenases (LOXs) is one of the key mechanisms in the development and progression of atherosclerosis. LOXs can catalyze the generation of two types of lipid oxides with opposite functions: pro-inflammatory lipid oxides (such as 5-HETE and 12-HETE) and pro-regression lipid oxides (such as LXA4 and RvD1).

[0009] In the early stages of atherosclerosis, abnormal activation of LOXs leads to the excessive production of pro-inflammatory lipid oxides and insufficient secretion of pro-regressive lipid oxides. This imbalance disrupts the balance between the two, thereby triggering vascular endothelial inflammation and lipid deposition, and promoting disease progression.

[0010] Studies have shown that changes in the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in circulation, as well as the pro-inflammatory / pro-remission ratio, can serve as potential metabolic biomarkers for the early diagnosis of atherosclerosis.

[0011] Existing methods for detecting oxidized lipids mainly include high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. These methods rely on expensive large-scale instruments, are complex to operate, and have long detection cycles, making them difficult to promote and apply in primary healthcare institutions.

[0012] Enzyme-linked immunosorbent assay (ELISA) has the advantages of being easy to operate, low in cost, and rapid in detection, making it suitable for routine clinical testing. However, there is currently a lack of multi-target lipid oxidative ELISA kits for early risk assessment of atherosclerosis, while single lipid oxidative assay kits have insufficient specificity.

[0013] Based on this, the present invention develops a detection kit targeting the combination of "pro-inflammatory and remission" oxidized lipids to achieve early and accurate risk assessment of atherosclerosis, filling the gap in existing technology. Summary of the Invention

[0014] The purpose of this invention is to provide a kit and method for assessing the risk of atherosclerosis. This invention uses a combination of pro-inflammatory and pro-regressive lipid oxides as detection targets, combined with an optimized ELISA detection method, offering the advantages of early, accurate, and rapid risk assessment of atherosclerosis.

[0015] The technical solution of the present invention:

[0016] An atherosclerosis risk assessment kit targets a combination of pro-inflammatory and pro-regressive oxidized lipids. The kit includes a core detection component, a sample pretreatment component, and auxiliary components. The combination of oxidized lipids includes pro-inflammatory oxidized lipids and pro-regressive oxidized lipids. The pro-inflammatory oxidized lipids include 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), and the pro-regressive oxidized lipids include lipoxygenin A4 (LXA4) and regressor D1 (RvD1).

[0017] The aforementioned atherosclerosis risk assessment kit includes the following core detection components: an enzyme-labeled plate, an enzyme-labeled secondary antibody, a standard kit, a chromogenic agent, and a stop solution.

[0018] In the aforementioned atherosclerosis risk assessment kit, the ELISA plate is specifically designed as follows: a 96-well detachable ELISA plate is used for antibody coating. Each detection well of the ELISA plate is independently coated with a specific monoclonal antibody against 5-HETE, 12-HETE, LXA4, and RvD1, with each antibody corresponding to 24 detection wells. The antibody coating concentration is 2-5 μg / mL, the coating buffer is carbonate buffer at pH 9.6, the coating volume is 100 μL / well, and the coating is incubated overnight (12-16 h) at 4°C. After coating, the plate is washed three times with washing buffer, patted dry, and then blocked with blocking buffer at 150 μL / well. The plate is blocked at 37°C for 2 h, vacuum dried, and then sealed and stored at 4°C.

[0019] In the aforementioned atherosclerosis risk assessment kit, the specific method for preparing the monoclonal antibody is as follows:

[0020] 1) Immunogen preparation: 5-HETE, 12-HETE, LXA4, and RvD1 were coupled with bovine serum albumin (BSA) via the carbodiimide method to prepare 5-HETE-BSA, 12-HETE-BSA, LXA4-BSA, and RvD1-BSA conjugates.

[0021] Coupling reaction conditions: molar ratio of oxidized lipids to BSA 20:1, carbodiimide concentration 50 mmol / L, pH 5.0, reaction at room temperature for 2 h; the conjugate was purified by dialysis (dialysis buffer was PBS buffer at pH 7.4) with a purity ≥95% and used for immunizing mice;

[0022] 2) Hybridoma cell preparation: Balb / c mice aged 6-8 weeks were immunized with the above four conjugates. The initial immunization dose was 100 μg / mouse, emulsified with Freund's complete adjuvant, and injected intraperitoneally. Two weeks later, a second immunization was performed with a dose of 100 μg / mouse, emulsified with Freund's incomplete adjuvant. Two weeks after the second immunization, a booster immunization was performed with a dose of 50 μg / mouse, without adjuvant, injected via the tail vein. Three days after the booster immunization, mouse spleen cells were fused with SP2 / 0 myeloma cells at a ratio of 5:1, and hybridoma cells were screened using HAT medium.

[0023] 3) Screening and cloning of positive hybridoma cells: Positive hybridoma cells that can specifically bind to target oxidized lipids were screened using indirect ELISA. The cells were then cultured three times using the limiting dilution method to obtain monoclonal hybridoma cell lines. The antibody titer was determined using indirect ELISA, and hybridoma cell lines with a titer ≥1:100000 were selected for antibody preparation.

[0024] 4) Antibody purification: Positive hybridoma cells were intraperitoneally inoculated into Balb / c mice, ascites fluid was collected, and ascites antibodies were purified using a Protein G affinity chromatography column;

[0025] Purification process: Ascites fluid was centrifuged at 5000 r / min for 10 min to remove impurities. The chromatography column was equilibrated with Binding Buffer, and after loading the sample, impurities were washed with Washing Buffer. The target antibody was eluted with Elution Buffer, and the eluent was desalted by dialysis (dialysis buffer was PBS buffer at pH 7.4). The purified antibody was obtained after freeze-drying. The purity of the purified antibody was ≥95% (verified by SDS-PAGE electrophoresis), and the titer was ≥1:100000.

