LL-37-ApoB-100 biomarker and its application

Abstract

The present invention relates to the field of biomarker technology, and in particular to the LL‑37‑ApoB‑100 biomarker and its application. The present invention has discovered a biomarker LL‑37‑ApoB‑100 complex in blood and / or atheromatous plaques related to the prediction and diagnosis of coronary atherosclerosis / coronary heart disease. Antibodies are prepared using the marker, and coronary atherosclerosis or coronary atherosclerosis-related diseases can be predicted and diagnosed by enzyme-linked immunosorbent assay, and different subtypes of coronary heart disease such as coronary atherosclerosis and stable angina, unstable angina and acute myocardial infarction can be effectively identified. Compared with traditional coronary atherosclerosis or coronary heart disease markers, the biomarker has significant advantages such as higher sensitivity, higher specificity, faster and simpler detection, lower cost and less trauma.

Description

Technical Field

[0001] The present invention relates to the technical field of biomarkers, in particular to an LL-37-ApoB-100 biomarker and applications thereof. Background Art

[0002] Coronary artery disease (CAD) is a disease in which atherosclerotic changes occur in the coronary arteries of the heart, causing narrowing or obstruction of the coronary artery lumen. Under the influence of various factors, myocardial ischemia, hypoxia and even necrosis occur, leading to a series of clinical symptoms.

[0003] Currently, coronary angiography is the gold standard for the diagnosis of coronary heart disease. However, coronary angiography is an invasive coronary interventional examination method and is not suitable for people with coagulation disorders, bleeding diseases, or allergies to contrast agents. A variety of risk factors or biomarkers, such as C-reactive protein (CRP), lipoprotein (a), fibrinogen, and homocysteine, have been used to predict the risk of atherosclerotic vascular disease [Hackam DG and Anand SS. Emerging risk factors for atherosclerotic vascular disease: a critical review of the evidence. Jama. 2003; 290: 932-40.]. However, intervention studies that reduce risk factors such as C-reactive protein do not completely reduce disease risk [Ridker PM. From C-Reactive Protein to Interleukin-6 to Interleukin-1: Moving Upstream To Identify Novel Targets for Atheroprotection. Circulation research. 2016; 118: 145-56.].

[0004] To date, blood biomarkers for coronary atherosclerosis are still lacking. Conventional risk factors (Hcy, hsCRP, TG, TC, ApoB-100, LDL-C and HDL-C) have low sensitivity and specificity in diagnosing coronary atherosclerotic heart disease. Therefore, it is urgent to identify and develop new biomarkers. Summary of the invention

[0005] In order to solve the above problems, the present invention provides LL-37-ApoB-100 biomarkers and their applications. The LL-37-ApoB-100 complex provided by the present invention is used as a marker for coronary atherosclerosis or coronary heart disease related to coronary atherosclerosis, and can effectively identify coronary atherosclerosis and different subtypes of coronary heart disease such as stable angina pectoris, unstable angina pectoris and acute myocardial infarction. Compared with traditional coronary atherosclerosis or coronary heart disease markers, it has significant advantages such as higher sensitivity, higher specificity, faster and simpler detection, lower cost and less trauma.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides the use of a reagent for detecting an LL-37-ApoB-100 complex in preparing a diagnostic kit for coronary atherosclerosis or a disease related to coronary atherosclerosis. The LL-37-ApoB-100 complex consists of an antimicrobial peptide LL-37 and apolipoprotein B-100.

[0008] Preferably, the disease associated with coronary atherosclerosis includes coronary heart disease.

[0009] Preferably, the reagent for detecting the LL-37-ApoB-100 complex comprises an anti-LL-37-ApoB-100 complex antibody.

[0010] The invention provides a diagnostic kit for coronary atherosclerosis or diseases related to coronary atherosclerosis, comprising an anti-LL-37-ApoB-100 complex antibody, wherein the LL-37-ApoB-100 complex consists of an antimicrobial peptide LL-37 and apolipoprotein B-100.

[0011] Preferably, the anti-LL-37-ApoB-100 complex antibody comprises a polyclonal antibody.

[0012] Preferably, the polyclonal antibody comprises a rabbit-derived polyclonal antibody.

[0013] Preferably, the preparation method of the LL-37-ApoB-100 complex comprises: mixing the antimicrobial peptide LL-37 and apolipoprotein B-100, and letting the mixture stand to obtain the LL-37-ApoB-100 complex.

[0014] Preferably, the mass ratio of the antimicrobial peptide LL-37 to apolipoprotein B-100 is 250-300:100-150; and the standing time is 25-40 minutes.

[0015] Preferably, the anti-LL-37-ApoB-100 complex antibody is labeled with biotin.

[0016] Preferably, the diagnostic kit further comprises an enzyme-linked immunosorbent assay reagent.

