A biomarker for diagnosing rheumatoid arthritis with interstitial lung disease and related products thereof

Abstract

The application discloses a biomarker for diagnosing rheumatoid arthritis combined with interstitial lung disease and related products thereof. The application first discovers a serum biomarker, anti-EXOSC4 autoantibody, which is related to the diagnosis of rheumatoid arthritis combined with interstitial lung disease, and the serum biomarker has high diagnostic efficiency. The application provides a basis for clinical decision-making related to rheumatoid arthritis combined with interstitial lung disease, lays a foundation for subsequent clinical research, and has good application prospect and important conversion significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to a biomarker for diagnosing rheumatoid arthritis complicated with interstitial lung disease and related products. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease. Approximately 50% of RA patients develop extra-articular manifestations, the most common of which is interstitial lung disease (ILD). Evidence suggests that the mortality rate for patients with rheumatoid arthritis and interstitial lung disease (RA-ILD) is 2–10 times higher than that for RA patients without ILD. The median survival for RA-ILD patients is 2–14 years, with a 5–10-year survival rate of approximately 50%. Therefore, early diagnosis of RA-ILD is crucial for identifying disease progression and optimizing treatment strategies, thereby improving patients' quality of life.

[0003] Currently, high-resolution computed tomography (HRCT) is considered the gold standard for diagnosing and monitoring the progression of rheumatoid arthritis-infectious disease (RA-ILD). However, by the time RA-ILD is definitively diagnosed by HRCT, patients may already have impaired lung function. Furthermore, the clinical use of HRCT is limited by factors such as ionizing radiation and cost. Therefore, increasing research is exploring new diagnostic methods, including lung ultrasound, biomarkers, and novel autoantibodies. Autoantibodies play a crucial role in the diagnosis, classification, and monitoring of rheumatic and immune diseases. They not only serve as biomarkers to help physicians determine the presence of the disease but also reflect its activity and prognosis. Early diagnosis of RA-ILD through autoantibody detection is of great significance for clinical treatment.

[0004] EXOSC4 is a key component of the exosome complex, playing a crucial role in RNA processing and degradation. This complex, composed of multiple subunits, is essential for maintaining stable intracellular RNA levels. It also influences various biological processes, including gene expression, cell growth, and programmed cell death. Currently, EXOSC4 is considered a therapeutic target for diseases such as pancreatic cancer, lung adenocarcinoma, and ovarian cancer. However, no studies or reports have been found regarding EXOSC4 as a diagnostic biomarker for RA-ILD. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a biomarker and related products for diagnosing rheumatoid arthritis complicated with interstitial lung disease.

[0006] This invention is the first to discover a serum biomarker, anti-EXOSC4 autoantibody, associated with the diagnosis of rheumatoid arthritis with interstitial lung disease (RA-ILD). By detecting the expression level of the anti-EXOSC4 autoantibody (e.g., using the EXOSC4 antigen) in subjects, rheumatoid arthritis with interstitial lung disease can be diagnosed rapidly and accurately. Based on this pioneering research result, this invention is proposed.

[0007] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0008] A first aspect of the present invention provides a biomarker for diagnosing or screening rheumatoid arthritis complicated with interstitial lung disease.

[0009] Furthermore, the biomarker is an anti-EXOSC4 autoantibody.

[0010] Furthermore, the anti-EXOSC4 autoantibody is an autoantibody found in the subject's serum, whole blood, or plasma;

[0011] Optionally, the subject is a human being.

[0012] In this invention, EXOSC4 is referred to as exosome component 4 [Homo sapiens (human)], and its Gene ID in NCBI is 54512. The amino acid sequence corresponding to EXOSC4 is shown in SEQ ID NO:1. Currently, there are no studies or reports on the use of anti-EXOSC4 autoantibodies as biomarkers for the diagnosis or screening of rheumatoid arthritis complicated with interstitial lung disease.

[0013] In a specific embodiment of the present invention, the present invention for the first time uses a reagent for detecting the anti-EXOSC4 autoantibody (e.g., EXOSC4 antigen) in the diagnosis of rheumatoid arthritis complicated with interstitial lung disease. Validation in real clinical samples (training set and validation set) collected in the present invention revealed that the serum biomarker has high diagnostic efficacy (AUC value greater than 0.80), high accuracy, sensitivity and specificity, and can be used for the effective diagnosis of rheumatoid arthritis complicated with interstitial lung disease.