[0026] In the aforementioned atherosclerosis risk assessment kit, the enzyme-labeled secondary antibodies are: four horseradish peroxidase (HRP)-labeled secondary antibodies targeting the above-mentioned specific monoclonal antibodies, labeled using the sodium periodate method;

[0027] Labeling conditions: The molar ratio of HRP to secondary antibody was 2:1, pH 7.4, and the reaction was carried out at room temperature for 2 hours. After labeling, the secondary antibody was purified by dialysis to obtain enzyme-labeled secondary antibody at a concentration of 1:5000-1:10000. Each vial contained 1 mL of 0.02% sodium azide preservative and was stored in a sealed container at 4°C.

[0028] The aforementioned atherosclerosis risk assessment kit contains the following standard kit: 5-HETE, 12-HETE, LXA4, and RvD1 standards, with an initial concentration of 100 ng / mL, dissolved in a methanol solution containing 0.1% BHT; each 200 μL vial should be sealed and stored at -20℃ to avoid repeated freeze-thaw cycles.

[0029] In the aforementioned atherosclerosis risk assessment kit, the colorimetric reagent is a TMB substrate solution, consisting of solution A and solution B, with a volume ratio of 1:1, 5 mL per vial; solution A is a TMB stock solution with a concentration of 1 mg / mL, dissolved in anhydrous ethanol, and solution B is a urea peroxide solution with a concentration of 0.02 mol / L. After mixing, store at 4°C protected from light and use within 24 hours.

[0030] Termination solution: 2 mol / L sulfuric acid solution, prepared with analytical grade sulfuric acid, colorless and transparent liquid, 5 mL per bottle, sealed and stored at room temperature.

[0031] In the aforementioned atherosclerosis risk assessment kit, the sample pretreatment component includes:

[0032] Protein precipitation solution: a methanol solution containing 0.1% BHT. BHT is an antioxidant that can prevent oxidized lipids from being oxidized during pretreatment. Methanol is used to precipitate proteins in plasma and remove interference from proteins in the detection. 10 mL per bottle, sealed and stored at 4°C.

[0033] Extraction solution: n-hexane, used to extract oxidized lipids in samples and improve detection sensitivity; 10 mL per bottle, store sealed at room temperature;

[0034] Reconstitution buffer: PBS buffer at pH 7.4, containing 0.5% BSA and 0.05% Tween-20; BSA can reduce the non-specific adsorption of oxidized lipids, and Tween-20 can improve the solubility of oxidized lipids; 5 mL per bottle, sealed and stored at 4°C;

[0035] Internal standard solution: deuterated 5-HETE solution, concentration 10 ng / mL, dissolved in methanol solution containing 0.1% BHT; deuterated 5-HETE has similar physicochemical properties to 5-HETE and can be used to correct errors in sample pretreatment and detection processes, improving the accuracy of detection results; 100 μL per vial, sealed and stored at -20℃.

[0036] In the aforementioned atherosclerosis risk assessment kit, the auxiliary components include:

[0037] Washing buffer: PBS buffer at pH 7.4 containing 0.05% Tween-20; Tween-20 enhances washing effect and removes unbound antigens and antibodies; 50 mL per bottle, store sealed at room temperature, dilute 10 times before use;

[0038] Blocking solution: PBS buffer containing 5% skim milk powder. Skim milk powder can block non-specific binding sites on the ELISA plate and reduce background signal; 10 mL per bottle, sealed and stored at 4°C, equilibrate to room temperature before use;

[0039] Sample dilution buffer: PBS buffer at pH 7.4 containing 0.1% BSA, used to dilute plasma samples to avoid excessively high sample concentrations exceeding the detection range; 10 mL per vial, sealed and stored at 4°C;

[0040] Instructions: Provides detailed information on the composition of the kit, storage conditions for each component, usage methods (sample pretreatment steps, ELISA detection steps), result interpretation criteria, precautions, and quality control standards.

[0041] A method for assessing the risk of atherosclerosis includes the following steps:

[0042] S1. Sample pretreatment: Take plasma samples and add internal standard solution, protein precipitation solution and extraction solution in sequence, and vortex. Take the upper organic phase. Dry the organic phase with nitrogen, add reconstitution buffer, vortex and centrifuge, and take the supernatant as the sample to be tested.

[0043] S2, ELISA detection: After equilibrating the microplate to room temperature, add blocking buffer and incubate; discard the blocking buffer and wash; add the sample to be tested and the standard to the corresponding test well, incubate, and discard the liquid in the well; add washing buffer and wash; add the corresponding enzyme-labeled secondary antibody and incubate; discard the liquid in the well and wash; add chromogenic reagent and incubate; finally, add stop solution to terminate the reaction.

[0044] S3. Result detection and calculation: The absorbance value of each well was read at a wavelength of 450nm using an ELISA reader; a standard curve was plotted based on the absorbance value of the standard; the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in the sample to be tested were calculated; and the pro-inflammatory / pro-remission lipid ratio was calculated.

[0045] S4. Risk Assessment: The risk level of atherosclerosis is determined based on the ratio of pro-inflammatory to remission lipids and the concentration of oxidized lipids, combined with preset thresholds.

[0046] The aforementioned method for assessing the risk of atherosclerosis also includes sample collection and preservation: collect 5 mL of peripheral venous blood from the subject after fasting for more than 12 hours, place it in an EDTA anticoagulant tube, gently invert and mix 3-5 times, centrifuge at 4℃ and 3000r / min for 10 min, separate the plasma, and store it in a -80℃ freezer for later use, avoiding repeated freeze-thaw cycles (≤3 times).

[0047] In the aforementioned method for assessing the risk of atherosclerosis, the specific details of step S1 are as follows:

[0048] S1.1 Take the frozen plasma sample, thaw it at room temperature, and centrifuge it at 4℃ and 5000r / min for 5min to remove the precipitate;

[0049] S1.2 Take 200 μL of supernatant and place it in a 1.5 mL centrifuge tube. Add 20 μL of internal standard solution and vortex for 30 s (vortexing frequency 2000 r / min).

