Biomarkers for idiopathic inflammatory myopathies and uses thereof

Abstract

The application provides a biomarker for idiopathic inflammatory myopathy and application thereof, and the biomarker is an anti-HDGFL1 antibody. The application further discloses application of the anti-HDGFL1 antibody as the biomarker for the idiopathic inflammatory myopathy, application of an anti-HDGFL1 antibody detection agent in preparation of a product related to diagnosis of the idiopathic inflammatory myopathy, a kit and a system for diagnosing the idiopathic inflammatory myopathy, evaluating a prognosis of a patient with the idiopathic inflammatory myopathy, or determining a type of the idiopathic inflammatory myopathy. The application realizes effective diagnosis, treatment, prognosis evaluation and disease typing for a patient with idiopathic inflammatory myopathy who is specific to serum myositis and negative to an autoantibody through the anti-HDGFL1 antibody.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to biomarkers for idiopathic inflammatory myopathy and their applications. Background Technology

[0002] Idiopathic inflammatory myopathy (IIM) is a group of autoimmune diseases primarily affecting skeletal muscle, often accompanied by muscle weakness, rash, interstitial lung disease, and involvement of multiple internal organs. Myositis-specific autoantibodies (MSA) are crucial for the diagnosis and treatment of IIM. Current research shows that up to 60%-80% of IIM patients have MSA, and each MSA appears to be associated with a specific clinical phenotype and is almost exclusively present in IIM patients. For example, MDA5 antibody-positive patients often have rapidly progressing interstitial lung disease, while TIF1-γ antibodies are associated with an increased risk of cancer. However, statistics show that 20%-30% of IIM patients do not have detectable known myositis-specific autoantibodies, i.e., they are MSA-negative IIM patients. This poses a significant challenge to the diagnosis, prognosis, and progression of IIM. Therefore, for MSA-negative IIM patients, the search for novel myositis autoantibodies can help identify other disease subgroups in the myositis spectrum and is of great significance for the early diagnosis, early treatment, prognosis, and research on potential mechanisms of IIM. Summary of the Invention

[0003] The purpose of this invention is to provide a biomarker for characterizing idiopathic inflammatory myopathy and its application, for effective diagnosis, treatment, monitoring of idiopathic inflammatory myopathy, as well as its prognostic assessment and disease classification, especially for MSA-negative IIM patients.

[0004] To achieve the above objectives, the present invention adopts the following technical solution.

[0005] According to a first aspect of the invention, the use of an anti-HDGFL1 (Hepatoma derived growth factor-like 1) antibody as a biomarker for idiopathic inflammatory myopathy is provided.

[0006] Furthermore, the application of anti-HDGFL1 antibody as a biomarker for characterizing idiopathic inflammatory myopathy in patients with MSA-negative idiopathic inflammatory myopathy and myositis-specific autoantibodies.

[0007] Furthermore, the application of anti-HDGFL1 antibody as a biomarker for characterizing idiopathic inflammatory myopathy in patients with MSA-negative idiopathic inflammatory myopathy in the preparation of diagnostic products for idiopathic inflammatory myopathy.

[0008] According to a second aspect of the present invention, an anti-HDGFL1 antibody detection reagent is provided for use in the preparation of products related to the diagnosis of idiopathic inflammatory myopathy.

[0009] Furthermore, products related to the diagnosis of idiopathic inflammatory myopathy include products for diagnosing idiopathic inflammatory myopathy, products for assessing the prognosis of patients with idiopathic inflammatory myopathy, or products for determining the subtype of idiopathic inflammatory myopathy.

[0010] Furthermore, products related to the diagnosis of idiopathic inflammatory myopathy include at least one of reagents, kits, and chips.

[0011] Furthermore, the anti-HDGFL1 antibody detection reagent includes a reagent for detecting the expression level of anti-HDGFL1 antibody by at least one of the following methods: immunofluorescence assay, radioimmunoassay, colloidal gold method, antibody screening, complement fixation, and enzyme-linked immunosorbent assay.

[0012] According to a third aspect of the present invention, an anti-HDGFL1 antibody detection reagent is provided for use in the preparation of a diagnostic product for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the subtype of idiopathic inflammatory myopathy by means of the following steps:

[0013] Provide a serum sample from the individual to be tested;

[0014] Measure the level of anti-HDGFL1 antibody in serum samples;

[0015] The level of anti-HDGFL1 antibody measured can be used to diagnose, assess prognosis, or classify diseases in the individual being tested.

