A molecular marker for diagnosing anti-MDA5 positive dermatomyositis and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a molecular marker for diagnosing anti-MDA5 positive dermatomyositis and its application. The application of a molecular marker in the preparation of a diagnostic kit for diagnosing anti-MDA5 positive dermatomyositis, the molecular markers are serum soluble intercellular adhesion molecule-1 ICAM-1 and vascular cell adhesion molecule-1 VCAM-1. Experiments have shown that the levels of ICAM-1 and VCAM-1 in dermatomyositis patients are higher than those in normal people, and the levels of both anti-MDA5 antibody positive dermatomyositis serum are higher than those in the anti-MDA5 antibody negative group. ICAM-1 and VCAM-1 are used as markers for diagnosing anti-MDA5 positive dermatomyositis, which solves the problems that the detection method of anti-MDA5 antibodies in the prior art is complicated, the preparation of the kit and the selection of standard products are difficult, the operation of the immunoprecipitation method is complicated, and it cannot be popularized in clinical applications. Early detection of elevated adhesion molecule levels is conducive to the diagnosis of anti-MDA5 antibody positive dermatomyositis, which is conducive to guiding the next step of treatment and reducing the risk of disease deterioration and death in dermatomyositis patients.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a molecular marker for diagnosing anti-MDA5 positive dermatomyositis and an application thereof. Background Art

[0002] Idiopathic inflammatory myopathy (IIM) is a systemic disease that mainly attacks skeletal muscle, with clinical features of skeletal muscle weakness, skin lesions, systemic organ damage, and the production of specific antibodies. Polymyositis (PM) and dermatomyositis (DM) are the two most common types. Interstitial lung disease (ILD) is a common complication of PM / DM and the main cause of death in PM / DM patients, with a mortality rate of approximately 50%. Among them, rapidly progressive interstitial lung disease (RP-ILD) has a higher mortality rate, accounting for approximately 50-75%. RP-ILD is defined as acute interstitial pneumonia, with dyspnea or worsening cough within weeks or months. Therefore, early diagnosis and active treatment of RP-ILD are crucial to improving patient prognosis.

[0003] In 2005, Japanese scholar Sato used immunoprecipitation to first discover a new type of antibody in the serum of CADM patients. It is a polypeptide of about 140kd and is called anti-CADM-140 antibody. In 2009, Sato confirmed that the target antigen recognized by anti-CADM-140 antibody is the cytoplasmic protein melanoma differentiation-associated gene 5 (MDA5), which is involved in the host's immune defense against double-stranded RNA viruses. The incidence of RP-ILD in anti-MDA5 antibody-positive dermatomyositis is higher than that in anti-MDA5-negative dermatomyositis, and anti-MDA5 antibody is an independent risk factor for poor prognosis in PM / DM. Therefore, early detection of anti-MDA5 antibody and the use of corresponding treatment plans are also crucial to improving patient prognosis.

[0004] There are two methods for detecting anti-MDA5 antibodies: immunoprecipitation and enzyme-linked immunosorbent assay. Immunoprecipitation is the gold standard for detecting anti-MDA5 antibodies, but its disadvantage is that it cannot produce quantitative results. Therefore, an enzyme-linked immunosorbent assay using recombinant MDA5 protein as an antigen was developed for routine detection, and the standard curve was obtained based on serum with high titer anti-MDA5 antibodies.

[0005] Adhesion molecules are involved in leukocyte transendothelial migration. The main players in the leukocyte adhesion cascade reaction include the selectin family, integrin family, and immunoglobulin superfamily. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are the main members of the immunoglobulin superfamily. ICAM-1 is expressed on the surface of activated endothelial cells, leukocytes, fibroblasts, myeloid cells, and some tumor cells, and VCAM-1 is expressed on activated endothelial cells. The increased expression of both and the increased levels of serum free soluble ICAM-1 and VCAM-1 are related to autoinflammatory and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, etc. There are no reports on the use of ICAM-1 and VCAM-1 as molecular markers for the diagnosis of anti-MDA5-positive dermatomyositis. Thoroughly achieving early diagnosis and early effective treatment in a practical sense, further comprehensively and systematically studying the full picture of PM / DM serum antibodies, and finding markers related to PM / DM disease diagnosis are the focus of PM / DM disease research and clinical issues that need to be urgently addressed. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker for diagnosing anti-MDA5 positive dermatomyositis and its application. The present invention has found that the serum soluble ICAM-1 and VCAM-1 levels of dermatomyositis patients are higher than those of normal people, and the levels of the two in anti-MDA5 antibody positive dermatomyositis serum are higher than those in the anti-MDA5 antibody negative group. Serum soluble intercellular adhesion molecule-1 ICAM-1 and vascular cell adhesion molecule-1 VCAM-1 are used as markers for diagnosing anti-MDA5 positive dermatomyositis, which solves the problems of complex detection methods of anti-MDA5 antibodies in the prior art, difficult kit preparation and standard product selection, and complex immunoprecipitation operation, which cannot be popularized in clinical applications. Early detection of elevated adhesion molecule levels is conducive to diagnosing anti-MDA5 antibody positive dermatomyositis, which is conducive to guiding the next step of treatment and reducing the risk of disease deterioration and death in dermatomyositis patients.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] The invention discloses an application of molecular markers in preparing a diagnostic kit for diagnosing anti-MDA5 positive dermatomyositis. The molecular markers are serum soluble intercellular adhesion molecule-1 ICAM-1 and vascular cell adhesion molecule-1 VCAM-1.

