Application of MXRA5 as marker in preparation of reagent and kit for diagnosing calcified aortic valve disease

Abstract

The invention relates to the technical field of calcified aortic valve disease reagents and kits, in particular to application of MXRA5 as a marker in preparation of a calcified aortic valve disease diagnosis reagent and kit, and finds that the level of MXRA5 in a calcified aortic valve disease patient is remarkably increased for the first time, and the metabolite MXRA5 has a very high area under a curve, so that the calcified aortic valve disease diagnosis reagent can be applied to diagnosis of calcified aortic valve diseases. The metabolite can effectively distinguish healthy control and CAVD groups, so that application of MXRA5 to CAVD has high diagnostic value, and the metabolite has great significance in diagnosis of early-stage calcified aortic valve diseases and guidance of standardized treatment; the diagnostic index provided by the invention is plasma metabolite, and can be detected only by taking a small amount of blood, so that the kit is basically noninvasive.

Description

Technical Field

[0001] The present invention relates to the technical field of reagents and kits for calcific aortic valve disease, and is an application of MXRA5 as a marker in the preparation of reagents and kits for diagnosing calcific aortic valve disease. Background Art

[0002] Calcified aortic valve disease (CAVD) is a chronic condition affecting the aortic valve, characterized by thickening of the valve leaflets, calcified nodules, and fibrosis. As the disease progresses, it can lead to aortic valve stenosis and altered hemodynamics, leading to structural changes in the left ventricle and ultimately, chronic heart failure. According to the American Heart Association, the prevalence of CAVD in individuals aged 65 and older is 20% to 30%, while the prevalence in individuals aged 85 and older is 48% to 57%. From 1990 to 2019, the global incidence, prevalence, and mortality rates of CAVD increased by 3.51-fold, 4.43-fold, and 1.38-fold, respectively. As China enters an aging society, CAVD will become a significant burden of disease in the future. However, there are currently no effective medications to mitigate or slow the progression of CAVD, and surgery or percutaneous aortic valve replacement remain the only treatment options for CAVD. Understanding the risk factors for CAVD is fundamental to identifying and implementing interventions. The particularity of CAVD risk factors and the diagnostic and therapeutic value of various inflammatory indicators and new inflammatory markers in CAVD patients are worthy of our in-depth discussion and research.

[0003] Extensive research is currently underway to identify blood biomarkers for calcific aortic valve disease to improve diagnosis and facilitate personalized treatment. In-depth studies have focused on genetic polymorphisms, miRNAs, and immune cells, but their widespread clinical application is often limited by complex assays and limited practicality. ELISA, or enzyme-linked immunosorbent assay, is a commonly used biochemical analytical method, particularly important in immunological testing. In ELISA, an antigen or antibody is bound to the surface of a solid support (such as a microplate). The sample to be tested is then added, allowing a specific immune reaction to occur between the antigen or antibody on the support. Next, an enzyme-labeled antigen or antibody is added to form an antigen-antibody-enzyme-labeled antigen / antibody complex. Finally, a substrate is added, which catalyzes the color development of the substrate, and the concentration of the analyte is quantitatively or qualitatively determined based on the color intensity or optical density. This method is simple to operate, cost-effective, and can rapidly and simultaneously measure multiple samples with high specificity. Studying differential metabolites in plasma and exploring metabolic markers are of great significance for achieving early diagnosis of CAVD.

[0004] Human matrix-remodeling associated protein 5 (MXRA5) is a matrix-remodeling protein. MXRA5 is a 312 kDa protein with a vascular endothelial growth factor receptor and a member of the MXRA family. The MXRA family's primary role in the human body is in cell adhesion and matrix remodeling. Its expression is elevated in the cartilage of patients with osteoarthritis, making MXRA5 widely used in the diagnosis and treatment evaluation of orthopedic diseases (Lin Qiaofeng et al. Expression of human matrix-remodeling associated protein in the feces of patients with colon cancer and its clinical significance [J]. Shenzhen Journal of Integrated Traditional Chinese and Western Medicine, Vol. 27, No. 8, April 2017, p. 3-4).