[0017] Beneficial effects:

[0018] The present invention provides the use of a reagent for detecting LL-37-ApoB-100 complex in the preparation of a diagnostic kit for coronary atherosclerosis or diseases related to coronary atherosclerosis, wherein the LL-37-ApoB-100 complex is composed of antimicrobial peptide LL-37 and apolipoprotein B-100. The present invention has discovered a biomarker LL-37-ApoB-100 complex in blood and / or atheromatous plaques related to the prediction and diagnosis of coronary atherosclerosis / coronary heart disease, and antibodies are prepared using the marker, and coronary atherosclerosis or diseases related to coronary atherosclerosis can be predicted and diagnosed by an enzyme-linked immunosorbent assay method, and coronary atherosclerosis and different subtypes of coronary heart disease such as stable angina pectoris, unstable angina pectoris and acute myocardial infarction can be effectively identified, and compared with traditional coronary atherosclerosis or coronary heart disease markers, the present invention has significant advantages such as higher sensitivity, higher specificity, faster and simpler detection, lower cost and lower trauma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 The result diagram is that LL-37 and ApoB-100 form a stable complex;

[0021] Figure 2 The figure is the result of purification and titer detection of rabbit polyclonal antibody of Cramp-ApoB-100 complex;

[0022] Figure 3 The figure is the result of purification and titer detection of rabbit polyclonal antibody of LL-37-ApoB-100 complex;

[0023] Figure 4 To build Apoe - / - Results of the mouse coronary atherosclerosis animal model;

[0024] Figure 5 This is a fluorescence detection result of atherosclerotic plaques in patients with coronary atherosclerosis;

[0025] Figure 6 The results of fluorescence detection of thoracic aorta plaque and plasma detection in mice fed with normal diet and high fat diet are shown in the figure;

[0026] Figure 7 Apoe induced by high-fat diet for 2, 4, and 6 weeks- / - The result graph shows that the Cramp-ApoB-100 complex content in mouse plasma is positively correlated with the area of ​​coronary atherosclerotic plaques;

[0027] Figure 8 Apoe induced by high-fat diet for 2, 4, and 6 weeks - / - Results of the correlation analysis between Cramp and blood lipid levels in mouse plasma and the area of ​​coronary atherosclerotic plaques;

[0028] Fig. 9 The results of the plasma levels of LL-37-ApoB-100 complex in the control group, coronary atherosclerosis group and patients with different types of coronary heart disease are shown in the figure;

[0029] Fig.10 The ROC curve results of LL-37-ApoB-100 complex, LL-37 and conventional risk factors in diagnosing coronary atherosclerotic heart disease. DETAILED DESCRIPTION

[0030] The present invention provides the use of a reagent for detecting LL-37-ApoB-100 complex in the preparation of a diagnostic kit for coronary atherosclerosis or diseases related to coronary atherosclerosis, wherein the LL-37-ApoB-100 complex is composed of an antimicrobial peptide LL-37 and apolipoprotein B-100. The LL-37 of the present invention binds to ApoB-100 through electrostatic interaction and hydrophobic interaction. In the present invention, the diseases related to coronary atherosclerosis preferably include coronary heart disease, and more preferably include stable angina pectoris, unstable angina pectoris and acute myocardial infarction; the reagent for detecting LL-37-ApoB-100 complex preferably includes an anti-LL-37-ApoB-100 complex antibody.

[0031] The present invention finds that the LL-37-ApoB-100 complex can be used as a marker for coronary atherosclerosis / coronary heart disease, and can effectively identify coronary atherosclerosis and different coronary heart disease pressure types such as stable angina, unstable angina and acute myocardial infarction. Compared with traditional coronary atherosclerosis / coronary heart disease markers, it has significant advantages such as higher sensitivity, higher specificity, faster and simpler detection, lower cost and less trauma. The LL-37-ApoB-100 complex can be used as a marker for coronary atherosclerosis / coronary heart disease, and can also be used as a predictor for predicting the onset of early coronary atherosclerosis and the degree of development of coronary heart disease.