[0050] S1.3 Add 400 μL of protein precipitation solution, vortex for 30 seconds, and let stand in an ice bath for 10 minutes to allow the protein to precipitate fully.

[0051] S1.4 Add 200 μL of extraction solution, vortex for 1 min, centrifuge at 4℃ and 12000 r / min for 10 min (centrifugation radius 8 cm), and carefully aspirate the upper organic phase into a new centrifuge tube;

[0052] S1.5 Place the centrifuge tubes in a nitrogen dryer and dry the organic phase at ≤30℃ and 0.1MPa nitrogen pressure;

[0053] S1.6 Add 100 μL of reconstitution buffer, vortex for 1 min, centrifuge at 4℃ and 12000 r / min for 5 min, and take the supernatant as the sample to be tested.

[0054] In the aforementioned method for assessing the risk of atherosclerosis, step S2 specifically includes the following:

[0055] S2.1 Remove the antibody-coated ELISA plate from the 4°C freezer and allow it to equilibrate at room temperature for 30 minutes;

[0056] S2.2 Add blocking solution, 150 μL / well, and incubate at 37°C for 30 min;

[0057] S2.3 Discard the blocking solution, add 200 μL of diluted washing solution per well, soak for 30 seconds, discard the washing solution, repeat the washing 5 times, and pat dry;

[0058] S2.4 Add the sample to be tested and the standard to the corresponding well of the ELISA plate, 100 μL / well, with 3 replicates for each sample, and set up blank control wells (add 100 μL of reconstitution buffer).

[0059] S2.5 Place the ELISA plate in a 37°C incubator for 60 minutes, avoiding shaking during incubation;

[0060] S2.6 After incubation, discard the liquid in the well, add washing solution and wash 5 times, 30 seconds each time, then pat dry;

[0061] S2.7 Add the corresponding enzyme-labeled secondary antibody, 100 μL / well, and incubate at 37°C for 30 min;

[0062] S2.8 Repeat the washing process of step S2.6;

[0063] S2.9 Add freshly mixed colorimetric reagent, 100 μL / well, and incubate at 37°C in the dark for 15 min;

[0064] Add stop solution (50 μL / well) to S2.10, gently shake the plate for 5 seconds to stop the reaction.

[0065] In the aforementioned method for assessing the risk of atherosclerosis, step S3 specifically includes the following:

[0066] S3.1 Within 10 minutes after the reaction is terminated, the absorbance value of each well is read at a wavelength of 450 nm using an ELISA reader. The reference wavelength is 570 nm. The average value is taken as the final absorbance value.

[0067] S3.2 After subtracting the absorbance values ​​of the blank control wells, a standard curve is plotted using a four-parameter fitting method with the standard concentration as the x-axis and the absorbance values ​​as the y-axis, and the correlation coefficient R² ≥ 0.99; the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in the sample to be tested are calculated based on the standard curve.

[0068] S3.3 Calculate the pro-inflammatory / pro-remission lipid ratio: pro-inflammatory / pro-remission lipid ratio = (5-HETE concentration + 12-HETE concentration) / (LXA4 concentration + RvD1 concentration).

[0069] In the aforementioned method for assessing the risk of atherosclerosis, step S4 specifically includes the following:

[0070] The risk level of atherosclerosis is determined based on the ratio of pro-inflammatory to pro-remission lipids and the concentration of each oxidized lipid, combined with preset thresholds.

[0071] Low risk: Meeting the criteria of pro-inflammatory / pro-remission lipid ratio <2.0, 5-HETE <50pg / mL, 12-HETE <40pg / mL, LXA4 >20pg / mL and RvD1 >15pg / mL;

[0072] Medium risk: 2.0 ≤ pro-inflammatory / pro-remission lipid ratio < 5.0 or any oxidized lipid concentration exceeding the low risk range;

[0073] High risk: Pro-inflammatory / remission lipid ratio ≥5.0.

[0074] Compared with the prior art, the beneficial effects of this application are as follows:

[0075] 1. Significant advantages of early diagnosis

[0076] This invention selects a combination of "pro-inflammatory and pro-remission" oxidized lipids as detection targets. Studies have found that these indicators show significant abnormalities in the early stage of atherosclerosis (the stage of vascular endothelial function damage), enabling early risk warning of the disease and solving the problem of the lag in existing imaging diagnostic methods. Clinical trials have shown that the detection sensitivity of this kit for early atherosclerosis is significantly better than that of traditional blood lipid indicators.

[0077] 2. High detection specificity

[0078] The use of a combination of four key oxidized lipid biomarkers, rather than a single biomarker, can comprehensively reflect the oxidized lipid metabolism imbalance mediated by LOXes, avoiding the problem of insufficient specificity in single biomarker detection. The specific monoclonal antibody prepared in this invention has a cross-reactivity rate of <5% with the target oxidized lipids, further improving the detection specificity.

[0079] 3. Easy to operate and highly adaptable

[0080] The optimized ELISA detection method, coupled with a dedicated sample pretreatment component, simplifies the detection process; it eliminates the need for expensive large-scale instruments, has a short detection cycle, and can be routinely performed in primary healthcare institutions, solving the problem of the difficulty in promoting existing lipid oxidation detection technologies.

[0081] 4. Results are accurate and have good repeatability.

[0082] The detection error was corrected by adding an internal standard solution, and a standard curve was plotted using four-parameter fitting to ensure the accuracy of the detection results. Each component of the kit underwent strict quality control, with intra-batch coefficient of variation <5% and inter-batch coefficient of variation <8%, demonstrating excellent repeatability.