[0005] Patent publication number CN118542923A discloses a traditional Chinese medicine composition for treating calcific aortic valve disease, as well as its preparation method and use. The composition is made from the following raw medicinal materials in parts by weight: raw astragalus root, red peony root, stir-fried white atractylodes, Polygonatum sibiricum (wine-fermented), and Curcuma zedoaria (vinegar-peony root). The composition intervenes in calcific aortic valve disease by invigorating qi and promoting blood circulation, strengthening the body's resistance and eliminating symptoms. It improves ultrasound findings of localized or diffusely increased valve echogenicity, valve thickening, and aortic valve area stenosis, reduces peak flow velocity, and decreases the degree of aortic valve calcification. The composition is used to treat chest tightness and pain, shortness of breath, fatigue, and palpitations caused by qi deficiency and blood stasis in patients with aortic valve calcification. The composition is particularly suitable for patients with calcific aortic valve disease exhibiting the aforementioned symptoms.

[0006] Patent document with publication number CN113667733A discloses the application of circRNA PRDM5 in the development of diagnostic kits and therapeutic drugs for calcific aortic valve disease. By screening a circRNA PRDM5 related to the osteogenic differentiation of human valvular interstitial cells and verifying its application, by detecting the expression level of circRNA PRDM5 in normal and calcified aortic valve tissues, and conducting cellular functional studies on the circRNA PRDM5 gene, it was found that overexpression of circRNA PRDM5 can significantly promote the osteogenic differentiation ability of human valvular interstitial cells, proving that circRNA PRDM5 and its expression products can be used as markers for diagnosing calcific aortic valve disease, and can also be used as target genes for the preparation of drugs for treating calcific aortic valve disease, providing a new non-surgical treatment option for the treatment of aortic valve calcification.

[0007] There are currently no reports on MXRA5 as a marker for calcific aortic valve disease. Summary of the Invention

[0008] The present invention provides an application of MXRA5 as a marker in the preparation of reagents and kits for diagnosing calcific aortic valve disease. It discloses for the first time that the level of human matrix remodeling-associated protein 5 (hereinafter referred to as MXRA5) is significantly elevated in patients with calcific aortic valve disease, and that the metabolite (i.e., MXRA5) has a very high area under the curve. This metabolite can effectively distinguish between healthy controls and CAVD groups, suggesting that the application of MXRA5 in CAVD has a high diagnostic value.

[0009] One of the technical solutions of the present invention is achieved through the following measures: an application of MXRA5 as a marker in the preparation of a reagent for diagnosing calcific aortic valve disease.

[0010] The second technical solution of the present invention is achieved through the following measures: an application of MXRA5 as a marker in the preparation of a kit for diagnosing calcific aortic valve disease.

[0011] The following are further optimizations and / or improvements to the above technical solutions: The sample source of the above-mentioned MXRA5 is one or more of blood, serum, plasma, lymph, sweat, saliva, vitreous humor, synovial fluid, lymph, semen, cerebrospinal fluid, urine, cell culture fluid, cells, in vitro tissue samples and organ samples.

[0012] Preferably, the sample source of the MXRA5 is plasma.

[0013] In the above samples, the MXRA5 level was significantly elevated.

[0014] The MXRA5 level is significantly elevated in the plasma of the above-mentioned patients with calcific aortic valve disease.

[0015] The present invention discovered for the first time that the level of MXRA5 is significantly elevated in patients with calcific aortic valve disease, and the metabolite MXRA5 has a very high area under the curve. This metabolite can effectively distinguish between healthy controls and CAVD groups, suggesting that the application of MXRA5 in CAVD has a high diagnostic value and is of great significance for the diagnosis of early calcific aortic valve disease and guiding standardized treatment. Moreover, the diagnostic indicator provided by the present invention is a plasma metabolite, which only requires a small amount of blood for detection and is basically non-invasive. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Attachment Figure 1 Comparison of MXRA5 levels between the healthy control group and the CAVD group.

[0017] Attachment Figure 2 This is the ROC curve analysis diagram of MXRA5 for detecting CAVD. DETAILED DESCRIPTION

[0018] The present invention is not limited to the following embodiments, and specific implementation methods can be determined based on the technical solutions of the present invention and actual conditions.

[0019] Unless otherwise specified, the instruments, reagents, and materials used in this invention are all existing instruments, reagents, and materials in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples are all conventional experimental methods and detection methods in the prior art unless otherwise specified. Normal temperature and room temperature in this invention generally refer to temperatures between 15°C and 25°C, and are generally defined as 25°C.

[0020] The present invention will be further described below in conjunction with the embodiments: Example 1: Use of MXRA5 as a marker in the preparation of a reagent for diagnosing calcific aortic valve disease.

[0021] Example 2: Use of MXRA5 as a marker in the preparation of a kit for diagnosing calcific aortic valve disease.

[0022] The kit includes reagents for detecting the content of MXRA5 in a sample and instructions for using the kit to assess whether a subject has or is susceptible to calcific aortic valve disease.