[0032] The present invention provides a diagnostic kit for coronary atherosclerosis or a disease related to coronary atherosclerosis, comprising an anti-LL-37-ApoB-100 complex antibody, wherein the LL-37-ApoB-100 complex is composed of an antimicrobial peptide LL-37 and apolipoprotein B-100. In the present invention, the anti-LL-37-ApoB-100 complex antibody preferably comprises a polyclonal antibody; the polyclonal antibody preferably comprises a rabbit-derived polyclonal antibody; the anti-LL-37-ApoB-100 complex antibody is preferably labeled with biotin; the antigen for preparing the polyclonal antibody is preferably the LL-37-ApoB-100 complex; the preparation method of the LL-37-ApoB-100 complex preferably comprises: mixing the antimicrobial peptide LL-37 and apolipoprotein B-100, standing, and obtaining the LL-37-Apo B-100 complex; the mass ratio of the antimicrobial peptide LL-37 and apolipoprotein B-100 is preferably 250-300:100-150, further preferably 260-290:110-120, and more preferably 286:114; the standing time is preferably 25-40 minutes, and more preferably 30 minutes; the diagnostic kit preferably also includes an enzyme-linked immunosorbent reagent; the enzyme-linked immunosorbent reagent preferably includes a coating solution, a blocking solution, a washing solution, a streptavidin-horseradish peroxidase conjugate, a chromogenic substrate, and a stop solution.

[0033] The present invention preferably also provides a method for using the diagnostic kit described in the above technical solution, preferably comprising: the sample to be tested is diluted with a coating solution and then coated in a 96-well plate, the sample wells are sealed and then adsorbed on the LL-37-ApoB-100 complex in the sample by using a biotin-labeled anti-LL-37-ApoB-100 complex antibody, and finally a color reaction is established by a streptavidin-horseradish peroxidase conjugate. The concentration of the LL-37-ApoB-100 complex in the tested sample is calculated by comparing with the standard curve. In the present invention, the sample to be tested preferably includes a plasma sample. The present invention can diagnose the severity of coronary heart disease according to the concentration of the LL-37-ApoB-100 complex, thereby achieving the purpose of predicting and diagnosing coronary heart disease.

[0034] To further illustrate the present invention, the LL-37-ApoB-100 biomarker and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Cramp (SEQ ID NO.3), LL-37 (SEQ ID NO.1) and its scrambled control peptide GL-37 (SEQ ID NO.2) were synthesized by Gill Biochemical (Shanghai) Co., Ltd., using a peptide synthesizer model 433A (ABI, USA). The synthesized peptides were verified by reverse phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and the sequence was correct with a purity greater than 98%. The synthesized peptide samples were sealed in the form of dry powder and stored at -20°C. The synthesized peptide sequences are as follows:

[0037] LL-37: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, SEQ ID NO.1;

[0038] GL-37: GLKLRFESKIKGEFLKTPEVRFRDIKLKDFNRISVQR, SEQ ID NO.2;

[0039] Cramp: GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE, SEQ ID NO.3.

[0040] Example 2

[0041] Surface Plasmon Resonance

[0042] The surface plasmon resonance method was used to analyze the interaction between LL-37 and ApoB-100. The experiment used a BIAcoreS200 (GE) instrument and a CM 5 chip (GE). The specific steps are as follows:

[0043] (1) Activation of CM5 chip: 0.1 M N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloric acid (EDC) and 0.1 M N-hydroxysuccinimide (NHS) were mixed in a volume ratio of 1:1 and flowed over the surface of the CM5 chip at a flow rate of 5 μl / min to activate the carboxyl groups on its surface.

[0044] (2) Covalent coupling: A 10 μg / ml LL-37 or GL-37 aqueous solution was flowed over the chip surface at a flow rate of 5 μl / min. Coupling was stopped when the expected response value was reached.

[0045] (3) Blocking excess active sites: 40 μl of 1 M ethanolamine was flowed over the chip surface at a flow rate of 5 μl / min to block excess active sites.

[0046] (4) Equilibrium system: HEPS-EP buffer was flowed through the chip surface at a flow rate of 5 μl / min until the displayed response value returned to the baseline level.

[0047] (5) Protein binding experiment: ApoB-100 (RayBiotech, MD-26-0010P) solutions with concentrations from low to high (12.5, 25, 50, 100 μg / ml) were prepared with HEPS-EP buffer, and 20 μl of sample was set in sequence and flowed over the chip surface at a flow rate of 5 μl / min. When detecting the next concentration gradient, a low concentration NaOH solution was used to wash the bound protein, and then the system was equilibrated with HEPS-EP buffer before injection.

[0048] (6) Process the data, plot using graphics software, and calculate the binding and dissociation constants. Figure 1 .

[0049] Using surface plasmon resonance (SPR) technology, LL-37 is coated onto a CM5 chip as a stationary phase; ApoB-100 is used as a mobile phase and flows through the chip surface. If there is an interaction between the two proteins being detected, the metal surface refractive index of the chip changes when the mobile phase flows through the chip surface, and the sensing curve will present an upward curve. The more obvious the refractive index change and the greater the amplitude of the curve change, the greater the direct interaction strength between the surface stationary phase and the mobile phase. If there is no interaction between the two proteins being detected, the sensor curve will be consistent with the baseline and there will be no obvious changes.

