Young coronary heart disease marker HTRA1 and application thereof

Abstract

The invention discloses a young coronary heart disease marker HTRA1 and application thereof, and belongs to the technical field of biomarkers. The expression level of the HTRA1 in serum of young coronary heart disease patients is obviously increased, the ROC value of the HTRA1 in the young coronary heart disease patients is high, healthy control and young coronary heart disease groups can be effectively distinguished, it is prompted that the HTRA1 has high value for predicting and diagnosing the young coronary heart disease, and the HTRA1 can be used for developing corresponding diagnostic reagents and kits and has good application prospects. The kit is used for detecting the content of HTRA1 in a human body sample, so that auxiliary diagnosis of the young coronary heart disease is realized. The diagnostic reagent and the kit have the advantages of being high in specificity, high in sensitivity, easy and convenient to operate and the like, and important reference bases can be provided for early discovery and treatment of the young coronary heart disease.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology, and in particular to HTRA1, a biomarker for coronary heart disease in young people, and its applications. Background Technology

[0002] Coronary artery disease (CAD) in young adults refers to coronary heart disease occurring in people under the age of 45, and its incidence has been increasing year by year in recent years. According to the latest data from the World Health Organization (WHO), the number of young CAD patients worldwide is increasing at a rate of approximately 5% per year. In epidemiological surveys in some Chinese cities, the incidence rate of CAD in young adults has risen from less than 1% ten years ago to 3%-5% currently. Young CAD patients often experience acute onset, severe symptoms, and poor prognosis due to their young age of onset and frequently combined with various unhealthy lifestyle habits (such as prolonged sleep deprivation, smoking, excessive alcohol consumption, and lack of exercise). Some young CAD patients experience acute myocardial infarction as their first symptom, and some even die suddenly due to delayed diagnosis and treatment, placing a heavy burden on their families and society.

[0003] Currently, commonly used clinical diagnostic methods for coronary heart disease include electrocardiography (ECG) and coronary angiography, but these methods have certain limitations. While ECG is the most basic diagnostic tool for coronary heart disease, its sensitivity for diagnosing early-stage coronary artery disease is relatively low. For example, in some young patients with coronary heart disease, the degree of myocardial ischemia is mild in the early stages, and the ECG may only show nonspecific ST-T changes, making it difficult to distinguish from physiological changes or other heart diseases, leading to a high rate of missed diagnoses. Although coronary angiography is considered the "gold standard" for diagnosing coronary heart disease, it is an invasive procedure with risks such as vascular injury, contrast agent allergy, and arrhythmia during the operation. Furthermore, the examination is expensive and not suitable for large-scale screening. In addition, other imaging examinations such as echocardiography and cardiac magnetic resonance imaging mainly focus on assessing cardiac structure and function, and have limited value in the early diagnosis of coronary artery lesions.

[0004] HTRA1 (Heat-Demanding Factor A1) is a serine protease that plays a crucial role in various physiological and pathological processes. Studies have shown that HTRA1 is essential in the development and progression of atherosclerosis, influencing the progression of coronary artery disease by participating in multiple pathological processes such as extracellular matrix remodeling, regulation of inflammatory responses, and migration and proliferation of vascular smooth muscle cells. Furthermore, its expression level is closely related to the severity of coronary artery disease. However, there are currently no reports on the application of HTRA1 in the diagnosis of coronary artery disease in young adults. Existing research mainly focuses on elderly patients with coronary artery disease, lacking in-depth exploration and application development of specific biomarkers for young adults. There is an urgent need to find a highly specific and sensitive biomarker and develop corresponding diagnostic reagents and kits for the early diagnosis of coronary artery disease in young adults. Summary of the Invention

[0005] The purpose of this invention is to provide a biomarker for coronary heart disease in young people, HTRA1, and its application, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention is the application of HTRA1 as a biomarker in the preparation of products for the diagnosis or monitoring of coronary heart disease in young people.

[0008] The second technical solution of the present invention is a product for diagnosing or monitoring coronary heart disease in young people, including a specific antibody against the HTRA1 protein.

[0009] Based on the above technical solution, the present invention has the following technical effects:

[0010] 1. High specificity: Specific antibodies against HTRA1 protein can accurately identify HTRA1 protein in samples, reducing cross-reaction with other proteins.