[0009] Furthermore, the diagnosis is to distinguish anti-MDA5 positive dermatomyositis from healthy people.

[0010] Furthermore, serum soluble intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were quantitatively analyzed using enzyme-linked immunosorbent assay and predicted the presence of anti-MDA5 antibodies.

[0011] Compared with the prior art, the present invention has the following beneficial effects.

[0012] Serum soluble intercellular adhesion molecule-1 ICAM-1 and vascular cell adhesion molecule-1 VCAM-1 are markers for diagnosing anti-MDA5 positive dermatomyositis, which solve the problems in the prior art of complex anti-MDA5 antibody detection methods, difficulty in kit preparation and standard selection, and complex immunoprecipitation operations, which cannot be popularized in clinical applications.

[0013] The clinical features of anti-MDA5 antibody-positive dermatomyositis are severe skin and lung involvement with mild myositis or no muscle involvement. Abnormal infiltration of leukocytes and increased expression of adhesion molecules ICAM-1 and VCAM-1 can be found in local tissue inflammation such as synovial tissue in rheumatoid arthritis, renal tissue in systemic lupus erythematosus, and lung tissue in acute lung injury. Therefore, the levels of ICAM-1 and VCAM-1 can predict the degree of local tissue involvement in patients with dermatomyositis and assist in the diagnosis of anti-MDA5-positive dermatomyositis. Serum soluble adhesion molecules can be quantitatively analyzed using enzyme-linked immunosorbent assay and predict the presence of anti-MDA5 antibodies, with good diagnostic accuracy.

[0014] The detection process of serum soluble adhesion molecules is not complicated, and the test results can be obtained using an ELISA instrument. The kit production method is mature and easy to purchase, and can be popularized in clinical applications. Early detection of elevated adhesion molecule levels is conducive to the diagnosis of anti-MDA5 antibody-positive dermatomyositis, which is conducive to guiding the next step of treatment and reducing the risk of disease progression and death in patients with dermatomyositis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The changes of serum soluble ICAM-1 and VCAM-1 levels in healthy controls and anti-MDA5 positive and negative dermatomyositis. (A). The serum soluble ICAM-1 level in anti-MDA5 negative dermatomyositis (n=18) was higher than that in healthy controls (n=21), and in anti-MDA5 positive dermatomyositis (n=16) was higher than that in healthy controls and anti-MDA5 negative dermatomyositis groups. The statistical method was one-way analysis of variance with Bonferroni correction. t (A). The serum soluble VCAM-1 level was higher in anti-MDA5-negative dermatomyositis (n=25) than in healthy controls (n=21), and higher in anti-MDA5-positive dermatomyositis (n=20) than in healthy controls and anti-MDA5-negative dermatomyositis groups. Statistical method used one-way analysis of variance with Bonferroni correctiont test.

[0016] Figure 2 The results of ROC curve analysis are shown in Figure 3. A When the diagnostic threshold of serum sICAM-1 was 243.4635 ng / ml, the sensitivity of identifying anti-MDA5 antibody-positive dermatomyositis was 81.2%, the specificity was 66.7%, and the area under the curve was 0.767, p=0.001. B When the diagnostic threshold of serum sVCAM-1 was 730.4086 ng / ml, the sensitivity of identifying anti-MDA5 antibody-positive dermatomyositis was 75%, the specificity was 84%, and the area under the curve was 0.78, p<0.001. C The sensitivity of sICAM-1 and sVCAM-1 combined to identify anti-MDA5 antibody-positive dermatomyositis was 80%, the specificity was 76.9%, and the area under the curve was 0.779, p=0.004. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments and drawings.

[0018] Example 1 Detection of serum soluble ICAM-1 and VCAM-1 levels in patients with dermatomyositis.