[0050] When ApoB-100 was used as the mobile phase to flow over the LL-37-CM 5 chip surface, the results were as follows Figure 1 As shown in A, ApoB-100 can bind to LL-37 in a concentration gradient-dependent manner. When ApoB-100 is used as the mobile phase to flow through the scrambled control peptide GL-37-CM 5 chip surface, the results are as follows Figure 1 As shown in B, there is no obvious interaction between ApoB-100 and the scrambled control peptide GL-37.

[0051] Example 3

[0052] Preparation and purification of rabbit polyclonal antibodies

[0053] Rabbit polyclonal antibodies against LL-37-ApoB-100 complex and Cramp-ApoB-100 complex were prepared as follows:

[0054] Add 286 μg of LL-37 and 114 μg of ApoB-100 to 1 ml of water, incubate at room temperature for 30 minutes to obtain LL-37-ApoB-100 complex; add 286 μg of Cramp and 114 μg of ApoB-100 to 1 ml of water, incubate at room temperature for 30 minutes to obtain Cramp-ApoB-100 complex; add 286 μg of Cramp and 114 μg of LDL to 1 ml of water, incubate at room temperature for 30 minutes to obtain Cramp-LDL complex.

[0055] 400 μg of LL-37-ApoB-100 complex and Cramp-ApoB-100 complex were dissolved in 1 ml of Freund's complete adjuvant, and the mixture was thoroughly mixed by vortexing. Then, 2 ml of the mixed adjuvant was injected subcutaneously into New Zealand white rabbits by multi-point injection on the back. 14 days after the first immunization, the second immunization was carried out in the same way, and the antigen mass was halved. At 28 and 42 days after the first immunization, the antigen was dissolved in Freund's incomplete adjuvant, and the antigen mass was halved compared with the first immunization. On the seventh day after the last booster immunization, blood was collected from the ear vein to detect the antibody titer. Rabbits with an antibody titer of more than 1:1000 were anesthetized (2% pentobarbital, intravenous injection of 1 ml / kg body weight), and rabbit blood was obtained by cannulation of the jugular vein, left to stand overnight at 4°C, and centrifuged at 3500 r / min for 30 minutes to obtain antiserum containing rabbit polyclonal antibodies. The antibody titer was tested using the indirect ELISA method. When the antibody titer of the rabbit antiserum was greater than 1:1000, the antibody was purified using a rabbit-specific purification kit (CellBiolabs, AKR-160). The purified antibody was labeled with long-arm biotin and stored at -80°C for future use.

[0056] The results of purification and titer detection of rabbit polyclonal antibody against Cramp-ApoB-100 complex are shown in Figure 2 ,in, Figure 2 A in the figure shows the purified antibody after 12% SDS-PAGE and Coomassie Brilliant Blue staining, where two bands of IgG heavy chain and light chain are clearly seen; Figure 2B in the figure is the titer test of the Cramp-ApoB-100 complex antibody, which compares the specificity of the antibody in recognizing Cramp, LDL (Yeasen, 20613ES05), ApoB-100, Cramp-ApoB-100, and Cramp-LDL. The absorbance values ​​at 450nm were 0.11±0.02, 0.13±0.02, 0.15±0.05, 1.14±0.13, and 0.95±0.05 (mean±SD values), respectively. ***p<0.001. The absorbance values ​​of the Cramp-ApoB-100 complex rabbit polyclonal antibody at 450nm for Cramp, LDL, and ApoB-100 were significantly lower than those for the complex, which indicates that the antibody can specifically recognize the Cramp-ApoB-100 and Cramp-37-LDL complexes.

[0057] The results of purification and titer detection of rabbit polyclonal antibody against LL-37-ApoB-100 complex are shown in Figure 3 , Figure 3 A in the figure shows the purified antibody after 12% SDS-PAGE and Coomassie Brilliant Blue staining, where two bands of IgG heavy chain and light chain are clearly seen. This indicates that the antibody purification kit has a good effect in purifying IgG. Figure 3 B in the figure is the titer test of the antibody against the LL-37-ApoB-100 complex, and the specificity of the antibody in recognizing LL-37, LDL, ApoB-100, LL-37-ApoB-100, and LL-37-LDL was compared. The absorbance values ​​at 450 nm were 0.11±0.01, 0.10±0.004, 0.23±0.03, 1.22±0.11, and 1.08±0.09 (mean±SD values), respectively. ***p<0.001. The absorbance values ​​of the LL-37-ApoB-100 complex rabbit polyclonal antibody against LL-37, LDL, and ApoB-100 at 450 nm were significantly lower than those for the complex, which indicates that the antibody can specifically recognize the LL-37-ApoB-100 and LL-37-LDL complexes.