[0011] 2. High sensitivity: It can detect low concentrations of HTRA1 protein in samples, improving diagnostic sensitivity.

[0012] 3. Easy to use: The kit provides complete testing reagents and operating procedures, eliminating the need for complex instruments and equipment, making it convenient for clinical application.

[0013] 4. It provides a new method for the early diagnosis of coronary heart disease in young people, which helps to improve the early detection rate of coronary heart disease in young people and provides a basis for timely treatment.

[0014] This invention discovered that HTRA1 expression levels were significantly elevated in the serum of young patients with coronary heart disease (CHD), and that the ROC value was higher in these patients, effectively distinguishing them from healthy controls. This suggests that HTRA1 has high value in predicting and diagnosing CHD in young people and can be used to develop corresponding diagnostic reagents and kits for detecting HTRA1 levels in human samples, thereby enabling the auxiliary diagnosis of CHD in young people. The diagnostic reagents and kits of this invention have advantages such as high specificity, high sensitivity, and ease of operation, providing important reference for the early detection and treatment of CHD in young people. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a scatter plot of serum HTRA1 concentrations in the first group of young people with coronary heart disease and the healthy negative control group.

[0017] Figure 2 Scatter plot (a) of serum HTRA1 concentration in the second group of young people with coronary heart disease and healthy negative control group and Western Blot plot of HTRA1 expression (b).

[0018] Figure 3 This is an ROC curve analysis of HTRA1 used to detect coronary heart disease in young people. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0025] This invention provides the application of HTRA1 as a biomarker in the preparation of products for the diagnosis or monitoring of coronary heart disease in young people.

[0026] In some specific implementation schemes, the method for diagnosing or monitoring coronary heart disease in young people is as follows: quantitatively detect HTRA1 in the sample to be tested and compare it with the control sample or reference sample. If the level of HTRA1 in the sample to be tested is significantly upregulated, it indicates that the patient has coronary heart disease in young people.

[0027] In some specific implementation schemes, the sources of the samples to be tested include human body fluids, cell culture media, cells, tissue samples, and organ samples;

[0028] The human body fluids include blood, serum, plasma, lymph, sweat, saliva, vitreous fluid, synovial fluid, lymph, semen, cerebrospinal fluid, and urine.

[0029] In some specific implementations, the product includes reagents, kits, chips, test strips, or high-throughput sequencing platforms.

[0030] This invention also provides a product for diagnosing or monitoring coronary heart disease in young people, including a specific antibody against the HTRA1 protein.

[0031] In some specific implementations, sample processing solutions, calibrators, and quality control products are also included.

[0032] In some specific implementations, the sample processing solution contains Tris-HCl buffer, NaCl, and Tween-20.

[0033] This invention discovered that HTRA1 expression levels were significantly elevated in the serum of young patients with coronary heart disease (CHD), and that the ROC value was higher in these patients, effectively distinguishing them from healthy controls. This suggests that HTRA1 has high value in predicting and diagnosing CHD in young people and can be used to develop corresponding diagnostic reagents and kits for detecting HTRA1 levels in human samples, thereby enabling the auxiliary diagnosis of CHD in young people. The diagnostic reagents and kits of this invention have advantages such as high specificity, high sensitivity, and ease of operation, providing important reference for the early detection and treatment of CHD in young people.

[0034] In this application's description, the ROC curve, short for Receiver Operating Characteristic curve, is a curve plotted using a series of different binary classification methods (cutoff values ​​or decision thresholds), with the true positive rate (sensitivity) as the ordinate and the false positive rate (1-specificity) as the abscissa. In this application's description, the AUC (Area Under Curve) is defined as the area under the ROC curve and the coordinate axes; obviously, this area will not be greater than 1. Since the ROC curve is generally above the line y = x, the AUC value ranges between 0.5 and 1. An AUC < 0.5 indicates no diagnostic significance; an AUC = 0.5–0.7 indicates low diagnostic accuracy; an AUC = 0.7–0.9 indicates moderate diagnostic accuracy; and an AUC > 0.9 indicates high diagnostic accuracy. The closer the AUC is to 1.0, the higher the reliability of the detection method.