[0016] Among them, diagnosing the individual to be tested means determining whether the individual to be tested is a patient with myositis-specific autoantibody MSA negative idiopathic inflammatory myopathy;

[0017] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of patients with MSA-negative idiopathic inflammatory myopathy;

[0018] Among them, disease typing of the individuals to be tested refers to assessing the clinical phenotype of MSA-negative idiopathic inflammatory myopathy patients and determining the correlation between anti-HDGFL1 antibody and clinical phenotype.

[0019] According to a fourth aspect of the present invention, a kit is provided for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the subtype of idiopathic inflammatory myopathy, comprising an anti-HDGFL1 antibody detection reagent.

[0020] According to a fifth aspect of the present invention, a method is provided for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the classification of idiopathic inflammatory myopathy:

[0021] Provide a serum sample from the individual to be tested;

[0022] Measure the level of anti-HDGFL1 antibody in serum samples;

[0023] The level of anti-HDGFL1 antibody measured can be used to diagnose, assess prognosis, or classify diseases in the individual being tested.

[0024] Among them, diagnosing the individual to be tested means determining whether the individual to be tested is a patient with myositis-specific autoantibody MSA negative idiopathic inflammatory myopathy;

[0025] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of patients with MSA-negative idiopathic inflammatory myopathy;

[0026] Among them, disease typing of the individuals to be tested refers to assessing the clinical phenotype of MSA-negative idiopathic inflammatory myopathy patients and determining the correlation between anti-HDGFL1 antibody and clinical phenotype.

[0027] According to a sixth aspect of the present invention, a system is provided for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the classification of idiopathic inflammatory myopathy, comprising:

[0028] The sample acquisition module is used to provide serum samples from the individual to be tested;

[0029] The sample detection module is used to determine the level of anti-HDGFL1 antibody in serum samples;

[0030] The analysis and evaluation module is used to diagnose, assess prognosis, or classify diseases in individuals based on the measured levels of anti-HDGFL1 antibodies.

[0031] Among them, diagnosing the individual to be tested means determining whether the individual to be tested is a patient with MSA-negative idiopathic inflammatory myopathy;

[0032] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of patients with MSA-negative idiopathic inflammatory myopathy;

[0033] Among them, disease typing of the individuals to be tested refers to assessing the clinical phenotype of MSA-negative idiopathic inflammatory myopathy patients and determining the correlation between anti-HDGFL1 antibody and clinical phenotype.

[0034] The present invention has the following significant technical advantages over the prior art:

[0035] (1) The present invention provides a biomarker for characterizing idiopathic inflammatory myopathy, which can be used for the diagnosis, prognostic assessment and disease classification of idiopathic inflammatory myopathy.

[0036] (2) The present invention can be used as a supplementary diagnostic biomarker in cases where MSA cannot be used to diagnose patients with idiopathic inflammatory myopathy, i.e., when MSA is negative.

[0037] (3) This invention helps to further identify other disease subgroups in the myositis spectrum, and is of great significance for the early diagnosis, early treatment, prognosis and potential mechanisms of IIM. Attached Figure Description

[0038] Figure 1 This is a heatmap showing the distribution of SNR values ​​of the top 50 candidate target proteins in Example 1, arranged in descending order of fold difference, in the MSA seronegative group (MSANeg), MSA seropositive group (MSAPos), systemic lupus erythematosus patients (SLE), systemic sclerosis patients (SSc), and healthy control group (HC).

[0039] Figure 2 The fluorescence signal of HDGFL1 protein in the MSA serum-negative group (MSA-) and the healthy control group (HC) in Example 1 is shown.

[0040] Figure 3 The images show the fluorescence bands of the MSA-negative group (MSA-) and the healthy control group (HC) in Example 2. Detailed Implementation

[0041] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] In one embodiment of the present invention, an application of an anti-HDGFL1 antibody as a biomarker for idiopathic inflammatory myopathy is provided.

