CN-122017261-A - Atherosclerosis risk assessment kit and assessment method

CN122017261ACN 122017261 ACN122017261 ACN 122017261ACN-122017261-A

Abstract

The invention discloses an atherosclerosis risk assessment kit and an atherosclerosis risk assessment method, wherein the kit takes pro-inflammatory and pro-fading oxidized lipid combination as a detection target, and comprises a core detection component, a sample pretreatment component and an auxiliary component, wherein a specific monoclonal antibody in the core detection component has high specificity and high sensitivity, the concentration of 4 key oxidized lipids in blood plasma can be accurately and quantitatively detected, and the sample pretreatment component can effectively remove impurities such as proteins in the blood plasma and improve the detection accuracy. The risk assessment method is used for realizing early risk early warning of atherosclerosis by calculating the ratio of pro-inflammatory lipid to pro-regressive lipid and combining the concentration threshold values of each index. The invention takes the pro-inflammatory and pro-fading oxidized lipid combination as a detection target spot, combines an optimized ELISA detection method, and has the advantages of early, accurate and rapid risk assessment on atherosclerosis.

Inventors

HuangFu Ning
CHEN YIJU
MAO HUIJUAN
JI NA
XUE JING
SHEN QING

Assignees

宁波大学附属第一医院(宁波市第一医院)
杭州市临平区中西医结合医院
浙江工商大学

Dates

Publication Date: 20260512
Application Date: 20260209

Claims (10)