[0035] This invention experimentally demonstrates that HTRA1 expression is significantly upregulated in young patients with coronary heart disease, and can be applied in the preparation of diagnostic products for coronary heart disease in young people. This molecular marker can be used for early diagnosis of coronary heart disease in young people, which is rapid and effective. This is not only significant for early treatment of coronary heart disease in young people and saving medical costs, but also provides therapeutic targets and important evidence for clinical applications such as gene therapy and drug therapy.

[0036] Example 1

[0037] ELISA detection of HTRA1 expression in serum of young people with coronary heart disease and healthy negative controls

[0038] 1. Study subjects: Peripheral blood samples were collected from 20 young patients diagnosed with coronary heart disease and 20 healthy controls admitted to the Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All patients signed informed consent forms, and the study was approved by the review committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

[0039] Inclusion criteria:

[0040] Young patients with coronary heart disease:

[0041] The patient must have been diagnosed with at least one coronary artery that showed a diameter reduction of ≥50% from two or more different angles via coronary angiography; have complete medical records and coronary angiography results; and be under 45 years of age.

[0042] Health control group: Recruit age-matched healthy individuals.

[0043] Exclusion criteria: recent surgery, trauma, acute infection, or coexisting chronic diseases such as hematological diseases, immune system diseases, and malignant tumors.

[0044] 2. Collect serum: Centrifuge the collected peripheral blood samples at 3000 r / min for 15 min and collect the supernatant to obtain the serum sample.

[0045] 3. Sample Detection: ELISA kits (purchased from Huamei Biotechnology Co., Ltd., specifically the Human Serine Protease HTRA1 Enzyme-Linked Immunosorbent Assay Kit, catalog number: CSB-EL010901HU) were used to validate serum samples from healthy individuals and young patients with coronary heart disease. Figure 1 The figure shows the statistical graph of HTRA1 expression levels in the serum of the two groups. Figure 1 It can be seen that the expression was significantly higher in the young patients with coronary heart disease.

[0046] Example 2

[0047] Western blot analysis of HTRA1 expression in serum of young people with coronary heart disease and healthy negative controls.

[0048] Based on the results of Example 1, peripheral blood samples were collected from 105 young patients with coronary heart disease and 105 healthy controls.

[0049] Following the method described in Example 1, the serum HTRA1 expression levels in the two groups were further examined, confirming that the serum HTRA1 level in young patients with coronary heart disease was significantly higher than that in healthy controls. Figure 2 (a). Meanwhile, Western blot analysis revealed significantly increased expression of HTRA1 (HTRA1 monoclonal antibody purchased from Huamei Biotechnology Co., Ltd., catalog number: CSB-RA835695MA1HU) in young patients with coronary heart disease. Figure 2 (b) The specific method is as follows:

[0050] 1. Following the method described in Example 1, peripheral serum protein levels were collected from 105 young patients with coronary heart disease and 105 healthy controls. The inclusion and exclusion criteria were the same as in Example 1. All patients signed informed consent forms, and the data was approved by the review committee of Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology.

[0051] 2. Determination of total protein concentration

[0052] 2.1 Constructing a standard curve for protein concentration determination: This part of the experiment used the BCA protein concentration assay kit provided by Beyotime Biotechnology Co., Ltd., and the operation was performed according to the instructions included with the kit. The 2 mg / mL BSA standard was diluted to different concentration gradients as follows: 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, according to the product instructions.

[0053] 2.2 Prepare the AB mixture by mixing solutions A and B from the BCA kit at a ratio of 50:1. Take 95 μL of the AB mixture and mix it with 5 μL of standard diluents of different concentrations and the protein sample to be tested.

[0054] 2.3 Incubate at 37℃ for 30 minutes, then measure the OD value of each sample using a microplate reader. Plot a standard curve using the OD values ​​of the standard dilutions and the corresponding protein concentrations, and calculate the protein concentration of the sample to be tested.

[0055] 2.4 After aliquoting the protein samples, add the corresponding volume of protein loading buffer at a ratio of 5:1, boil in a 100°C metal bath for 5 minutes, and then immediately place on ice to cool fully to achieve the purpose of protein denaturation.

[0056] 2.5 Prepare SDS-PAGE gels. The formulations of separating gel solutions of different concentrations are shown in Table 1.

[0057] Table 1 Formulations of separating gel solutions at different concentrations

[0058]

[0059] 2.6 Slowly pour the prepared separating gel buffer solution into the assembled glass plate, leaving a liquid level of approximately 2-3 cm. Slowly and evenly fill the separating gel with deionized water. After the separating gel solidifies, pour out the deionized water from the glass plate, blot it dry with filter paper, and then slowly pour in the stacking gel and insert the comb. The stacking gel formula is shown in Table 2.