[0019] From October 2017 to December 2019, 45 patients with dermatomyositis were admitted to the Department of Rheumatology and Immunology, the First Affiliated Hospital of China Medical University. According to the presence of anti-MDA5 antibodies, they were divided into anti-MDA5-positive dermatomyositis (25 cases) and anti-MDA5-negative dermatomyositis (20 cases). Twenty-one healthy controls matched for gender and age were included in the same period. All subjects had 5 ml of venous blood collected at the time of enrollment, added to a vacuum glass tube without anticoagulant, centrifuged at 3000 r / min for 10 min, and serum samples were separated. The levels of serum sICAM-1 and sVCAM-1 were detected by enzyme-linked immunosorbent assay (ELISA) kits, and the operation was strictly carried out according to the instructions of the reagents. The operation steps were as follows: 100 ul of standard antigen, blank control and diluted serum to be tested were added to the microplate embedded with ICAM-1 (or VCAM-1) antibody, and then 50 ul of biotinylated detection antibody was added to each well and incubated at room temperature for 2 hours. Wash the plate 6 times, then add 100ul of horseradish peroxidase-labeled streptavidin and incubate at room temperature for 1 hour. Wash the plate 6 times, then add the colorimetric substrate tetramethylbenzidine (TMB) and color at room temperature for 5-30 min in the dark. Then add 100ul of stop solution to terminate the reaction. Measure the absorbance at a wavelength of 450 nm (reference wavelength 570 nm). Use the absorbance of the standard antigen and its corresponding concentration value to make a standard curve, compare the absorbance of the test serum with the standard curve, and calculate the concentration value of the test serum. Each standard antigen and test serum were made into two wells and the average value was taken. The concentration values ​​of the two indicators were in line with the normal distribution, and the results were expressed as mean ± standard deviation. The statistical method used one-way analysis of variance and Bonferroni correction. t Multiple comparisons were performed using SPSS 20.0, and all tests were two-sided. p < 0.05 was considered statistically significant. The serum soluble ICAM-1 and VCAM-1 levels of anti-MDA5 antibody-positive dermatomyositis patients were higher than those of the anti-MDA5 antibody-negative group. Figure 1 As shown. A. The serum soluble ICAM-1 level was higher in anti-MDA5-negative dermatomyositis (n=18) than in healthy controls (n=21), and higher in anti-MDA5-positive dermatomyositis (n=16) than in healthy controls and anti-MDA5-negative dermatomyositis groups. B. The serum soluble VCAM-1 level was higher in anti-MDA5-negative dermatomyositis (n=25) than in healthy controls (n=21), and higher in anti-MDA5-positive dermatomyositis (n=20) than in healthy controls and anti-MDA5-negative dermatomyositis groups.

[0020] Example 2 Serum soluble adhesion molecules can be quantitatively analyzed using enzyme-linked immunosorbent assay to predict the presence of anti-MDA5 antibodies.

[0021] The ROC curve was drawn to analyze the predictive value of the serum sICAM-1 and sVCAM-1 levels detected in Example 1 for anti-MDA5 antibody-positive dermatomyositis. SPSS 20.0 and MedCalc 17.0 were used for drawing and statistical analysis. Binomial classification logistic regression was used to output the predicted probabilities of sICAM-1, sVCAM-1 and sICAM-1 combined with sVCAM-1, respectively, and then the three predicted probabilities were subjected to ROC curve analysis with anti-MDA5 antibodies. The cut-off value, sensitivity and specificity of serum sICAM-1, sVCAM-1 and sICAM-1 combined with sVCAM-1 for diagnosing anti-MDA5 positive dermatomyositis were analyzed. The area under the curve between 0.5-0.7 indicates a low diagnostic value; between 0.7-0.9 indicates a medium diagnostic value; and above 0.9 indicates a high diagnostic value. The results of the ROC curve analysis show that Figure 2 As shown. A When the diagnostic threshold of serum sICAM-1 was 243.4635 ng / ml, the sensitivity of identifying anti-MDA5 antibody-positive dermatomyositis was 81.2%, the specificity was 66.7%, and the area under the curve was 0.767, p=0.001. B When the diagnostic threshold of serum sVCAM-1 was 730.4086 ng / ml, the sensitivity of identifying anti-MDA5 antibody-positive dermatomyositis was 75%, the specificity was 84%, and the area under the curve was 0.78, p<0.001. C The sensitivity of sICAM-1 and sVCAM-1 combined to identify anti-MDA5 antibody-positive dermatomyositis was 80%, the specificity was 76.9%, and the area under the curve was 0.779, p=0.004.

Claims

1. Use of a molecular marker in the preparation of a diagnostic kit for diagnosing anti-MDA5 positive dermatomyositis, characterized in that: The molecular markers are serum soluble intercellular adhesion molecule-1 ICAM-1 and vascular cell adhesion molecule-1 VCAM-1; the diagnosis is to distinguish anti-MDA5 positive dermatomyositis from healthy people.

2. The use of a molecular marker as claimed in claim 1 in preparing a diagnostic kit for diagnosing anti-MDA5 positive dermatomyositis, characterized in that: Serum soluble intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were quantified using enzyme-linked immunosorbent assay and predicted the presence of anti-MDA5 antibodies.

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