[0043] HDGFL1 is a nucleoprotein containing 251 amino acids with a molecular weight of approximately 27 kDa. It may activate double-stranded DNA binding, transcriptional co-regulatory factor activity, and participate in the transcriptional regulation of RNA polymerase II. The inventors have discovered for the first time that anti-HDGFL1 antibodies can be used to characterize idiopathic inflammatory myopathy, particularly in identifying MSA-negative IIM patients. In this invention, anti-HDGFL1 antibodies refer to immunoglobulins that are spontaneously generated in the target subject (e.g., an MSA-negative individual) or produced under stress, primarily present in body fluids such as serum, and that specifically bind to and react with the HDGFL1 protein. Specifically, anti-HDGFL1 antibodies refer to specific autoantibodies against the HDGFL1 protein. Furthermore, since the serum of healthy individuals does not contain anti-HDGFL1 antibodies, and for some patients with idiopathic inflammatory myopathy, especially MSA-negative IIM patients, there are currently no reports of the presence of autospecific antibodies in their bodies, anti-HDGFL1 antibodies can be used as biomarkers to specifically characterize idiopathic inflammatory myopathy, thereby avoiding invasive damage to individuals during myositis diagnosis.

[0044] Meanwhile, the biomarkers for characterizing idiopathic inflammatory myopathy provided by this invention can help to further identify other disease subgroups in the myositis spectrum, and are of great significance for the early diagnosis, early treatment, prognosis and potential mechanisms of idiopathic inflammatory myopathy.

[0045] In this invention, an IIM patient can be an MSA-negative patient. For example, an IIM patient is an individual in whom MSA has not yet been detected or determined. Specifically, this individual is a human individual.

[0046] The inventors' research found that 33.3% of MSA-negative IIM patients had anti-HDGFL1 antibodies in their serum. Therefore, in situations where the lack of an effective MSA biomarker makes it impossible to diagnose IIM patients promptly and accurately, i.e., when patients are MSA-negative, anti-HDGFL1 antibodies can be used as a supplementary biomarker.

[0047] In one embodiment of the present invention, a method for screening and determining biomarkers characterizing idiopathic inflammatory myopathy is provided, specifically including the following steps:

[0048] (1) Chip preprocessing steps: The proteomics chip is sealed;

[0049] (2) Chip incubation steps: Add serum samples from patients with idiopathic inflammatory myopathy to the pretreated proteome chip for incubation, wash, add fluorescent secondary antibody for incubation, and then wash to obtain the incubated proteome chip.

[0050] (3) Chip detection steps: Place the incubated proteome chip in a chip scanner, read the signal of the secondary antibody fluorescence on the incubated proteome chip, and save the image;

[0051] (4) Data processing steps: Based on the saved images and the target protein coating array list of the proteome chip, the analysis software GenePix Pro is used to segment and extract each protein signal pixel according to the protein point matrix parameters in the proteome chip. At the same time, manual screening is performed one by one to check the signal intensity of the corresponding signal points of all protein points on the chip, and the signals are extracted for further analysis to obtain the detection signal and relative quantitative basis.

[0052] Specifically, prior to the chip preprocessing in step (1), a step of obtaining serum samples is also included. These serum samples include serum from MSA-negative IIM patients, serum from MSA-positive IIM patients, and serum from healthy individuals serving as controls. Preferably, the serum samples include serum from MSA-negative IIM patients, serum from MSA-positive IIM patients, serum from autoimmune control groups (serum from patients with systemic lupus erythematosus and systemic sclerosis), and serum from healthy individuals serving as controls, to increase comparability between samples and the accuracy of target protein antibody screening.

[0053] Optionally, the serum samples mentioned above can be three-in-one, four-in-one, or five-in-one serum samples.

[0054] Specifically, in the chip preprocessing step, human proteome chips, such as HuProt, are used. TM Human proteome microarray. Specifically, in the (1) microarray preprocessing step, HuProt... TM The human proteome chip was removed from a -80°C freezer and placed in a 4°C freezer for resuscitation. After one day, it was removed and allowed to equilibrate at room temperature for 1-2 hours. The equilibrated proteome chip was then placed in an incubation chamber, and 5-10 ml of 2-4% bovine serum albumin (BSA) solution was added as a blocking solution. The chip was then blocked at room temperature (20°C-25°C) using a shaker for 1-2 hours.