[0058] Example 4

[0059] Mouse model of coronary atherosclerosis

[0060] Select 6-week-old male Apoe - / -Mice were randomly divided into groups of 6 in each group and fed a high-fat diet (containing 21% fat and 0.15% cholesterol) to induce the formation of coronary atherosclerosis in mice. At 0, 2, 4, and 6 weeks of feeding the high-fat diet, the mice were anesthetized with 2% (w / w) sodium pentobarbital, with a dosage of 40 mg / kg body weight. The anesthetized mice were fixed on the dissecting table, the chest cavity was opened, the heart was exposed, and cardiac perfusion was performed. First, the blood of the mouse was perfused with normal saline, and then the whole body of the mouse was fixed by perfusion with 4% paraformaldehyde. For the fixed mice, the heart and thoracic aorta were carefully peeled off, and the excess fat tissue was removed. The peeled heart and thoracic aorta were stored in the fixative and placed at 4°C for later use.

[0061] Example 5

[0062] Tissue cryosectioning and Oil Red-O staining

[0063] The mouse thoracic aorta tissue sample fixed with 4% paraformaldehyde was taken out, dehydrated with 40% sucrose solution (prepared with PBS), and left to stand at 4°C overnight. The next day, the dehydrated sample was embedded with OCT embedding medium, and then made into 8 μm tissue frozen sections using a cryostat (Leica, CM 3050S). The mouse thoracic aorta frozen sections were stained with Oil Red-O. The specific steps are as follows:

[0064] (1) Prepare Oil Red O staining solution: Mix Oil Red O stock solution and water in a ratio of 3:2, filter with a 0.22 μm filter, and store in a dark place for later use.

[0065] (2) Soak the sections with 60% isopropanol for 1 min, discard the isopropanol, and allow to dry naturally. This step is conducive to Oil Red-O staining.

[0066] (3) Add an appropriate amount of Oil Red-O staining solution and stain for 15 minutes in the dark.

[0067] (4) After staining, discard the staining solution and rinse with 60% isopropanol for 5 seconds.

[0068] (5) Rinse immediately with clean water and allow to dry naturally.

[0069] (6) After staining, the images were recorded using a microscope (Nikon Eclipse 80i, Japan), and quantitative image analysis was performed using software (Image).

[0070] By feeding Apoe - / -The mice were fed a high-fat diet to induce a model of coronary atherosclerosis. After two, four, and six weeks of high-fat diet feeding, the thoracic aorta root of the mice was taken, fixed with paraformaldehyde, and frozen sections were made. The atheromatous plaques at the root of the thoracic aorta were marked by Oil Red-O staining, and the area of ​​the plaques after staining was used to measure the severity of coronary atherosclerosis. The results are shown in Figure 4 , where A is a schematic diagram of Oil Red-O staining results of mouse thoracic aorta root (n=6), and B is the statistical result of plaque area in A. ***p<0.001.

[0071] Depend on Figure 4 It can be seen from Oil Red-O staining that Apoe - / - The thoracic aortic root of mice had different degrees of atherosclerotic plaques, and the Oil Red-O-positive areas accounted for 0.85%, 4.86%, and 9.95% of the area of ​​the entire thoracic aortic root lumen section, respectively. - / - The longer the mice were fed a high-fat diet, the more severe their coronary atherosclerosis became.

[0072] Example 6

[0073] Observational case-control study

[0074] A total of 635 patients with coronary heart disease who underwent coronary angiography at the First Affiliated Hospital of Kunming Medical University from June 2022 to December 2023 and 227 healthy controls were recruited. The subjects' gender, age, systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), homocysteine ​​(Hcy), high-sensitivity C-reactive protein (hsCRP) and other clinical data were recorded. The clinical characteristics of the healthy control group (NCA) and the coronary heart disease group (CAD) are shown in Table 1, and the values ​​are expressed as mean ± SD or median (interquartile range).

[0075] Table 1 Basic clinical data of healthy control group (NCA) and coronary artery disease group (CAD)

[0076]

[0077] Inclusion criteria: Patients with stenosis of the main coronary artery ≤50% in coronary angiography were diagnosed with coronary atherosclerosis (i.e., NOCA group). Patients with stenosis of at least one main coronary artery ≥50% in coronary angiography were diagnosed with coronary heart disease (i.e., CAD group); based on symptoms, signs, and related examination results, CAD patients were further divided into stable angina pectoris (SAP), unstable angina pectoris (UAP), and acute myocardial infarction (AMI). The control group consisted of subjects with negative coronary angiography results (i.e., NCA group).

[0078] Exclusion criteria: Subjects were excluded if they had myocarditis, fatty liver, cerebral infarction, peripheral coronary atherosclerosis, cervical coronary atherosclerosis, cerebral coronary atherosclerosis, malignant tumors, autoimmune diseases, and infectious diseases.