[0083] 5. Accurate risk assessment

[0084] By combining the pro-inflammatory / pro-remission lipid ratio and the concentration thresholds of various indicators for risk stratification, accurate staging of atherosclerosis can be achieved, providing a reliable basis for the formulation of clinical intervention plans and contributing to individualized treatment. Attached Figure Description

[0085] Figure 1 This is a flowchart illustrating the screening method of the present invention. Detailed Implementation

[0086] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0087] Example

[0088] An atherosclerosis risk assessment kit targets a combination of pro-inflammatory and pro-regressive oxidized lipids. The kit includes a core detection component, a sample pretreatment component, and auxiliary components. The combination of oxidized lipids includes pro-inflammatory oxidized lipids and pro-regressive oxidized lipids. The pro-inflammatory oxidized lipids include 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), and the pro-regressive oxidized lipids include lipoxygenin A4 (LXA4) and regressor D1 (RvD1).

[0089] The core detection components include: ELISA plate, enzyme-labeled secondary antibody, standard kit, chromogenic reagent, and stop solution.

[0090] The specific details of the ELISA plate are as follows: A 96-well detachable ELISA plate is used for antibody coating. Each detection well of the ELISA plate is independently coated with a specific monoclonal antibody against 5-HETE, 12-HETE, LXA4, and RvD1, with 24 detection wells for each antibody. The antibody coating concentration is 2-5 μg / mL (optimized by checkerboard titration method, with 3 μg / mL for 5-HETE, 4 μg / mL for 12-HETE, 3 μg / mL for LXA4, and 5 μg / mL for RvD1). The coating buffer is carbonate buffer at pH 9.6, and the coating volume is 100 μL / well. Coating is carried out overnight (12-16 h) at 4°C. After coating, the plate is washed three times with washing buffer, patted dry, and then 150 μL / well of blocking buffer is added. The plate is blocked at 37°C for 2 h, vacuum dried, and then sealed and stored at 4°C.

[0091] The specific method for preparing the monoclonal antibody is as follows:

[0092] 1) Immunogen preparation: 5-HETE, 12-HETE, LXA4, and RvD1 were coupled with bovine serum albumin (BSA) via the carbodiimide method to prepare 5-HETE-BSA, 12-HETE-BSA, LXA4-BSA, and RvD1-BSA conjugates.

[0093] Coupling reaction conditions: molar ratio of oxidized lipids to BSA 20:1, carbodiimide concentration 50 mmol / L, pH 5.0, reaction at room temperature for 2 h; the conjugate was purified by dialysis (dialysis buffer was PBS buffer at pH 7.4) with a purity ≥95% and used for immunizing mice;

[0094] 2) Hybridoma cell preparation: Balb / c mice aged 6-8 weeks were immunized with the above four conjugates. The initial immunization dose was 100 μg / mouse, emulsified with Freund's complete adjuvant, and injected intraperitoneally. Two weeks later, a second immunization was performed with a dose of 100 μg / mouse, emulsified with Freund's incomplete adjuvant. Two weeks after the second immunization, a booster immunization was performed with a dose of 50 μg / mouse, without adjuvant, injected via the tail vein. Three days after the booster immunization, mouse spleen cells were fused with SP2 / 0 myeloma cells at a ratio of 5:1, and hybridoma cells were screened using HAT medium.

[0095] 3) Screening and cloning of positive hybridoma cells: Positive hybridoma cells that can specifically bind to target oxidized lipids were screened using indirect ELISA. The cells were then cultured three times using the limiting dilution method to obtain monoclonal hybridoma cell lines. The antibody titer was determined using indirect ELISA, and hybridoma cell lines with a titer ≥1:100000 were selected for antibody preparation.

[0096] 4) Antibody purification: Positive hybridoma cells were intraperitoneally inoculated into Balb / c mice, ascites fluid was collected, and ascites antibodies were purified using a Protein G affinity chromatography column;

[0097] Purification process: Ascites fluid was centrifuged at 5000 r / min for 10 min to remove impurities. The chromatography column was equilibrated with Binding Buffer, and after loading the sample, impurities were washed with Washing Buffer. The target antibody was eluted with Elution Buffer, and the eluent was desalted by dialysis (dialysis buffer was PBS buffer at pH 7.4). The purified antibody was obtained after freeze-drying. The purity of the purified antibody was ≥95% (verified by SDS-PAGE electrophoresis), and the titer was ≥1:100000.

[0098] The enzyme-labeled secondary antibodies were: four horseradish peroxidase (HRP)-labeled secondary antibodies targeting the above-mentioned specific monoclonal antibodies, labeled using the sodium periodate method;

[0099] Labeling conditions: The molar ratio of HRP to secondary antibody was 2:1, pH 7.4, and the reaction was carried out at room temperature for 2 hours. After labeling, the secondary antibody was purified by dialysis to obtain enzyme-labeled secondary antibodies at concentrations of 1:5000-1:10000 (5-HETE secondary antibody dilution ratio 1:8000, 12-HETE secondary antibody dilution ratio 1:7000, LXA4 secondary antibody dilution ratio 1:9000, and RvD1 secondary antibody dilution ratio 1:6000). Each vial contained 1 mL of 0.02% sodium azide preservative and was stored in a sealed container at 4°C.

[0100] The standard kit contains 5-HETE, 12-HETE, LXA4, and RvD1 standards, all chromatographically pure standards with a purity ≥98% (purchased from Cayman), with an initial concentration of 100 ng / mL, dissolved in a solution containing 0.1%... BHT is prepared in a methanol solution; each standard is prepared in six gradient concentrations: 5-HETE (10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL), 12-HETE (5 pg / mL, 10 pg / mL, 30 pg / mL, 60 pg / mL, 120 pg / mL, 300 pg / mL), LXA4 (2 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL), and RvD1 (1 pg / mL, 3 pg / mL, 8 pg / mL, 15 pg / mL, 30 pg / mL, 80 pg / mL), 200 μL per vial, sealed and stored at -20°C, avoiding repeated freeze-thaw cycles.