[0060] Table 2 Concentrated Gel Formulation

[0061]

[0062] 2.7 After the stacking gel has solidified, install the gel glass plate into the electrophoresis apparatus. Carefully remove the comb and gently rinse the sample wells with deionized water. Pour 1L of 1×Tris-glycine electrophoresis buffer into the electrophoresis tank, ensuring the wells are submerged and air bubbles are expelled. Then, use a pipette or micropipette to inject the corresponding protein sample and protein marker into the wells. The formulation of 1×Tris-glycine electrophoresis buffer is shown in Table 3.

[0063] Table 31 × Tris-glycine electrophoresis buffer formulation

[0064]

[0065]

[0066] 2.8 Protein gel electrophoresis

[0067] After loading the sample, connect the corresponding positive and negative electrodes to the power supply. Then adjust the power supply voltage to 80V. Once the protein marker bands have separated, adjust the voltage to 120V. After 1.5-2 hours, when the blue loading buffer has reached near the bottom of the separating gel, immediately turn off the power and stop electrophoresis. Remove the gel plate from the electrophoresis apparatus and proceed with the subsequent membrane transfer steps.

[0068] 2.9 Transfer of film

[0069] Wet transfer was performed using 2L of 1× transfer buffer. A PVDF membrane of appropriate size was cut according to the area of ​​the sample gel, and then the cut PVDF membrane was activated by immersing it in methanol solution for 1 minute. With the black side of the transfer clamp facing down, the following items were placed in order: sponge pad, 2 sheets of filter paper, protein gel, activated PVDF membrane, 2 sheets of filter paper, sponge pad. The clamp was then secured, ensuring no air bubbles were removed between the PVDF membrane and the gel. The clamp was placed into the transfer tank with the black side facing down, and the protein was transferred to the PVDF membrane at a current of 200mA per tank. The transfer time depended on the molecular weight of the transferred protein, with a transfer time of 30 minutes per 50kD. The 1× transfer buffer formulation is shown in Table 4.

[0070] Table 41 × Transfer Buffer Formulation

[0071]

[0072] 2.10 Closed

[0073] After the transfer was complete, the PVDF membrane was removed from the transfer clamp and placed in an antibody incubation box containing 10 ml of protein blocking buffer. The incubator was then placed on a horizontal shaker and gently shaken for 2 hours at room temperature. The protein blocking buffer formulation is shown in Table 5.

[0074] Table 5 Protein Blocking Solution Formulation

[0075]

[0076] 2.11 Primary Antibody Incubation and Washing: After blocking, rinse with 1×TBST, then add the corresponding primary antibody working solution (dilute the primary antigen solution with protein blocking solution at a ratio of 1:1500) and incubate overnight at 4°C with gentle shaking. The next day, recover the primary antibody and rinse 6 times with 1×TBST with gentle shaking for 10 minutes each time. The 1×TBST formulation is shown in Table 6.

[0077] Table 61 × TBST Formulation

[0078]

[0079] 2.12 Secondary Antibody Incubation and Rinsing: After the final rinse with the primary antibody, dilute the secondary antibody with 10 ml of 1×TBST at a ratio of 1:2000. Incubate on a shaker at room temperature for 2 hours. After incubation, rinse 6 times with 1×TBST, shaking gently for 10 minutes each time. After rinsing with the secondary antibody, add an appropriate amount of 1×TBST to soak the PVDF membrane for subsequent experiments.

[0080] 2.13 Exposure: Prepare the exposure solution by mixing ECL chemical discovery color developer solution A and solution B in a 1:1 ratio. After adding the developer solution to the film, place it in an exposure machine for color development.

[0081] like Figure 2 As shown in Figure b, in multiple samples, the protein expression level of HTRA1 in young patients with coronary heart disease was significantly higher than that in the healthy control group.

[0082] Example 3

[0083] Preparation of HTRA1 Diagnostic Kit for Coronary Artery Disease in Young People

[0084] 1. HTRA1 biomarker

[0085] In this embodiment, a protein biomarker for coronary heart disease risk assessment and / or diagnosis is first provided. This protein biomarker is HTRA1 (NCBI website: serineproteaseHTRA1precursor[Homosapiens]-Protein- NCBI ).