[0079] Example 7

[0080] Tissue cryosectioning and immunofluorescence staining

[0081] Human coronary artery atherosclerotic plaques and normal artery inner wall samples or mouse thoracic aorta tissue samples fixed with 4% paraformaldehyde were taken out, dehydrated with 40% sucrose solution (prepared in PBS), and left to stand overnight at 4°C. The next day, the dehydrated samples were embedded with OCT embedding medium, and then 8 μm tissue frozen sections were made using a cryostat (Leica, CM 3050S).

[0082] Perform immunofluorescence on frozen tissue samples. The specific steps are as follows:

[0083] (1) Use an immunohistochemistry pen to draw a circle around the tissue, add an appropriate amount of PBS to wash the tissue, and gently dry it with filter paper.

[0084] (2) Membrane disruption: Add an appropriate amount of 0.3% (w / w) Triton X-100 to the encircled tissue and incubate at room temperature for 30 min.

[0085] (3) Washing: Wash three times with appropriate amount of PBS, 5 min each time.

[0086] (4) Blocking: Add an appropriate amount of 2% BSA-PBS solution and incubate at room temperature for 1 h.

[0087] (5) Incubation with primary antibody: Add the diluted anti-LL-37-ApoB-100 or Cramp-ApoB-100 complex rabbit polyclonal antibody prepared in Example 3 and incubate overnight at 4°C. The antibody is diluted with the above blocking solution (1:3000). If two antibodies need to be incubated at the same time, mix the two antibodies at a ratio of 1:1 and add them to the tissue.

[0088] (6) Cleaning: Same as step (3).

[0089] (7) Incubation with fluorescent secondary antibody: Add 1:200 diluted anti-rabbit fluorescent secondary antibody (Thermo Fisher Scientific, A11008) and incubate at 37°C in the dark for 1 h. Select anti-rabbit secondary antibodies with different fluorescent labels according to the desired fluorescent color.

[0090] (8) Cleaning: Same as step (3).

[0091] (9) Sealing: Add a small amount of mounting medium containing DAPI onto the tissue and gently cover it with a coverslip.

[0092] (10) The sealed tissue sections were placed on a laser confocal microscope for observation.

[0093] The frozen sections of atherosclerotic plaques and normal artery samples were examined by immunofluorescence to detect the localization of LL-37 and ApoB-100 in atherosclerotic plaques and normal arterial walls. LL-37 was labeled with green fluorescence, ApoB-100 with red fluorescence, and DAPI (blue fluorescence, specifically recognizing double-stranded DNA) marked the location of cell nuclei in tissues. The results are shown in Figure 5 , scale bar is 30 μm.

[0094] Depend on Figure 5 It can be seen that there are a large number of LL-37 and ApoB-100 in the atheromatous plaques of patients with coronary atherosclerosis, and most of the LL-37 and ApoB-100 in the plaques are obviously co-localized. However, there is almost no LL-37 and ApoB-100 in the artery samples of normal people.

[0095] Figure 6 The figure shows the results of the presence of a large amount of Cramp-ApoB-100 complexes in mouse coronary atherosclerotic plaques and plasma. Figure 6 A in the figure indicates that the Apoe1 gene was found in the rats fed with high-fat diet by immunofluorescence detection. - / - There is a large amount of Cramp-ApoB-100 complex in the thoracic aortic plaques of mice, while ApoE - / - There is no obvious presence of the complex in the mouse thoracic aorta. Green fluorescence marks the Cramp-ApoB-100 complex, and blue fluorescence marks the location of the cell nucleus in the tissue. The scale bar is 100 μm. Figure 6 B is Apoe in the experiment that was fed a high-fat diet for two, four, and six weeks. - / - The concentrations of Cramp-ApoB-100 complex in mouse plasma were 0.85±0.34, 4.86±1.43, and 9.95±2.57 (mean±SD, n=20), respectively. **p<0.01, ***p<0.001.

[0096] Depend on Figure 6 Apoe - / - After mice were induced with coronary atherosclerosis, a large number of Cramp-ApoB-100 complexes appeared in the thoracic aorta atherosclerotic plaques. As the high-fat diet induction time increased (the area of ​​thoracic aortic atherosclerotic plaques in mice gradually increased), the ApoE - / - The level of Cramp-ApoB-100 complex in mouse plasma also gradually increased.

[0097] Example 8

[0098] Concentration determination of LL-37-ApoB-100 complex and Cramp-ApoB-100 complex

[0099] The determination of the concentration of LL-37-ApoB-100 complex in the plasma of patients with coronary atherosclerotic heart disease and the control group, the determination of the concentration of Cramp-ApoB-100 complex in the plasma of coronary atherosclerosis model mice, and the determination of the titer of rabbit polyclonal antibodies were all determined by indirect ELISA method. The specific steps are as follows:

[0100] (1) Coating antigen: Use coating solution to dilute different concentrations of LL-37, Cramp, ApoB-100 and their complexes as standards, dilute plasma with coating solution, and add 100 μl to each well of the ELISA plate. Incubate at 37°C for 1 hour, then coat overnight at 4°C. The coating solution is 0.05M carbonate buffer at pH 9.6, and the preparation method is: 1.59g sodium carbonate and 2.93g sodium bicarbonate are dissolved in 1L deionized water.