[0014] In this invention, AUC refers to the area under the receiver operating characteristic (ROC) curve, which is well known in the art. AUC measurement is useful for comparing the accuracy of classifiers across the entire data range. Classifiers with higher AUCs have a greater ability to correctly classify unknown groups among two or more target groups. ROC curves are useful for depicting the performance of specific features (e.g., any biomarkers described herein and / or any entries of other biomedical information) when distinguishing between two groups.

[0015] Typically, characteristic data are selected across the entire population in ascending order based on the values ​​of a single characteristic. Then, for each value of that characteristic, the true positive rate and false positive rate of the data are calculated. The true positive rate is determined by counting the number of cases with values ​​higher than that characteristic and dividing by the total number of cases. The false positive rate is determined by counting the number of controls with values ​​higher than that characteristic and dividing by the total number of controls.

[0016] ROC curves can be generated about individual features or about other individual outputs. For example, combinations of two or more features can be combined mathematically (e.g., addition, subtraction, multiplication, etc.) to provide a single sum value, which can be plotted on the ROC curve. Furthermore, any combination of multiple features derived from individual output values ​​can be plotted on the ROC curve, which can be used to analyze the accuracy of diagnostics.

[0017] In this invention, the sample refers to a composition obtained from or derived from the target subject, which contains cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics.

[0018] In some embodiments, the sample may be obtained from a subject (including humans or non-human mammals, preferably humans) and may be a blood sample, tissue sample, or other fluid sample of biological origin, such as a biopsy tissue sample or tissue culture or cells derived therefrom. The source of the tissue sample may be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluids; cells from any stage of an individual's pregnancy or development; or plasma. The term "sample" includes biological samples that have been processed in any way after their acquisition, such as by reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. The present invention does not particularly limit the specific type of sample.

[0019] Exemplarily, the samples include, but are not limited to: serum, whole blood, plasma, tissue, tissue-derived cells, blood-derived cells, lymph, synovial fluid, exosomes, cell extracts, feces, urine, saliva, sputum, synovial fluid, pleural effusion, peritoneal effusion, serous cavity effusion, lymph, cerebrospinal fluid, uterine fluid, digestive juices, bile, pulmonary endobronchial lavage fluid, or organs, or any combination thereof. In a specific embodiment of the present invention, the sample is a serum sample derived from the subject.

[0020] In this invention, the subject refers to any animal, including both human and non-human animals. The term non-human animals includes all vertebrates, such as mammals, including non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of this invention, the subject is preferably a human.

[0021] A second aspect of the present invention provides a reagent for detecting biomarkers as described in the first aspect of the present invention.

[0022] Furthermore, the reagent is used for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker.

[0023] Furthermore, the reagent is used to detect the biomarker via an antigen-antibody reaction;

[0024] Optionally, the reagent is used to detect the biomarker by ELISA, fluorescence immunoassay, or chemiluminescence immunoassay;

[0025] Optionally, the reagent is the EXOSC4 antigen;

[0026] Optionally, the amino acid sequence of the EXOSC4 antigen is as shown in SEQ ID NO:1 or has at least 80% sequence homology with SEQ ID NO:1 and is capable of recognizing the anti-EXOSC4 autoantibody.

[0027] In this invention, homology refers to sequence homology. The homology of amino acid sequences is expressed as the similarity between sequences, and sequence homology is often measured as a percentage of homology (or similarity or identity); the higher the percentage, the more similar the two sequences are. When compared using standard methods, homologs or variants of peptides will have a relatively high degree of sequence homology.

[0028] In some embodiments, the sequence of the EXOSC4 antigen is selected from any one of the following (1)-(3):

[0029] (1) The amino acid sequence as shown in SEQ ID NO:1;

[0030] (2) An amino acid sequence that has at least 80% sequence homology with SEQ ID NO:1 and is capable of recognizing the anti-EXOSC4 autoantibody;

[0031] (3) The amino acid sequence that can recognize the anti-EXOSC4 autoantibody obtained by modifying or mutating the amino acid sequence in (1) or (2) above.

[0032] In this invention, the reagent is used to detect the expression level of the anti-EXOSC4 autoantibody as described above.

[0033] A third aspect of the invention provides the use of reagents for detecting biomarkers as described in the first aspect of the invention in the preparation of diagnostic products for the diagnosis or screening of rheumatoid arthritis complicated with interstitial lung disease.

[0034] Furthermore, the sample is the subject's serum, whole blood, or plasma.