[0055] Specifically, in the chip pretreatment step, the blocking solution is removed from the blocked chip, serum sample is added, and it is incubated at room temperature (20℃-25℃) for 1-3 hours. The chip is then washed with 0.2% tris(hydroxymethyl)aminomethane buffer (TBST), and fluorescent secondary antibody is added. The chip is then incubated in the dark for 1.0-1.5 hours. After incubation, the chip is washed again with 0.2% TBST solution, and then with ultrapure water.

[0056] Specifically, in the chip detection step, fluorescence signals are read using a GenePix 4000 chip scanner and GenePix Pro software, and TIFF images are saved. The scanner parameters are set as follows: photomultiplier tube (PMT) is set to 600, scan power (ScanPower) is set to 10, and scan pixel size is 10μm.

[0057] Specifically, after the chip preprocessing step and preferably before chip incubation, a chip quality assessment step is also included, in which an anti-GST antibody is used to hybridize with the proteomics chip to verify the number of effective protein spots on the proteomics chip and the correlation of signal intensity between parallel protein spots.

[0058] Specifically, in the data processing step, the signal value corresponding to the target protein is the average signal-to-noise ratio (SNR) of two parallel signal values ​​(F635medium / B635medium). When this value is greater than 2, it is determined that the autoantibody against the target protein can be detected. The cut-off point for each target protein is the mean of the signal value (F635mean) + 3SD of the healthy control group. The positive rate of immune response of each target protein on the serum sample and the proteomic chip is calculated. High-throughput bioinformatics analysis tools are used to perform chip significance analysis to analyze the differentially expressed protein information between groups. Candidate target proteins are selected with a q value of less than 5% and a fold change of more than 3.

[0059] Finally, based on the fluorescence intensity and the target protein coating array list of the proteome chip, the antibody with the highest fluorescence intensity was identified as the biomarker for characterizing idiopathic inflammatory myopathy.

[0060] In embodiments of the present invention, target proteins on a proteome chip can capture specific autoantibodies in serum samples from IIM patients, forming target protein-specific autoantibody complexes. These specific autoantibodies can then bind to a second antibody (such as a fluorescent antibody), forming a target protein-specific autoantibody-second antibody complex. Since the second antibody carries a detectable fluorescent group, the level of specific autoantibodies can be determined by detecting the fluorescence intensity. Higher fluorescence intensity indicates a higher level of specific autoantibodies in the serum samples of IIM patients. Based on the target protein coating array list of the proteome chip, the target protein with the highest fluorescence intensity can be identified, thereby determining the antibody targeting that protein and obtaining a biomarker characterizing idiopathic inflammatory myopathy.

[0061] In this invention, the method for determining biomarkers characterizing idiopathic inflammatory myopathy is implemented using proteomics chips. It is a high-throughput protein immunoassay analysis technology that has advantages over traditional detection methods, including high throughput, high sensitivity, small sample volume, and high automation.

[0062] In one embodiment of the present invention, an anti-HDGFL1 antibody is provided as a biomarker for characterizing idiopathic inflammatory myopathy in MSA-negative IIM patients in the preparation of products related to the diagnosis of idiopathic inflammatory myopathy.

[0063] In one embodiment of the present invention, an anti-HDGFL1 antibody detection reagent is provided for use in the preparation of diagnostic products for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the subtype of idiopathic inflammatory myopathy.

[0064] Anti-HDGFL1 antibodies can be used as biomarkers for the diagnosis, prognostic assessment, or disease classification of idiopathic inflammatory myopathy. Therefore, by detecting anti-HDGFL1 antibodies, idiopathic inflammatory myopathy can be diagnosed, assessed for prognosis, or classified.

[0065] Furthermore, products related to the diagnosis of idiopathic inflammatory myopathy include at least one of reagents, kits, and chips.

[0066] In one embodiment of the present invention, an anti-HDGFL1 antibody detection reagent is provided for use in the preparation of a diagnostic product for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the subtype of idiopathic inflammatory myopathy by means of the following steps:

[0067] Provide a serum sample from the individual to be tested;

[0068] Measure the level of anti-HDGFL1 antibody in serum samples;

[0069] The level of anti-HDGFL1 antibody measured can be used to diagnose, assess prognosis, or classify diseases in the individual being tested.