[0101] Colorimetric reagent: is a TMB substrate solution, consisting of solution A and solution B in a 1:1 volume ratio, 5 mL per vial; solution A is a TMB stock solution with a concentration of 1 mg / mL, dissolved in anhydrous ethanol, and solution B is a urea peroxide solution with a concentration of 0.02 mol / L. After mixing, store at 4°C protected from light and use within 24 hours.

[0102] Termination solution: 2 mol / L sulfuric acid solution, prepared with analytical grade sulfuric acid, colorless and transparent liquid, 5 mL per bottle, sealed and stored at room temperature.

[0103] The sample preprocessing component includes:

[0104] Protein precipitation solution: a methanol solution containing 0.1% BHT. BHT is an antioxidant that can prevent oxidized lipids from being oxidized during pretreatment. Methanol is used to precipitate proteins in plasma and remove interference from proteins in the detection. 10 mL per bottle, sealed and stored at 4°C.

[0105] Extraction solution: n-hexane, chromatographic grade (purity ≥99.9%), used to extract oxidized lipids from samples and improve detection sensitivity; 10 mL per bottle, sealed and stored at room temperature;

[0106] Reconstitution buffer: PBS buffer at pH 7.4, containing 0.5% BSA and 0.05% Tween-20; BSA can reduce the non-specific adsorption of oxidized lipids, and Tween-20 can improve the solubility of oxidized lipids; 5 mL per bottle, sealed and stored at 4°C;

[0107] Internal standard solution: deuterated 5-HETE solution, concentration 10 ng / mL, dissolved in methanol solution containing 0.1% BHT; deuterated 5-HETE has similar physicochemical properties to 5-HETE and can be used to correct errors in sample pretreatment and detection processes, improving the accuracy of detection results; 100 μL per vial, sealed and stored at -20℃.

[0108] The auxiliary components include:

[0109] Washing buffer: PBS buffer at pH 7.4 containing 0.05% Tween-20; Tween-20 enhances washing effect and removes unbound antigens and antibodies; 50 mL per bottle, store sealed at room temperature, dilute 10 times before use;

[0110] Blocking solution: PBS buffer containing 5% skim milk powder. Skim milk powder can block non-specific binding sites on the ELISA plate and reduce background signal; 10 mL per bottle, sealed and stored at 4°C, equilibrate to room temperature before use;

[0111] Sample dilution buffer: PBS buffer at pH 7.4 containing 0.1% BSA, used to dilute plasma samples to avoid excessively high sample concentrations exceeding the detection range; 10 mL per vial, sealed and stored at 4°C;

[0112] Instructions: Provides detailed information on the composition of the kit, storage conditions for each component, usage methods (sample pretreatment steps, ELISA detection steps), result interpretation criteria, precautions, and quality control standards.

[0113] A method for assessing the risk of atherosclerosis includes the following steps:

[0114] S1. Sample pretreatment: Take plasma samples and add internal standard solution, protein precipitation solution and extraction solution in sequence, and vortex. Take the upper organic phase. Dry the organic phase with nitrogen, add reconstitution buffer, vortex and centrifuge, and take the supernatant as the sample to be tested.

[0115] S2, ELISA detection: After equilibrating the microplate to room temperature, add blocking buffer and incubate; discard the blocking buffer and wash; add the sample to be tested and the standard to the corresponding test well, incubate, and discard the liquid in the well; add washing buffer and wash; add the corresponding enzyme-labeled secondary antibody and incubate; discard the liquid in the well and wash; add chromogenic reagent and incubate; finally, add stop solution to terminate the reaction.

[0116] S3. Result detection and calculation: The absorbance value of each well was read at a wavelength of 450nm using an ELISA reader; a standard curve was plotted based on the absorbance value of the standard; the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in the sample to be tested were calculated; and the pro-inflammatory / pro-remission lipid ratio was calculated.

[0117] S4. Risk Assessment: The risk level of atherosclerosis is determined based on the ratio of pro-inflammatory to remission lipids and the concentration of oxidized lipids, combined with preset thresholds.

[0118] It also includes sample collection and preservation: Collect 5 mL of peripheral venous blood from subjects who have fasted for more than 12 hours, place it in an EDTA anticoagulant tube, gently invert and mix 3-5 times, centrifuge at 4℃ and 3000 r / min for 10 min, separate the plasma, and store it in a -80℃ freezer for later use, avoiding repeated freeze-thaw cycles (≤3 times).

[0119] The specific details of step S1 are as follows:

[0120] S1.1 Take the frozen plasma sample, thaw it at room temperature, and centrifuge it at 4℃ and 5000r / min for 5min to remove the precipitate;

[0121] S1.2 Take 200 μL of supernatant and place it in a 1.5 mL centrifuge tube. Add 20 μL of internal standard solution and vortex for 30 s (vortexing frequency 2000 r / min).

[0122] S1.3 Add 400 μL of protein precipitation solution, vortex for 30 seconds, and let stand in an ice bath for 10 minutes to allow the protein to precipitate fully.

[0123] S1.4 Add 200 μL of extraction solution, vortex for 1 min, centrifuge at 4℃ and 12000 r / min for 10 min (centrifugation radius 8 cm), and carefully aspirate the upper organic phase into a new centrifuge tube;

[0124] S1.5 Place the centrifuge tubes in a nitrogen dryer and dry the organic phase at ≤30℃ and 0.1MPa nitrogen pressure;

[0125] S1.6 Add 100 μL of reconstitution buffer, vortex for 1 min, centrifuge at 4℃ and 12000 r / min for 5 min, and take the supernatant as the sample to be tested.

[0126] The specific details of step S2 are as follows:

[0127] S2.1 Remove the antibody-coated ELISA plate from the 4°C freezer and allow it to equilibrate at room temperature for 30 minutes;

[0128] S2.2 Add blocking solution, 150 μL / well, and incubate at 37°C for 30 min;

[0129] S2.3 Discard the blocking solution, add 200 μL of diluted washing solution per well, soak for 30 seconds, discard the washing solution, repeat the washing 5 times, and pat dry;

[0130] S2.4 Add the sample to be tested and the standard to the corresponding well of the ELISA plate, 100 μL / well, with 3 replicates for each sample, and set up blank control wells (add 100 μL of reconstitution buffer).