[0035] Furthermore, the diagnostic product is a diagnostic kit, a detection chip, or a detection strip;

[0036] Optionally, the diagnostic kit is a diagnostic kit for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker;

[0037] Optionally, the diagnostic kit is used to detect the biomarker via an antigen-antibody reaction;

[0038] Optionally, the diagnostic kit is an ELISA detection kit, a fluorescence immunoassay kit, or a chemiluminescent immunoassay kit;

[0039] Optionally, the diagnostic kit contains the EXOSC4 antigen;

[0040] Optionally, the amino acid sequence of the EXOSC4 antigen is as shown in SEQ ID NO:1 or has at least 80% sequence homology with SEQ ID NO:1 and is capable of recognizing the anti-EXOSC4 autoantibody.

[0041] Furthermore, the reagent is used for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker;

[0042] Optionally, the reagent is used to detect the biomarker via an antigen-antibody reaction;

[0043] Optionally, the reagent is used to detect the biomarker by ELISA, fluorescence immunoassay, or chemiluminescence immunoassay;

[0044] Optionally, the reagent is the EXOSC4 antigen;

[0045] Optionally, the amino acid sequence of the EXOSC4 antigen is as shown in SEQ ID NO:1 or has at least 80% sequence homology with SEQ ID NO:1 and is capable of recognizing the anti-EXOSC4 autoantibody.

[0046] In some embodiments, the method for detecting the biomarker as described in the first aspect of the present invention is not limited to enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, or chemiluminescence immunoassay. Any prior art method disclosed that can be used to detect the expression level or content of the biomarker (anti-EXOSC4 autoantibody) is within the scope of protection of the present invention.

[0047] Exemplary methods for detecting biomarkers as described in the first aspect of the present invention include, but are not limited to: enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, chemiluminescence immunoassay, radioimmunoassay (RIA), colloidal gold immunochromatography (GICA), immunoturbidimetry, latex agglutination test (LAT), surface plasmon resonance (SPR), etc.

[0048] A fourth aspect of the present invention provides a diagnostic or screening product.

[0049] Furthermore, the diagnostic or screening product comprises the reagents described in the second aspect of the present invention;

[0050] Optionally, the diagnostic or screening product is a diagnostic kit, a detection chip, or a detection strip;

[0051] Optionally, the diagnostic kit is a diagnostic kit for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker.

[0052] In some implementations, the diagnostic or screening product is used to diagnose or screen for rheumatoid arthritis with interstitial lung disease by quantitatively detecting anti-EXOSC4 autoantibodies in test samples from subjects.

[0053] In some embodiments, the diagnostic kit is an ELISA kit containing reagents for detecting the anti-EXOSC4 autoantibody. In a specific embodiment of the invention, the reagent for detecting the anti-EXOSC4 autoantibody is an EXOSC4 antigen. In a specific embodiment of the invention, the amino acid sequence of the EXOSC4 antigen is as shown in SEQ ID NO:1 or has at least 80% sequence homology with SEQ ID NO:1 and is capable of recognizing the anti-EXOSC4 autoantibody.

[0054] In some embodiments, the ELISA test kit may further include elements necessary for performing an ELISA. Exemplarily, the ELISA test kit may further include: a solid-phase carrier, enzyme markers, sample processing and reaction reagents, colorimetric and termination reagents, calibration and quality control elements, and auxiliary materials.

[0055] In some implementations, the solid-phase support is used to immobilize antigens and provide a reaction interface. Common types include: 96-well microplates (made of polystyrene, with strong adsorption and chemically treated surface to enhance protein binding ability) and magnetic beads (suitable for magnetic separation ELISA, such as chemiluminescent immunoassay).

[0056] In some implementations, the enzyme label is used to bind to the antibody to be detected, and signal amplification is achieved through enzyme-catalyzed substrate color development (or luminescence). Common enzyme-substrate systems include: horseradish peroxidase (HRP)-tetramethylbenzidine (TMB, commonly used) / o-phenylenediamine (OPD), and alkaline phosphatase (AP)-p-nitrophenyl phosphate (pNPP). Label forms include: enzyme-labeled secondary antibodies (e.g., goat anti-human IgG-HRP (used for indirect antibody detection)) and enzyme-labeled antigens (used for competitive antibody detection (if the antibody in the sample binds to the solid-phase antigen, the free antibody competes with the enzyme-labeled antigen for binding to a limited number of sites)).

[0057] In some embodiments, the sample processing and reaction reagents include a sample diluent, a washing buffer, and a blocking buffer. The sample diluent is used to dilute serum samples and reduce matrix interference (such as nonspecific proteins in serum). Common components include phosphate-buffered saline (PBS), Tris-HCl buffer, and solutions containing bovine serum albumin (BSA) or gelatin to block nonspecific binding sites. The washing buffer is used to wash away unbound substances and reduce background signal. Common components include PBS or TBS (Tris-buffered saline) + 0.05% Tween-20 (a surfactant to enhance washing effect). The blocking buffer is used to block unbound antigen sites on the solid-phase support and reduce nonspecific adsorption. Common reagents include 5% skim milk powder and 1% BSA (dissolved in PBS or TBS).