1. The atherosclerosis risk assessment kit is characterized by comprising a core detection component, a sample pretreatment component and an auxiliary component, wherein the oxidized lipid combination comprises pro-inflammatory oxidized lipids and pro-fading oxidized lipids, the pro-inflammatory oxidized lipids comprise 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), and the pro-fading oxidized lipids comprise lipoxin A4 (LXA 4) and resolubin D1 (RvD 1).
2. The atherosclerosis risk assessment kit according to claim 1, wherein the core detection component comprises: The ELISA plate is used for coating antibodies, each detection hole of the ELISA plate is independently coated with a specific monoclonal antibody aiming at one oxidized lipid, antibodies corresponding to 4 oxidized lipids are respectively coated with a plurality of detection holes, the coating concentration of the antibodies is 2-5 mug/mL, and the coating volume is 100 mug/hole; enzyme-labeled secondary antibodies, namely a plurality of horseradish peroxidase (HRP) labeled secondary antibodies respectively aiming at the specific monoclonal antibodies, wherein the dilution ratio of stock solution of the enzyme-labeled secondary antibodies is 1:5000-1:10000, and the concentration after dilution is working concentration; standard substance sets, wherein the standard substance sets comprise 5-HETE, 12-HETE, LXA4 and RvD1 standard substances, the initial concentration is 100ng/mL, and each standard substance has 6 gradient concentration gradients; the color reagent is TMB substrate solution, which consists of solution A and solution B, wherein the volume ratio of the solution A to the solution B is 1:1, the solution A is TMB stock solution, and the solution B is carbamide peroxide solution; Stop solution, 2mol/L sulfuric acid solution.
3. The atherosclerosis risk assessment kit according to claim 1, wherein the sample pretreatment assembly comprises: protein precipitation solution, which is methanol solution containing 0.1% BHT and 10mL each for precipitating protein in plasma sample; Extracting liquid, namely n-hexane, for extracting oxidized lipid in a sample; a re-dissolving buffer solution, namely PBS buffer solution with pH of 7.4, containing 0.5 percent of BSA and 0.05 percent of Tween-20, and is used for dissolving the extracted oxidized lipid; the internal standard solution is deuterated 5-HETE solution with the concentration of 10ng/mL and is used for correcting the accuracy of the detection result.
4. An atherosclerosis risk assessment kit according to claim 1, wherein said accessory component comprises: The washing solution is PBS buffer solution with pH of 7.4 and contains 0.05 percent of Tween-20, and is used for washing the ELISA plate; blocking solution, which is PBS buffer solution containing 5% skimmed milk powder and used for blocking nonspecific binding sites of the ELISA plate; sample dilutions PBS buffer, pH 7.4, containing 0.1% BSA was used to dilute the plasma samples.
5. An atherosclerosis risk assessment method according to any of claims 1-4, characterized by comprising the steps of: s1, sample pretreatment, namely sequentially adding an internal standard solution, a protein precipitation solution and an extraction solution into a plasma sample, carrying out vortex oscillation, and taking an upper organic phase; s2, ELISA detection, namely balancing an ELISA plate to room temperature, adding a sealing liquid for incubation, discarding the sealing liquid and washing, respectively adding a sample to be detected and a standard substance into corresponding detection holes, discarding liquid in the holes after incubation, adding a washing liquid for washing, adding a corresponding enzyme-labeled secondary antibody for incubation, discarding liquid in the holes and washing, adding a color reagent for incubation, and finally adding a stopping solution for stopping reaction; S3, detecting and calculating results, namely reading absorbance values of all holes at the wavelength of 450nm by adopting an enzyme-labeled instrument, drawing a standard curve according to the absorbance values of a standard substance, calculating the concentrations of 5-HETE, 12-HETE, LXA4 and RvD1 in a sample to be detected, and calculating the pro-inflammatory/pro-fading lipid ratio; and S4, risk assessment, namely judging the atherosclerosis risk level according to the pro-inflammatory/pro-fading lipid ratio and the concentration of the oxidized lipid combination and combining with a preset threshold value.
6. The method according to claim 5, wherein the specific content of step S1 is as follows: S1.1, taking 200 mu L of a fasting plasma sample of a subject, adding 20 mu L of an internal standard solution, and carrying out vortex oscillation for 30S; S1.2, adding 400 mu L of protein precipitation liquid, carrying out vortex oscillation for 30S, and standing in an ice bath for 10min; S1.3, adding 200 mu L of extract, vortex oscillating for 1min, centrifuging for 10min at 12000r/min at 4 ℃, and taking an upper organic phase; s1.4, drying the organic phase by nitrogen, adding 100 mu L of redissolution buffer, vortex oscillating for 1min, centrifuging for 5min at 12000r/min at 4 ℃, and taking the supernatant as a sample to be detected.
7. The method for assessing risk of atherosclerosis according to claim 6 wherein said nitrogen is blown dry at a temperature of 30 ℃ or less and a nitrogen pressure of 0.1MPa.
8. The method according to claim 5, wherein the specific content of step S2 is as follows: s2.1, taking the ELISA plate coated with the antibody out of a refrigerator at 4 ℃ and balancing the ELISA plate at room temperature for 30min; S2.2 adding a blocking solution, 150 mu L/hole and incubating at 37 ℃ for 30min; s2.3, discarding the sealing solution, adding 200 mu L/hole of diluted washing solution, soaking for 30S, discarding the washing solution, repeatedly washing for 5 times, and drying; s2.4, respectively adding a sample to be detected and a standard substance into detection holes corresponding to the ELISA plates, wherein each sample is provided with 3 compound holes and blank control holes at the same time, wherein each hole is 100 mu L/hole; s2.5, placing the ELISA plate in a 37 ℃ incubator for incubation for 60min, and avoiding oscillation in the incubation process; s2.6, after the incubation is finished, removing liquid in the hole, adding a washing liquid to wash for 5 times, and drying by beating every 30 seconds; S2.7, adding a corresponding enzyme-labeled secondary antibody, and incubating for 30min at 37 ℃ with 100 mu L/hole; s2.8, repeating the washing process of the step S2.6; S2.9, adding a freshly mixed color reagent, and incubating for 15min at 37 ℃ in a dark place at 100 mu L/hole; S2.10, adding a stop solution, 50 mu L/hole, gently shaking the ELISA plate for 5S, and stopping the reaction.
9. The method according to claim 5, wherein the specific content of step S3 is as follows: s3.1, reading absorbance values of all holes by adopting an enzyme-labeled instrument at the wavelength of 450nm, taking an average value as a final absorbance value, and referencing the wavelength of 570nm; S3.2, drawing a standard curve according to the absorbance value of the standard substance, and calculating the concentration of 5-HETE, 12-HETE, LXA4 and RvD1 in a sample to be detected, wherein the standard curve is fitted by four parameters, and the correlation coefficient R 2 is more than or equal to 0.99; S3.3, calculating the pro-inflammatory/pro-resolution lipid ratio = (5-HETE concentration +12-HETE concentration)/(lxa4 concentration + RvD1 concentration).
10. The method according to claim 5, wherein the specific content of step S4 is as follows: Low risk of meeting a pro-inflammatory/pro-resolution lipid ratio <2.0, 5-HETE <50pg/mL, 12-HETE <40pg/mL, LXA4>20pg/mL and RvD1>15pg/mL; a risk in the middle of 2.0≤pro-inflammatory/pro-resolution lipid ratio <5.0 or any oxidized lipid concentration outside the low risk range; High risk that the ratio of pro-inflammatory/pro-resolution lipids is not less than 5.0.