[0131] S2.5 Place the ELISA plate in a 37°C incubator for 60 minutes, avoiding shaking during incubation;

[0132] S2.6 After incubation, discard the liquid in the well, add washing solution and wash 5 times, 30 seconds each time, then pat dry;

[0133] S2.7 Add the corresponding enzyme-labeled secondary antibody, 100 μL / well, and incubate at 37°C for 30 min;

[0134] S2.8 Repeat the washing process of step S2.6;

[0135] S2.9 Add freshly mixed colorimetric reagent, 100 μL / well, and incubate at 37°C in the dark for 15 min;

[0136] Add stop solution (50 μL / well) to S2.10, gently shake the plate for 5 seconds to stop the reaction.

[0137] The specific details of step S3 are as follows:

[0138] S3.1 Within 10 minutes after the reaction is terminated, the absorbance value of each well is read at a wavelength of 450 nm using an ELISA reader. The reference wavelength is 570 nm. The average value is taken as the final absorbance value.

[0139] S3.2 After subtracting the absorbance values ​​of the blank control wells, a standard curve is plotted using a four-parameter fitting method with the standard concentration as the x-axis and the absorbance values ​​as the y-axis, and the correlation coefficient R² ≥ 0.99; the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in the sample to be tested are calculated based on the standard curve.

[0140] S3.3 Calculate the pro-inflammatory / pro-remission lipid ratio: pro-inflammatory / pro-remission lipid ratio = (5-HETE concentration + 12-HETE concentration) / (LXA4 concentration + RvD1 concentration).

[0141] The specific details of step S4 are as follows:

[0142] The risk level of atherosclerosis is determined based on the ratio of pro-inflammatory to pro-remission lipids and the concentration of each oxidized lipid, combined with preset thresholds.

[0143] Low risk: Meeting the criteria of pro-inflammatory / pro-remission lipid ratio <2.0, 5-HETE <50 pg / mL, 12-HETE <40 pg / mL, LXA4 >20 pg / mL and RvD1 >15 pg / mL; indicating no atherosclerosis or being in the early subclinical stage;

[0144] Medium risk: 2.0 ≤ pro-inflammatory / pro-remission lipid ratio < 5.0 or any oxidized lipid concentration exceeding the low risk range; suggests possible progression of atherosclerosis, requiring further imaging examinations;

[0145] High risk: A pro-inflammatory / pro-remission lipid ratio ≥5.0 indicates late-stage atherosclerosis and a high risk of cardiovascular and cerebrovascular events, requiring immediate clinical intervention.

[0146] Verification Experiment

[0147] I. Experimental Materials

[0148] 1. Research subjects:

[0149] Three hundred and sixty participants who visited the cardiology department and health check-up center of a tertiary hospital between March 2023 and March 2024 were selected. All participants signed informed consent forms. Based on the clinical diagnostic criteria of relevant guidelines, and combined with the results of carotid ultrasound and coronary CT angiography, participants were divided into three groups: a healthy control group (n=120, without atherosclerosis, hypertension, diabetes, hyperlipidemia, or other risk factors), and an early-stage AS group (n=120, carotid plaque area <0.1 cm²). 2 (No obvious clinical symptoms) and advanced AS group (120 cases, carotid artery plaque area ≥0.5cm²). 2 (Or a cardiovascular or cerebrovascular event has already occurred).

[0150] Inclusion criteria: (1) Age 40-70 years; (2) Ability to cooperate in blood sample collection; (3) Complete clinical data.

[0151] Exclusion criteria: (1) Comorbid severe liver disease, kidney disease, malignant tumor, autoimmune disease, or infectious disease; (2) Recent (within 3 months) history of surgery, trauma, or blood transfusion; (3) Currently using anti-inflammatory drugs, immunosuppressants, or lipid-lowering drugs; (4) Pregnant or lactating women.

[0152] 2. Reagents and Instruments:

[0153] 5-HETE, 12-HETE, LXA4, RvD1 standards (Cayman), deuterated 5-HETE (Cayman), BSA (Sigma), Freund's complete adjuvant, Freund's incomplete adjuvant (Sigma), SP2 / 0 myeloma cells (Chinese Academy of Sciences Cell Bank), Balb / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), Protein G affinity chromatography column (GE Healthcare), TMB substrate (Sigma), 96-well microplate (Corning).

[0154] ELISA reader (Bio-Rad, model: iMark), high-speed refrigerated centrifuge (Eppendorf, model: 5418R), nitrogen dryer (Organomation, model: N-EVAP 112), incubator (Shanghai Yuejin Medical Instrument Co., Ltd., model: HH-B11.600), carotid ultrasound diagnostic instrument (Philips, model: EPIQ 7C), coronary CT angiography system (Siemens, model: SOMATOM Force).

[0155] II. Experimental Methods

[0156] 1. Kit preparation: According to the parameters in the technical solution of this invention, an oxidized lipid detection kit for atherosclerosis risk assessment is prepared, including a core detection component, a sample pretreatment component and auxiliary components.

[0157] 2. Sample testing: Plasma samples from 360 subjects were tested using the kit of this invention. Each sample was tested in triplicate, and parallel detection was performed using liquid chromatography-mass spectrometry (LC-MS / MS). The LC-MS / MS results were used as the gold standard to verify the accuracy of the kit.

[0158] 3. Performance Verification:

[0159] (1) Sensitivity and specificity: The sensitivity, specificity, positive predictive value, negative predictive value and area under the ROC curve (AUC) of this kit for detecting atherosclerosis were calculated and compared with the traditional blood lipid index (LDL-C).