[0070] Among these, diagnosis of the individual to be tested refers to determining whether the individual being tested is an MSA-negative IIM patient; prognostic assessment of the individual to be tested refers to assessing the prognostic status of MSA-negative IIM patients; and disease subtyping of the individual to be tested refers to assessing the clinical phenotype of MSA-negative IIM patients and determining the correlation between anti-HDGFL1 antibody and clinical phenotype.

[0071] In this invention, diagnosing an individual involves determining whether they are an MSA-negative IIM patient based on the expression level of anti-HDGFL1 antibodies in their serum; or, if an individual is diagnosed with MSA-negative IIM, after treatment, the prognosis of myositis can be accurately and reliably assessed based on the degree of relief, reduction, worsening, or progression of myositis symptoms, providing a theoretical basis for further treatment and rehabilitation; or, if an individual is diagnosed with MSA-negative IIM, the correlation between anti-HDGFL1 antibody expression and clinical symptom phenotypes and myositis genotypes can be determined based on different anti-HDGFL1 antibody expression levels, combined with clinical symptom phenotypes and myositis genotypes, thus providing a theoretical basis for targeted therapies for different types of myositis.

[0072] In this invention, the prognosis of MSA-negative IIM patients is the degree of relief, reduction, worsening, or progression of myositis symptoms.

[0073] In this invention, the anti-HDGFL1 antibody detection agent refers to any compound, composition, solution, etc., capable of detecting anti-HDGFL1 antibody expression in vivo, in vitro, or ex vivo. For example, the anti-HDGFL1 antibody detection agent includes reagents that detect the expression and level of anti-HDGFL1 antibodies through immunofluorescence assay, radioimmunoassay, colloidal gold method, antibody screening, complement fixation, enzyme-linked immunosorbent assay (ELISA), etc. For instance, the anti-HDGFL1 antibody detection agent can capture anti-HDGFL1 antibodies in the serum sample of the individual to be tested, forming a detection agent conjugated with the anti-HDGFL1 antibody. Simultaneously, a second antibody can bind to the anti-HDGFL1 antibody, thus ultimately forming a detection agent-anti-HDGFL1 antibody-second antibody complex. Since the second antibody itself can be detected or carries detectable groups, or can react with other substrates to obtain detectable products, the level of anti-HDGFL1 antibodies in the serum sample of the individual to be tested can be obtained by quantitatively detecting the second antibody. Here, the anti-HDGFL1 antibody level can refer to the presence or absence of anti-HDGFL1 antibodies, or the level of anti-HDGFL1 antibodies.

[0074] The individual to be tested can be either an IIM individual or a non-IIM individual, which is understandable to those skilled in the art.

[0075] In this invention, the methods for obtaining serum samples, incubation, washing, and detection are conventional methods in the field of biological detection. Those skilled in the art, knowing that anti-HDGFL1 antibodies can serve as biomarkers for idiopathic inflammatory myopathy (IIM), can use conventional techniques to detect anti-HDGFL1 antibody levels to diagnose IIM, assess patient prognosis, or determine IIM classification. This is feasible for those skilled in the art. For example, in one specific embodiment of this invention, Western blotting is used to detect anti-HDGFL1 antibodies.

[0076] In one embodiment of the present invention, an apparatus / system is provided for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the classification of idiopathic inflammatory myopathy, comprising:

[0077] The sample acquisition module is used to provide serum samples from the individual to be tested;

[0078] The sample detection module is used to determine the level of anti-HDGFL1 antibody in serum samples;

[0079] The analysis and evaluation module is used to diagnose, assess prognosis, or classify diseases in individuals based on the measured levels of anti-HDGFL1 antibodies.

[0080] Among them, diagnosing the individual to be tested means determining whether the individual to be tested is an MSA-negative IIM patient;

[0081] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of MSA-negative IIM patients;

[0082] Among them, disease typing of the individuals to be tested refers to assessing the clinical phenotype of MSA-negative IIM patients and determining the correlation between anti-HDGFL1 antibody and clinical phenotype.

[0083] This invention, through the cooperation of a sample acquisition module, a sample detection module, and an analysis and evaluation module, can simply, quickly, and accurately determine whether an individual to be tested is an IIM individual or a non-IIM individual, thereby achieving the purpose of diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the classification of idiopathic inflammatory myopathy.

[0084] In one embodiment of the present invention, a computer-readable storage medium is provided having a computer program stored thereon, which, when executed by a processor, implements the apparatus / system described above.