[0023] Example 3: As an optimization of the above example, the sample source of MXRA5 is one or more of blood, serum, plasma, lymph, sweat, saliva, vitreous humor, synovial fluid, lymph, semen, cerebrospinal fluid, urine, cell culture fluid, cells, in vitro tissue samples and organ samples.

[0024] Example 4: As an optimization of the above example, the sample source of MXRA5 is plasma.

[0025] Example 5: As an optimization of the above example, the MXRA5 level in the sample is significantly increased.

[0026] Example 6: As an optimization of the above Example 5, the MXRA5 level in the plasma of patients with calcific aortic valve disease is significantly increased.

[0027] The research contents related to the application of MXRA5 as a marker in the preparation of reagents and kits for diagnosing calcific aortic valve disease described in the present invention are as follows: 1. Materials and Reagents Reagents Distilled or deionized water (H2O) Universal Diluent Standard Concentrated biotinylated detection antibody 100X (Biotin-antibody (100X)) Concentrated enzyme conjugate 100X (Streptavidin-HRP (100X)) Wash Buffer (20X) TMB Substrate Stop Solution Plate Sealer instrument microplate reader The standard in the reagent is the MXRA5 standard, which is the MXRA5 standard in the ELISA kit provided by Shanghai Jianglai Biotechnology Co., Ltd.

[0028] 37℃ constant temperature centrifuge 2. Experimental methods 2.1 Sample Collection Plasma samples were collected from 80 patients with calcific aortic valve disease and 80 healthy controls.

[0029] Collect specimens using EDTA as an anticoagulant and centrifuge them at 3500 rpm for 15 minutes at 2°C to 8°C within 30 minutes of collection. Take the supernatant or store it at -80°C to avoid repeated freezing and thawing.

[0030] Inclusion criteria for the calcific aortic valve disease group: 1) The subjects have signed the informed consent form.

[0031] 2) Echocardiography confirmed aortic valve thickening, restricted valvular activity, and localized hyperechoic calcification of the aortic valve.

[0032] 3) Exclusion criteria: patients with severe aortic regurgitation, severe liver and kidney damage, obvious hemodynamic abnormalities, malignant tumors, thyroid dysfunction, gout, congenital heart disease, infective endocarditis, etc. involving the aortic valve Inclusion criteria for healthy control group: 1) The subjects have signed the informed consent form.

[0033] 2) People in good health.

[0034] 3) Exclusion criteria: patients with severe heart valve disease, severe liver and kidney dysfunction, active malignant tumors, thyroid dysfunction, gout, congenital heart disease, infective endocarditis, etc.

[0035] All subjects collected 4 mL to 5 mL of fasting venous blood at 3500 rpm / 15 min at 4°C. After centrifugation, carefully remove the blood collection tube to avoid inversion, and immediately separate the upper plasma for later use.

[0036] 2.2 Preparation before metabolite (i.e., MXRA5) detection 1) Take out the concentrated biotinylated detection antibody and other reagents from the refrigerator and equilibrate them to room temperature.

[0037] 2) Preparation of standard working solution gradient: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 4000 pg / mL). Dilute to the following concentrations: 4000 pg / mL; 2000 pg / mL; 1000 pg / mL; 500 pg / mL; 250 pg / mL; 125 pg / mL; 62.5 pg / mL; 0 pg / mL.

[0038] Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 2000 pg / mL standard working solution into the first EP tube and mix thoroughly to make a 1000 pg / mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used as a blank well; there is no need to pipette liquid from the penultimate tube.

[0039] 3) Sample dilution: Pipette 100 μL of sample into an EP tube, add 100 μL of universal diluent (sample: diluent = 1:1 (V / V)), and vortex mix for 30 seconds.

[0040] 2.3 Metabolite Detection 1. Remove the required strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C.

[0041] 2. Sample Addition: Add 100 μl of sample (i.e., plasma sample collected in 2.1) or standard of varying concentrations to the corresponding wells. Add 100 μl of universal diluent to the blank wells. Cover the plate with a sealer and incubate at 37°C for 1 hour.

[0042] 3. Add biotinylated antibody: Remove the plate reader and discard the liquid without washing. Add 100 μL of biotinylated antibody working solution directly to each well. Cover the plate with a sealer and incubate at 37°C for 1 hour. (Dilute the 100X concentrated biotinylated antibody with universal diluent to a 1X working solution; for example: 10 μL of concentrated solution + 990 μL of universal diluent, prepared 15 minutes before use).