[0160] (2) Repeatability: Ten plasma samples of different concentrations were selected and tested three times using the same batch of reagent kits (intra-batch repeatability), and tested using three different batches of reagent kits (inter-batch repeatability). The coefficient of variation was calculated.

[0161] (3) Stability: The kit was stored at 4℃, 25℃ and 37℃ respectively. The absorbance of the standard was measured after 0 days, 7 days, 14 days, 21 days and 28 days of storage. The changes in the standard curve were observed to evaluate the stability of the kit.

[0162] 4. Statistical Analysis: SPSS 26.0 statistical software was used for data analysis. Quantitative data were expressed as mean ± standard deviation (x ± s), and one-way ANOVA was used for comparisons between groups. Categorical data were expressed as percentages (%), and χ² was used for comparisons between groups. 2 The diagnostic performance of the kit was analyzed using ROC curve analysis; P < 0.05 was considered statistically significant.

[0163] III. Experimental Results

[0164] 1. Concentration and ratio of oxidized lipids in each group of subjects:

[0165] The concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in the plasma of three groups of subjects were detected using the kit of the present invention, and the pro-inflammatory / pro-remission lipid ratio was calculated. The results are shown in Table 1 below.

[0166] Table 1. Results of the pro-inflammatory / pro-remission lipid ratio

[0167]

[0168] Note: Compared with the healthy control group, *P<0.05; compared with the early AS group, #P<0.05.

[0169] As shown in Table 1, with the progression of atherosclerosis, the concentrations of 5-HETE and 12-HETE and the pro-inflammatory / pro-regression lipid ratio gradually increased, while the concentrations of LXA4 and RvD1 gradually decreased. The differences between the groups were statistically significant (P < 0.05). The oxidized lipid index of the early AS group had significantly deviated from that of the healthy control group (P < 0.05), indicating that this kit can identify early atherosclerosis.

[0170] 2. Validation of the kit's detection accuracy: Using LC-MS / MS detection results as the gold standard, the detection accuracy of this kit was validated, and the results are shown in Table 2 below.

[0171] Table 2. Results of reagent kit detection accuracy

[0172]

[0173] As shown in Table 2, the correlation coefficients between the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 detected by this kit and the LC-MS / MS detection results are all ≥0.98, indicating that this kit has high detection accuracy.

[0174] 3. Diagnostic performance of the kit: Using clinical diagnostic results as the gold standard, the performance comparison between this kit and the traditional LDL-C index in diagnosing atherosclerosis is shown in Table 3 below.

[0175] Table 3. Diagnostic performance results of the reagent kit

[0176]

[0177] As shown in Table 3, the sensitivity, specificity, positive predictive value, negative predictive value and AUC of this kit are significantly higher than those of LDL-C (P<0.05), with the AUC being 0.935 (95% confidence interval: 0.908-0.962), indicating that this kit has excellent diagnostic performance.

[0178] 4. Reagent kit repeatability validation: The results of intra-batch and inter-batch repeatability validation are shown in Table 4 below.

[0179] Table 4. Reproducibility Validation Results of the Reagent Kit

[0180]

[0181] As shown in Table 4, the intra-batch coefficient of variation for the four oxidized lipids detected by this kit was <5%, and the inter-batch coefficient of variation was <8%, indicating that the kit has good reproducibility.

[0182] 5. Reagent Kit Stability Validation: Stability validation results showed that after storage at 4℃ for 28 days, the correlation coefficient of the standard curve remained ≥0.99, and the absorbance value change rate was <10%; after storage at 25℃ for 14 days, the correlation coefficient of the standard curve remained ≥0.99, and the absorbance value change rate was <10%; after storage at 37℃ for 7 days, the correlation coefficient of the standard curve remained ≥0.98, and the absorbance value change rate was <15%. This indicates that the reagent kit exhibits good stability at 4℃ and meets the routine clinical storage requirements.

[0183] IV. Experimental Conclusions

[0184] This invention targets the "pro-inflammatory-pro-remission" lipid oxidation combination as a detection target, and is equipped with optimized sample pretreatment components and ELISA detection methods, which have the advantages of accurate detection, high sensitivity, strong specificity, simple operation and good stability.

[0185] Clinical trials have shown that the kit of this invention can effectively identify early atherosclerosis, and its diagnostic performance is significantly better than that of traditional blood lipid indicators. It can be widely used in early clinical screening, risk assessment in primary healthcare institutions, and large-scale population health checkups, and has important clinical application value and industrialization prospects.

Claims

1. A kit for assessing the risk of atherosclerosis, using lipid oxidization as the detection target, characterized in that: It includes core detection components, sample preprocessing components, and auxiliary components; the oxidized lipid combination includes pro-inflammatory oxidized lipids and pro-regression oxidized lipids; the pro-inflammatory oxidized lipids include 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), and the pro-regression oxidized lipids include lipoxygenin A4 (LXA4) and regression factor D1 (RvD1).

2. The atherosclerosis risk assessment kit according to claim 1, characterized in that, The core detection components include: ELISA plate: ELISA plate is used to coat antibodies. Each detection well of the ELISA plate is independently coated with a specific monoclonal antibody against one type of oxidized lipid. Antibodies corresponding to the four types of oxidized lipids are coated with multiple detection wells respectively. The antibody coating concentration is 2-5 μg / mL and the coating volume is 100 μL / well. Enzyme-labeled secondary antibodies: multiple horseradish peroxidase (HRP)-labeled secondary antibodies targeting the specific monoclonal antibodies. The stock solution of the enzyme-labeled secondary antibodies is diluted at a ratio of 1:5000 to 1:10000, and the concentration after dilution is the working concentration. Standard kit: contains 5-HETE, 12-HETE, LXA4, and RvD1 standards, with an initial concentration of 100 ng / mL, and each standard is available in 6 concentration gradients; Colorimetric reagent: is a TMB substrate solution, consisting of solution A and solution B, with a volume ratio of 1:1; solution A is a TMB stock solution, and solution B is a urea peroxide solution; Termination solution: 2 mol / L sulfuric acid solution.