[0085] In one embodiment of the present invention, a method is provided for diagnosing idiopathic inflammatory myopathy, assessing the prognosis of patients with idiopathic inflammatory myopathy, or determining the classification of idiopathic inflammatory myopathy:

[0086] Provide a serum sample from the individual to be tested;

[0087] Measure the level of anti-HDGFL1 antibody in serum samples;

[0088] The level of anti-HDGFL1 antibody measured can be used to diagnose, assess prognosis, or classify diseases in the individual being tested.

[0089] Among them, diagnosing the individual to be tested means determining whether the individual to be tested is an MSA-negative IIM patient;

[0090] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of MSA-negative IIM patients;

[0091] Among them, disease typing of the individuals to be tested refers to assessing the clinical phenotype of MSA-negative IIM patients and determining the correlation between anti-HDGFL1 antibody and clinical phenotype.

[0092] In one embodiment of the invention, a diagnostic composition for idiopathic inflammatory myopathy is provided, comprising an anti-HDGFL1 antibody detection agent. Specifically, the diagnostic composition further comprises a pharmaceutically acceptable excipient or carrier.

[0093] In one embodiment of the present invention, a diagnostic kit for idiopathic inflammatory myopathy is provided, comprising an anti-HDGFL1 antibody detection reagent. The kit also includes adjuvants and instructions for use.

[0094] The kit also includes any of the above-mentioned types of anti-HDGFL1 antibody detection reagents.

[0095] The technical solution of this application will be further explained and illustrated below with reference to specific embodiments.

[0096] To facilitate understanding, the relevant technical terms involved in the embodiments of this application will first be explained and described. In this invention, HuProt TM The human proteome microarray is currently the largest human protein microarray in terms of capacity, containing the largest number of full-length human proteins (21,283 types) on a single slide. The full-length human proteins on the chip are purified from a eukaryotic model organism, yeast, and retain their native conformation and post-translational modifications such as phosphorylation and glycosylation. Each expressed protein contains a GST tag, allowing for quality control detection using an anti-GST antibody. The HuProt microarray of this invention… TM The human proteome chip originated from the CDI laboratory at Johns Hopkins University in the United States.

[0097] In addition, experimental methods in the following examples that do not specify specific conditions are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or publicly available.

[0098] Example 1: Screening of biomarkers

[0099] (1) Obtaining serum samples: Serum samples were obtained from 24 MSA-negative myositis patients as the MSA-negative group, serum samples were obtained from 15 MSA-positive myositis patients as the MSA-positive group, serum samples were obtained from 15 patients with systemic lupus erythematosus (SLE) and 15 patients with systemic sclerosis (SSc) as the autoimmune control group, and serum samples were obtained from 15 healthy individuals as the healthy control group (HC). Within each group, the serum samples of 5 patients were mixed to obtain a five-in-one serum sample with a total volume of 30 μl, of which the serum samples of the 5 patients were 6 μl each.

[0100] (2) Chip preprocessing: HuProt TM The human proteome chip was removed from a -80°C freezer and placed in a 4°C freezer for resuscitation. One day later, the proteome chip was removed from the 4°C freezer and placed at room temperature for 1 hour to equilibrate. The proteome chip was then removed and placed in an incubation box, and 6 ml of 3% bovine serum albumin (BSA) solution was added as a blocking solution. The chip was then blocked at 25°C using a shaker for 1 hour.

[0101] (3) Chip quality assessment: After sealing, the sealing solution was discarded, and the number of effective protein spots on the proteome chip and the correlation of signal intensity between parallel protein spots were verified by hybridization with anti-GST antibody.

[0102] (4) Chip incubation: After quality assessment, the proteome chip was washed to remove residual reagents. Then, 30 μl of a five-in-one serum sample was added, and the chip was incubated at 25°C for 2 hours. The chip was washed 5 times with 0.2% TBST solution as the washing buffer. After the last wash, the washing buffer was quickly discarded, and 6 ml of Alexa 647-labeled goat anti-human IgG diluted 500 times with 3% BSA solution was added. The chip was incubated at 25°C in the dark for 1 hour. After incubation, the chip was washed 4 times with 0.2% TBST solution as the washing buffer, and then washed 3 times with ultrapure water.