[0043] 4. Wash the plate: discard the liquid, add 300 μl 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 3 times.

[0044] 5. Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with a sealing mold, and incubate at 37°C for 30 minutes. (Dilute the 100X concentrated enzyme conjugate with universal diluent to a 1X working solution concentration; e.g., 10 μL of concentrate + 990 μL of universal diluent, prepared 15 minutes before use).

[0045] 6. Wash the plate: Discard the liquid and wash the plate 5 times according to the washing method in step 4.

[0046] 7. Add substrate: Add 90 μl of substrate (TMB) to each well, cover with a sealing mold, and incubate at 37°C in the dark for 15 minutes.

[0047] 8. Add stop solution: Take out the ELISA plate, add 50 μl of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm.

[0048] 2.4 Data Analysis Copy the original data from the microplate reader, convert it into Excel format, calculate the average OD value of the standard sample and the standard sample wells and subtract the blank well OD value as the correction value. Draw the standard curve with concentration as the vertical axis and OD value as the horizontal axis. 2 When the value is >0.99, this formula can be used to calculate the concentration.

[0049] If the OD value of the sample is higher than the upper limit of the standard curve, it should be appropriately diluted and re-measured and then multiplied by the corresponding dilution factor when calculating the sample concentration.

[0050] 3. Results After normality and homogeneity of variance tests were performed on the concentrations of MXRA5 in the calcific aortic valve disease group and the healthy control group, the t-test was used to compare whether there was a difference between the two groups. The results showed that MXRA5 was significantly increased in the calcific aortic valve disease group compared with the healthy control group (Table 1, Figure 1 ), and ROC curve analysis showed that the metabolite MXRA5 had good predictive value, with an AUC value of 0.929 (e.g. Figure 2 shown).

[0051] In summary, the present invention discovered for the first time that the level of MXRA5 is significantly elevated in patients with calcific aortic valve disease, and the metabolite (i.e., MXRA5) has a very high area under the curve. This metabolite can effectively distinguish between healthy controls and CAVD groups, suggesting that the application of MXRA5 in CAVD has a high diagnostic value and is of great significance for the diagnosis of CAVD and guiding standardized treatment. In addition, the diagnostic indicator provided by the present invention is a plasma metabolite, which only requires a small amount of blood for detection and is essentially non-invasive.

[0052] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

Claims

1. Use of MXRA5 as a marker in the preparation of a reagent for diagnosing calcific aortic valve disease.

2. The use of MXRA5 as a marker in the preparation of a reagent for diagnosing calcific aortic valve disease according to claim 1, characterized in that: The sample source of MXRA5 is one or more of blood, serum, plasma, lymph, sweat, saliva, vitreous humor, synovial fluid, lymph, semen, cerebrospinal fluid, urine, cell culture fluid, cells, isolated tissue samples and organ samples.

3. The use of MXRA5 as a marker in the preparation of a reagent for diagnosing calcific aortic valve disease according to claim 2, characterized in that: The sample source of MXRA5 is preferably plasma.

4. Use of MXRA5 as a marker in the preparation of a reagent for diagnosing calcific aortic valve disease according to claim 2 or 3, characterized in that: In the samples, the MXRA5 level was significantly elevated.

5. The use of MXRA5 as a marker in the preparation of a reagent for diagnosing calcific aortic valve disease according to claim 4, characterized in that: The MXRA5 level is significantly elevated in the plasma of patients with calcific aortic valve disease.

6. Use of MXRA5 as a marker in the preparation of a kit for diagnosing calcific aortic valve disease.

7. Use of MXRA5 as a marker in the preparation of a kit for diagnosing calcific aortic valve disease according to claim 6, characterized in that: The sample source of MXRA5 is one or more of blood, serum, plasma, lymph, sweat, saliva, vitreous humor, synovial fluid, lymph, semen, cerebrospinal fluid, urine, cell culture fluid, cells, isolated tissue samples and organ samples.

8. Use of MXRA5 as a marker in the preparation of a kit for diagnosing calcific aortic valve disease according to claim 7, characterized in that: The sample source of MXRA5 is preferably plasma.

9. Use of MXRA5 as a marker in the preparation of a kit for diagnosing calcific aortic valve disease according to claim 7 or 8, characterized in that: In the samples, the MXRA5 level was significantly elevated.

10. Use of MXRA5 as a marker in the preparation of a kit for diagnosing calcific aortic valve disease according to claim 9, characterized in that: The MXRA5 level is significantly elevated in the plasma of patients with calcific aortic valve disease.

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