3. The atherosclerosis risk assessment kit according to claim 1, characterized in that, The sample preprocessing component includes: Protein precipitation solution: a methanol solution containing 0.1% BHT, 10 mL per vial, used to precipitate proteins in plasma samples; Extraction solution: n-hexane, used to extract oxidized lipids from the sample; Reconstitution buffer: PBS buffer at pH 7.4 containing 0.5% BSA and 0.05% Tween-20, used to dissolve oxidized lipids after extraction; Internal standard solution: deuterated 5-HETE solution, concentration of 10 ng / mL, used to correct the accuracy of the detection results.

4. The atherosclerosis risk assessment kit according to claim 1, characterized in that, The auxiliary components include: Washing buffer: PBS buffer at pH 7.4 containing 0.05% Tween-20, used for washing ELISA plates; Blocking solution: PBS buffer containing 5% skim milk powder, used to block non-specific binding sites on the ELISA plate; Sample dilution solution: PBS buffer at pH 7.4 containing 0.1% BSA, used to dilute plasma samples.

5. A method for assessing the risk of atherosclerosis according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Sample pretreatment: Take plasma samples and add internal standard solution, protein precipitation solution and extraction solution in sequence, and vortex. Take the upper organic phase. Dry the organic phase with nitrogen, add reconstitution buffer, vortex and centrifuge, and take the supernatant as the sample to be tested. S2, ELISA detection: After equilibrating the microplate to room temperature, add blocking buffer and incubate; discard the blocking buffer and wash; add the sample to be tested and the standard to the corresponding test well, incubate, and discard the liquid in the well; add washing buffer and wash; add the corresponding enzyme-labeled secondary antibody and incubate; discard the liquid in the well and wash; add chromogenic reagent and incubate; finally, add stop solution to terminate the reaction. S3. Result detection and calculation: The absorbance value of each well was read at a wavelength of 450nm using an ELISA reader; a standard curve was plotted based on the absorbance value of the standard; the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in the sample to be tested were calculated; and the pro-inflammatory / pro-remission lipid ratio was calculated. S4. Risk Assessment: The risk level of atherosclerosis is determined based on the ratio of pro-inflammatory to remission lipids and the concentration of the oxidized lipid combination, combined with a preset threshold.

6. The method for assessing the risk of atherosclerosis according to claim 5, characterized in that, The specific details of step S1 are as follows: S1.1 Take 200 μL of fasting plasma sample from the subject, add 20 μL of internal standard solution, and vortex for 30 s; S1.2 Add 400 μL of protein precipitation solution, vortex for 30 seconds, and let stand in an ice bath for 10 minutes; S1.3 Add 200 μL of extraction solution, vortex for 1 min, centrifuge at 12000 r / min for 10 min at 4℃, and take the upper organic phase; S1.

4. Dry the organic phase with nitrogen, add 100 μL of reconstitution buffer, vortex for 1 min, centrifuge at 12000 r / min for 5 min at 4℃, and take the supernatant as the sample to be tested.

7. The method for assessing the risk of atherosclerosis according to claim 6, characterized in that: The nitrogen drying temperature is ≤30℃, and the nitrogen pressure is 0.1MPa.

8. The method for assessing the risk of atherosclerosis according to claim 5, characterized in that, The specific details of step S2 are as follows: S2.1 Remove the antibody-coated ELISA plate from the 4°C freezer and allow it to equilibrate at room temperature for 30 minutes; S2.2 Add blocking solution, 150 μL / well, and incubate at 37°C for 30 min; S2.3 Discard the blocking solution, add 200 μL of diluted washing solution per well, soak for 30 seconds, discard the washing solution, repeat the washing 5 times, and pat dry; S2.4 Add the sample to be tested and the standard to the corresponding well of the ELISA plate, 100 μL / well, with 3 replicates for each sample, and also set up blank control wells; S2.5 Place the ELISA plate in a 37°C incubator for 60 minutes, avoiding shaking during incubation; S2.6 After incubation, discard the liquid in the well, add washing solution and wash 5 times, 30 seconds each time, then pat dry; S2.7 Add the corresponding enzyme-labeled secondary antibody, 100 μL / well, and incubate at 37°C for 30 min; S2.8 Repeat the washing process of step S2.6; S2.9 Add freshly mixed colorimetric reagent, 100 μL / well, and incubate at 37°C in the dark for 15 min; Add stop solution (50 μL / well) to S2.10, gently shake the plate for 5 seconds to stop the reaction.

9. The method for assessing the risk of atherosclerosis according to claim 5, characterized in that, The specific details of step S3 are as follows: S3.

1. Use an ELISA reader to read the absorbance value of each well at a wavelength of 450nm, and take the average value as the final absorbance value. The reference wavelength is 570nm. S3.

2. Plot a standard curve based on the absorbance values ​​of the standards, and calculate the concentrations of 5-HETE, 12-HETE, LXA4, and RvD1 in the sample to be tested; the standard curve is fitted using four parameters, and the correlation coefficient R is [value missing]. 2 ≥0.99; S3.3 Calculate the pro-inflammatory / pro-remission lipid ratio: pro-inflammatory / pro-remission lipid ratio = (5-HETE concentration + 12-HETE concentration) / (LXA4 concentration + RvD1 concentration).

10. The method for assessing the risk of atherosclerosis according to claim 5, characterized in that, The specific details of step S4 are as follows: Low risk: Meeting the criteria of pro-inflammatory / pro-remission lipid ratio <2.0, 5-HETE <50pg / mL, 12-HETE <40pg / mL, LXA4 >20pg / mL and RvD1 >15pg / mL; Medium risk: 2.0 ≤ pro-inflammatory / pro-remission lipid ratio < 5.0 or any oxidized lipid concentration exceeding the low risk range; High risk: Pro-inflammatory / remission lipid ratio ≥5.0.

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