[0103] (5) Chip detection: The proteomics chip was placed in a dark place to dry, and the fluorescence signal was read in the GenePix 4000 chip scanner and GenePix Pro software. The TIFF image was saved. The scanner parameters were set as follows: photomultiplier tube (PMT) was set to 600, scan power (ScanPower) was set to 10, and scan pixel was 10μm.

[0104] (6) Data Processing: Based on the TIFF image from step (4) and the target protein coating array list of the proteome chip, the GenePix Pro analysis software was used to segment and extract the signal pixels of each protein according to the protein point matrix parameters in the proteome chip. At the same time, each protein point was manually screened to check the signal intensity of the corresponding signal points on the chip, and the signals were extracted for further analysis to obtain the detection signal and relative quantitative basis. The signal value corresponding to the target protein is the average signal-to-noise ratio (SNR) of two parallel point signal values ​​(F635medium / B635medium). When this value is greater than 2, it is determined that the autoantibody against the target protein can be detected. The cut-off point of each target protein is the mean + 3SD of the signal value (F635mean) of the healthy human control group. The positive rate of immune response of each target protein in the five-in-one serum sample and the proteome chip was calculated. High-throughput bioinformatics analysis tools were used to perform chip significance analysis to analyze the differential protein information between groups. Candidate target proteins were selected with a q value of less than 5% and a Fold change of more than 3.

[0105] The above analysis and screening yielded 118 candidate target proteins, the specific target antigen names of which are shown in Table 1. The distribution of the top 50 candidate target proteins, sorted by Fold change in descending order, across each group is as follows: Figure 1 As shown, HDGFL1 protein exhibits a significantly high signal (SNR value) in the MSA-negative group (MSANeg), while the signal is lower in other groups. Combined with the signal values ​​of the target proteins on the proteomic chip, such as... Figure 2 As shown, HDGFL1 protein exhibited a significantly high signal in the MSA-negative group (MSA-) and a low signal in the healthy control group (HC), suggesting that anti-HDGFL1 antibodies have the potential to serve as biomarkers for identifying patients with idiopathic inflammatory myopathy.

[0106] Table 1

[0107] SMCO3 EXOSC7 HK1 HDGFL1 THOP1 FERMT2 TXNDC2 DPY30 GSN NANP KLHL7 OTUD5 PARP1 OGDH MAGI1 KJ898030 BPIFA1 ENPP3 QPRT GAD2 C9orf142 VWA8 DGKQ POLB TPRX1 PGK1 BZW2 CAP1 DAPP1 THUMPD1 NUDT14 HGFAC MYLK PTMA C10orf25 N4BP1 COQ9 CDKN2C MAGEL2 LIPN Prdm9 TAF10 PRMT3 UBL3 HGS DMRT2 PLEKHM2_frag ITIH3 ZNF746 SUOX NRG3 FAM21A MAGEC2 ROPN1 DNAJB4 RAD23A GOT1 ACTN1 TXLNB MVK MYCL RPL18A A0jns7 ERICH5 NDEL1 PRMT7 BABAM1 ALOXE3 FAM29A TMEM163 VCP CAPRIN1 ELANE MESP2 TP53TG1 ZNF768 STAT4 TLK2 DGKK RBM24 RLN3 PRY TYMP E2F4 CLDN2 HBE1 ULBP2 IGLL5 NADSYN1 NVL PROL1 ZNF696 SASS6 ENPP4 GPD1 TRAP1 GCHFR TMX4 KLC2 KLHL26 BAX SLC1A5 PPP5C WASF2 RIMBP3_frag GDI2 RPL36AL SESN2 TCF24 SNX16 TSPYL6 SH3BGR AHCY NKX2-6 SEPHS1 AVP MLXIP UGT1A4

[0108] Example 2: Validation of screened biomarkers

[0109] (1) Obtaining serum samples: Serum samples were obtained from 42 MSA-negative myositis patients, forming the MSA-negative group (MSA group). - Serum samples from 149 MSA-positive myositis patients were obtained and designated as the MSA-positive group (MSA). + Serum samples from 21 patients with systemic lupus erythematosus and 20 patients with systemic sclerosis were obtained as the autoimmune disease control group, and serum samples from 73 healthy individuals were obtained as the healthy control group (HC).