[0086] 2. Preparation of HTRA1 diagnostic reagents: Dilute the HTRA1 monoclonal antibody with PBS buffer to an appropriate concentration to serve as the coating antibody and detection antibody.

[0087] Sample processing solution preparation: Weigh 12.1g of Tris and 8.76g of NaCl, dissolve them in 800mL of distilled water, adjust the pH to 7.4 with hydrochloric acid, add 1mL of Tween-20, and bring the volume to 1000mL to obtain the sample processing solution.

[0088] Calibrator preparation: The purified HTRA1 protein was diluted with sample processing solution to prepare calibrators of different concentrations, such as 0 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 400 ng / mL.

[0089] Preparation of quality control samples: Human samples with known HTRA1 content are selected and processed to serve as quality control samples, including low-concentration and high-concentration quality control samples.

[0090] Example 4

[0091] Methods for diagnosing coronary heart disease in young people using reagent kits

[0092] Sample collection: 5 mL of venous blood was collected from 105 young patients with coronary heart disease and healthy controls in the second group. The serum was separated by centrifugation and used as the test sample.

[0093] Sample processing: Add 100 μL of serum to 900 μL of sample processing solution, mix well, and incubate at 37°C for 10 minutes.

[0094] Testing steps:

[0095] (1) Take out the microplate coated with HTRA1 monoclonal antibody, add 50 μL of the treated sample and 50 μL of detection antibody, and incubate at 37°C for 1 hour.

[0096] (2) Wash the microplate three times, each time adding 300 μL of washing buffer (PBS containing 0.05% Tween-20).

[0097] (3) Add 100 μL of enzyme-labeled secondary antibody and incubate at 37°C for 30 minutes.

[0098] (4) Wash the microplate 5 times.

[0099] (5) Add 100 μL of substrate solution and incubate at 37°C in the dark for 15 minutes.

[0100] (6) Add 50 μL of stop solution and measure the absorbance at a wavelength of 450 nm.

[0101] Result Interpretation: Calculate the HTRA1 content in the sample based on the standard curve plotted using the calibrators. If the HTRA1 content in the sample is higher than the normal reference range, it is considered a possible diagnosis of coronary heart disease in young adults, requiring further confirmation based on clinical symptoms and other examination results.

[0102] like Figure 3 As shown, ROC analysis of data from young patients with coronary heart disease and healthy controls yielded an AUC of 0.8307, indicating that HTRA1 levels have high accuracy in diagnosing coronary heart disease in young people. Furthermore, by calculating the Youden index for all samples and taking the maximum value, the sensitivity of HTRA1 in detecting coronary heart disease in young people was 0.7238, and the specificity was 0.7048. This indicates that within a certain range, the kit can detect low concentrations of HTRA1 protein in the samples, and the HTRA1 protein-specific antibody shows minimal cross-reactivity with other proteins. This demonstrates the feasibility of using HTRA1 in diagnosing coronary heart disease in young people.

[0103] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of HTRA1 as a biomarker in the preparation of products for the diagnosis or monitoring of coronary heart disease in young people.

2. The application according to claim 1, characterized in that, The method for diagnosing or monitoring coronary heart disease in young people is as follows: quantitative detection of HTRA1 in the sample to be tested is performed and compared with the control sample or reference sample. If the level of HTRA1 in the sample to be tested is significantly upregulated, it indicates that the patient has coronary heart disease in young people.

3. The application according to claim 2, characterized in that, The sources of the samples to be tested include human body fluids, cell culture media, cells, tissue samples, and organ samples; The human body fluids include blood, serum, plasma, lymph, sweat, saliva, vitreous fluid, synovial fluid, lymph, semen, cerebrospinal fluid, and urine.

4. The application according to claim 1, characterized in that, The products include reagents, kits, chips, test strips, or high-throughput sequencing platforms.

5. A product for diagnosing or monitoring coronary heart disease in young people, characterized in that, Including specific antibodies against the HTRA1 protein.

6. The product according to claim 5, characterized in that, It also includes sample processing solutions, calibrators, and quality control products.

7. The product according to claim 6, characterized in that, The sample processing solution contains Tris-HCl buffer, NaCl, and Tween-20.

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