[0101] (2) Washing: After coating, pour out the coating solution in the plate wells and wash with washing solution, 250 μl per well, 3 to 5 minutes each time, 3 to 5 times. After the last wash, pat dry as much as possible. The washing solution is PBS buffer containing 0.1% Tween-20. The formula of PBS is: 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, 8.7g sodium chloride and 2.16g sodium dihydrogen phosphate dodecahydrate dissolved in 1L deionized water.

[0102] (3) Blocking: Add 200 μl of blocking solution to each well and block at 37°C for 1 h. The blocking solution is a 1% BSA solution prepared with washing solution.

[0103] (4) Adding primary antibody: After blocking, add the anti-LL-37-ApoB-100 or Cramp-ApoB-100 complex rabbit polyclonal antibody prepared in Example 3, 100 μl per well, and incubate at 37° C. for 1 h. The antibody is diluted with blocking solution.

[0104] When testing the antibody titer, add rabbit antiserum after blocking: dilute the rabbit antiserum in multiples (1:200, 1:400, ..., 1:102400) with blocking solution, 100 μl per well, incubate at 37°C for 1 hour. Set up blank control and negative control (rabbit IgG) at the same time.

[0105] (5) Washing: After the primary antibody incubation is completed, wash the product using the same steps as step (2) above.

[0106] (6) Adding enzyme-labeled antibody conjugate: After washing, add streptavidin-horseradish peroxidase conjugate (when testing antibody titer, add HPR-labeled anti-rabbit IgG antibody), 100 μl per well, and incubate at 37°C in the dark for 1 h.

[0107] (7) Washing: After the incubation is completed, wash with the same steps as above (2).

[0108] (8) Color development: Add 100 μl of TMB to each well and incubate at 37°C in the dark for 10 to 30 minutes.

[0109] (9) Stop: Add 100 μl of stop solution to each well to stop color development. The stop solution is 2 mol / l sulfuric acid, and the formula is: slowly add 21.7 ml of concentrated sulfuric acid to 78.3 ml of water and stir slowly to mix.

[0110] (10) Reading: Read the absorbance at 450 nm and 630 nm using an ELISA reader.

[0111] (11) Calculate the concentration based on the standard curve.

[0112] Figure 7 and Figure 8 Apoe induced by high-fat diet for 2, 4, and 6 weeks - / - The results of the positive correlation between the Cramp-ApoB-100 complex content in mouse plasma and the area of ​​coronary atherosclerotic plaques are shown in Figure 1. Figure 7 A to C in Figure 3 are the correlations between plasma Cramp-ApoB-100 complex or ApoB-100 levels and coronary atherosclerotic plaque area. Figure 8 A in the figure is the correlation between plasma Cramp level and coronary atherosclerotic plaque area. Figure 8 B-F in Figure 3 are the correlations between plasma lipid levels and coronary atherosclerotic plaque area. n = 20 mice per group. TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol.

[0113] Depend on Figure 7 and Figure 8 It can be seen that after two, four, and six weeks of high-fat diet, Apoe - / -The area of ​​atherosclerotic plaques in the thoracic main coronary arteries of mice gradually increased. By comparing the correlation between the aortic root plaque area and the levels of Cramp, ApoB-100, Cramp-ApoB-100 complex, ApoA, TG, TC, LDL-C, and HDL-C in plasma at different times, it was found that with the increase in the aortic root plaque area, the levels of Cramp, ApoB-100, Cramp-ApoB-100 complex, TG, TC, and LDL-C in mouse plasma had a trend of gradual increase (the correlation coefficient r value was positive and the value was getting larger and larger), and the levels of ApoA and HDL-C had a trend of gradual decrease (the correlation coefficient r value was negative and the value was getting smaller and smaller). However, the correlation coefficient r value of Cramp-ApoB-100 complex was higher than that of other groups, and there was a significant difference (p < 0.001), that is, the increase in the aortic root plaque area was more strongly correlated with the increase in the content of Cramp-ApoB-100 complex, and the increase in the content of this complex could better predict the increase in the area of ​​atherosclerotic plaques.