[0110] (2) Immunoblot method: 20 μl of recombinant HDGFL1 protein (0.75 μg / μl) was mixed with 60 μl of SDS-PAGE protein loading buffer (5×) and 220 μl of PBS, boiled at 100℃ for 10 minutes, and then added to a pre-prepared gel for SDS-PAGE electrophoresis. After electrophoresis, the protein in the gel was electrotransferred to a polyvinylidene fluoride (PVDF) membrane. The successfully transferred PVDF membrane was blocked for 2 hours, and the serum sample from step (1) was added. After incubation on a shaker for 2 hours, the membrane was washed, and then horseradish peroxidase (HRP)-labeled goat anti-human IgG was added for incubation. Finally, the fluorescence signal was detected by a fluorescence imager. The presence of a target band indicates a positive result, and the absence of a target band indicates a negative result. The specific band images can be seen in the image. Figure 3 The statistical results of the bands can be seen in Table 2.

[0111] like Figure 3 As shown in Table 2, anti-HDGFL1 antibodies were detectable in the MSA-negative group but not in the healthy control group. Further validation of the biomarker in a larger population revealed a positive rate of 33.3% in the MSA-negative group, 17.4% in the MSA-positive group, 0% in the healthy control group, and 24.4% in the autoimmune control group. This indicates that 33.3% of MSA-negative myositis patients had anti-HDGFL1 antibodies in their serum, while none of the serum of healthy individuals contained them. This suggests that anti-HDGFL1 antibodies can serve as a biomarker for characterizing idiopathic inflammatory myopathy. Furthermore, the positive rate of anti-HDGFL1 antibodies in MSA-negative patients was approximately twice that in MSA-positive patients. Therefore, for MSA-negative patients, given that currently known myositis-specific autoantibodies cannot be used as biomarkers to diagnose idiopathic inflammatory myopathy, anti-HDGFL1 antibodies can serve as a supplementary biomarker for the diagnosis and prognostic assessment of idiopathic inflammatory myopathy.

[0112] Table 2

[0113] Group Total number of samples (samples) Positive samples (number) Positive rate (%) MSA negative group 42 14 33.3％ healthy control group 73 0 0％ MSA positive group 149 26 17.4％ Autoimmune disease control group 41 10 24.4％

[0114] In summary, the anti-HDGFL1 antibody of the present invention can serve as a biomarker for characterizing idiopathic inflammatory myopathy (IIM) and can be used for the diagnosis, prognostic assessment, and classification of IIM. Furthermore, this biomarker can be used as a supplementary diagnostic biomarker in cases where MSA (microscopy-assay) is not available for diagnosing IIM, i.e., when MSA is negative. Therefore, the present invention contributes to the further identification of other disease subgroups within the myositis spectrum and is of significant importance for the early diagnosis, treatment, prognosis, and understanding of the underlying mechanisms of IIM.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an anti-HDGFL1 antibody detection agent in the manufacture of a kit for the diagnosis of idiopathic inflammatory myopathy with myositis-specific autoantibody MSA negativity.

2. Use according to claim 1, characterized in that, The anti-HDGFL1 antibody detection agent comprises a reagent for detecting the expression level of the anti-HDGFL1 antibody by at least one of an immunofluorescence assay, a radioimmunoassay, a colloidal gold method, and an enzyme-linked immunoassay method.

3. Use according to claim 1, characterized in that, The anti-HDGFL1 antibody detection agent is used for diagnosing idiopathic inflammatory myopathy with myositis-specific autoantibody MSA negativity by the following method: providing a serum sample from an individual to be detected; determining the level of anti-HDGFL1 antibody in the serum sample; diagnosing the individual to be detected according to the determined level of anti-HDGFL1 antibody; wherein the diagnosis of the individual to be detected refers to determining whether the individual to be detected is a patient with idiopathic inflammatory myopathy with myositis-specific autoantibody MSA negativity.

4. A system for diagnosing idiopathic inflammatory myopathy, characterized by, comprises: a sample obtaining module for providing a serum sample from an individual to be detected; a sample detecting module for determining the level of anti-HDGFL1 antibody in the serum sample; an analysis evaluating module for diagnosing the individual to be detected according to the determined level of anti-HDGFL1 antibody; wherein the diagnosis of the individual to be detected refers to determining whether the individual to be detected is a patient with idiopathic inflammatory myopathy with myositis-specific autoantibody MSA negativity.

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