Description

Atherosclerosis risk assessment kit and assessment method Technical Field The invention relates to the technical field of biological detection, in particular to an atherosclerosis risk assessment kit and an atherosclerosis risk assessment method. Background Atherosclerosis (AS) is a chronic vascular disease characterized by intimal lipid deposition, fibrous tissue hyperplasia and inflammatory response, and is the main pathological basis of serious cardiovascular and cerebrovascular events such AS coronary heart disease, cerebral infarction and the like. It is counted that more than 50% of all deaths from cardiovascular and cerebrovascular diseases are associated with atherosclerosis. Early identification of risk of atherosclerosis, timely intervention is critical in reducing the incidence of cardiovascular and cerebrovascular events. Currently, diagnosis and risk assessment of atherosclerosis rely mainly on imaging examinations (e.g. carotid ultrasound, coronary CT angiography) and traditional biochemical indicators (e.g. blood lipids, C-reactive proteins). However, there is obvious hysteresis in the imaging examination, and only the formed arterial plaque can be detected, so that the early stage of vascular endothelial function injury cannot be identified; The traditional biochemical index has low specificity, is easily interfered by various factors such as diet, exercise, liver diseases and the like, and is difficult to accurately reflect the early pathological process of atherosclerosis. Therefore, developing a detection technology capable of early and accurately assessing atherosclerosis risk is a problem to be solved in clinical urgent need. Lipoxygenase (Lipoxygenases, LOXs) mediated imbalance in the oxidative metabolism of polyunsaturated fatty acids is one of the key mechanisms in the development and progression of atherosclerosis. LOXs can catalyze the formation of two classes of oxidized lipids of opposite function, pro-inflammatory oxidized lipids (e.g., 5-HETE, 12-HETE) and pro-resolution oxidized lipids (e.g., LXA4, rvD 1). Abnormal activation of LOXs activities in the early stage of atherosclerosis leads to the generation of a large amount of pro-inflammatory oxidized lipid, and insufficient secretion of pro-fading oxidized lipid, so that balance between the pro-inflammatory oxidized lipid and the insufficient secretion of pro-fading oxidized lipid is broken, and vascular endothelial inflammation and lipid deposition are further initiated to promote disease progression. Studies have shown that changes in the concentrations of 5-HETE, 12-HETE, LXA4, rvD1 and pro-inflammatory/pro-resolution ratios in the circulation can be used as potential metabolic markers for early diagnosis of atherosclerosis. The existing lipid oxide detection methods mainly comprise high performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and the like, and the methods are required to depend on expensive large-scale instruments, are complex in operation, have long detection period and are difficult to popularize and apply in basic medical institutions. The ELISA has the advantages of simple operation, low cost and rapid detection, is suitable for clinical routine detection, but lacks a multi-target oxidized lipid ELISA detection kit aiming at early risk assessment of atherosclerosis at present, and has the problem of insufficient specificity of a single oxidized lipid detection kit. Based on the detection kit, the detection kit taking the pro-inflammatory and pro-fading oxidation lipid combination as a target spot is developed, so that early-stage accurate risk assessment of atherosclerosis is realized, and the blank of the prior art is filled. Disclosure of Invention The invention aims to provide an atherosclerosis risk assessment kit and an atherosclerosis risk assessment method. The invention takes the pro-inflammatory and pro-fading oxidized lipid combination as a detection target spot, combines an optimized ELISA detection method, and has the advantages of early, accurate and rapid risk assessment on atherosclerosis. The technical scheme of the invention is as follows: An atherosclerosis risk assessment kit takes a pro-inflammatory-pro-resolution oxidized lipid combination as a detection target, wherein the kit comprises a core detection component, a sample pretreatment component and an auxiliary component, the oxidized lipid combination comprises pro-inflammatory oxidized lipids and resolution-promoting oxidized lipids, the pro-inflammatory oxidized lipids comprise 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), and the resolution-promoting oxidized lipids comprise lipoxin A4 (LXa 4) and resolution-promoting element D1 (RvD 1). In the atherosclerosis risk assessment kit, the core detection component comprises an ELISA plate, an enzyme-labeled secondary antibody, a standard substance set, a color reagent and a stop solut