[0114] Fig. 9 The results of the plasma levels of LL-37-ApoB-100 complex in the control group, coronary atherosclerosis group and patients with different types of coronary heart disease are shown in the figure. In the NC, NOCA, SA, UA and AMI groups, the median (IQR) of the plasma concentration of LL-37-ApoB-100 complex was 31.74 (20.13-38.66) ng / ml, 38.73 (30.95-46.27) ng / ml, 45.75 (36.09-63.34) ng / ml, 49.00 (38.22-65.15) ng / ml and 52.61 (41.68-69.80) ng / ml. NC, control group (no coronary artery stenosis, n = 227); NOCA, coronary artery atherosclerosis group (arterial stenosis rate less than 50%, n = 92); SA, stable angina (n = 129); UA, unstable angina (n = 174); AMI, acute myocardial infarction (n = 240).

[0115] In this observational case-control study, based on the patient's symptoms and signs and related examination results, the severity of coronary atherosclerosis in patients was coronary atherosclerosis > stable angina > unstable angina > acute myocardial infarction. The median (IQR) plasma concentration of LL-37-ApoB-100 complex in these four groups of patients was higher than that in the control group, that is, the plasma concentration of LL-37-ApoB-100 complex increased with the severity of coronary atherosclerosis.

[0116] Fig.10The ROC curve results of LL-37-ApoB-100 complex, LL-37 and conventional risk factors for diagnosing coronary atherosclerotic heart disease (n=862). ***p<0.001. Homocysteine ​​(Hcy), high-sensitivity C-reactive protein (hsCRP), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C).

[0117] The area under the ROC curve (AUC) is shown in Table 2.

[0118] Table 2 Area under the ROC curve AUC results

[0119] Group AUC (95% CI) p-value LL-37-ApoB-100 0.82(0.81-0.85) 8.63E-48 LL-37 0.67(0.63-0.70) 1.64E-13 Hc 0.62(0.59-0.65) 8.39E-08 hsCRP 0.61(0.58-0.64) 8.82E-07 TG 0.56(0.52-0.59) 9.73E-03 TC 0.56(0.53-0.61) 4.00E-03 ApoB-100 0.54(0.51-0.57) 7.68E-02 LDL-C 0.52(0.49-0.56) 0.33 HDL-C 0.52(0.49-0.55) 0.4

[0120] The ROC curve and the area under the curve AUC values ​​can reflect the sensitivity and specificity of LL-37-ApoB-100 complex, LL-37 and conventional risk factors (Hcy, hsCRP, TG, TC, ApoB-100, LDL-C and HDL-C) in diagnosing coronary atherosclerotic heart disease. The area under the ROC curve AUC of LL-37-ApoB-100 complex is higher than that of other factors, which means that the complex has higher sensitivity and specificity than traditional coronary atherosclerosis / coronary heart disease markers.

[0121] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting LL-37-ApoB-100 complex in the preparation of a diagnostic kit for coronary atherosclerosis or a disease associated with coronary atherosclerosis, wherein the LL-37-ApoB-100 complex is composed of antimicrobial peptide LL-37 and apolipoprotein B-100; the disease associated with coronary atherosclerosis is coronary heart disease; the coronary heart disease is composed of stable angina pectoris, unstable angina pectoris and acute myocardial infarction; The preparation method of the LL-37-ApoB-100 complex comprises: The antimicrobial peptide LL-37 and apolipoprotein B-100 are mixed and allowed to stand to obtain the LL-37-ApoB-100 complex; The mass ratio of the antimicrobial peptide LL-37 to apolipoprotein B-100 is 250-300:100-150; and the standing time is 25-40 minutes.

2. The use according to claim 1, characterized in that: The reagent for detecting the LL-37-ApoB-100 complex comprises an anti-LL-37-ApoB-100 complex antibody.

3. A diagnostic kit for coronary atherosclerosis or a disease related to coronary atherosclerosis, characterized in that: The invention comprises an anti-LL-37-ApoB-100 complex antibody, wherein the LL-37-ApoB-100 complex is composed of the antimicrobial peptide LL-37 and apolipoprotein B-100; The preparation method of the LL-37-ApoB-100 complex comprises: mixing the antimicrobial peptide LL-37 and apolipoprotein B-100, and letting the mixture stand to obtain the LL-37-ApoB-100 complex; The mass ratio of the antimicrobial peptide LL-37 to apolipoprotein B-100 is 250-300:100-150; and the standing time is 25-40 minutes.

4. The diagnostic kit according to claim 3, characterized in that The anti-LL-37-ApoB-100 complex antibody includes a polyclonal antibody.

5. The diagnostic kit according to claim 4, characterized in that The polyclonal antibodies include rabbit-derived polyclonal antibodies.

6. The diagnostic kit according to claim 3 or 4, characterized in that The anti-LL-37-ApoB-100 complex antibody is labeled with biotin.

7. The diagnostic kit according to claim 6, characterized in that The diagnostic kit also includes an enzyme-linked immunosorbent assay reagent.

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