[0058] In some embodiments, the colorimetric and termination reagents include a colorimetric solution and a termination solution. The colorimetric solution corresponds to the substrate solution of the enzyme-labeled enzyme (e.g., the TMB solution corresponding to HRP), and is typically a colorless or light-colored liquid that develops color after reacting with the enzyme. The termination solution is used to terminate the enzymatic reaction and fix the color (suitable for substrates requiring termination, such as TMB). Common reagents include 2M sulfuric acid (HRP-TMB system) and 3M sodium hydroxide (AP-pNPP system).

[0059] In some implementations, the calibration and quality control elements include calibrators and quality control materials. The calibrators are used to establish a standard curve of antibody concentration versus absorbance for further quantitative detection. The quality control materials are used to monitor the accuracy of the experimental procedure and include a positive control (containing a known amount of anti-EXOSC4 antibody) and a negative control (normal serum without anti-EXOSC4 antibody).

[0060] In some implementations, the auxiliary materials include an ELISA plate cover (to prevent evaporation and contamination), a sealing film (for sealing the microplate during incubation), and instructions (containing operating steps, reagent composition, and result interpretation methods).

[0061] In some embodiments, the detection chip is a protein detection chip, which includes a solid support and EXOSC4 protein (i.e., EXOSC4 antigen) immobilized on the solid support. The solid support can be made of various commonly used materials in the chip industry, such as, but not limited to, plastic products, microparticles, membrane carriers, etc.

[0062] The fifth aspect of the present invention provides a system or apparatus for diagnosing or screening rheumatoid arthritis complicated with interstitial lung disease.

[0063] Furthermore, the system or device includes a processor, an input module, and an output module;

[0064] The processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the expression level of the biomarker described in the first aspect of the present invention in the subject sample, and a computer-readable medium containing instructions, which, when executed by the processor, execute an algorithm on the input expression level of the anti-EXOSC4 autoantibody; the output module is used to output whether the subject has rheumatoid arthritis with interstitial lung disease or the risk of having rheumatoid arthritis with interstitial lung disease.

[0065] In this invention, the system and / or apparatus is a method for distinguishing different components, elements, parts, sections, or assemblies at different levels. However, if other terms can achieve the same purpose, they can be replaced by other expressions. Those skilled in the art will know that this invention can be implemented as a device, method, or computer program product. Therefore, the disclosure of this invention can be specifically implemented in the following forms: entirely hardware, entirely software (including firmware, resident software, microcode, etc.), or a combination of hardware and software. Furthermore, in some specific embodiments, this invention can also be implemented as a computer program product in one or more computer-readable media containing computer-readable program code.

[0066] The present invention also provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the system or apparatus described in the fifth aspect of the present invention.

[0067] Furthermore, the present invention also provides a method for diagnosing or screening rheumatoid arthritis complicated with interstitial lung disease, the method comprising the following steps:

[0068] (1) Collect test samples from subject sources;

[0069] (2) Detect the expression level of anti-EXOSC4 autoantibodies in test samples from subjects;

[0070] (3) Diagnose or screen whether the subject has rheumatoid arthritis with interstitial lung disease or the risk of having rheumatoid arthritis with interstitial lung disease based on the expression level of the detected anti-EXOSC4 autoantibody.

[0071] In a specific embodiment of the present invention, the method for diagnosing or screening rheumatoid arthritis complicated with interstitial lung disease includes the following steps:

[0072] (1) Collect samples from the subjects to be tested and control samples; wherein the control samples are from healthy people and people with rheumatoid arthritis without interstitial lung disease;

[0073] (2) Detect and compare the expression levels of anti-EXOSC4 autoantibodies in the test subject samples and control samples;

[0074] (3) If the expression level of anti-EXOSC4 autoantibody in the sample of the subject to be tested is significantly upregulated compared with the expression level of anti-EXOSC4 autoantibody in the control sample, then the subject to be tested is judged to have a high risk of having rheumatoid arthritis with interstitial lung disease or rheumatoid arthritis with interstitial lung disease.

[0075] Furthermore, the sample from the subject to be tested is a serum sample, plasma sample, or whole blood sample from the subject to be tested.

[0076] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0077] (1) This invention is the first to discover a serum biomarker, anti-EXOSC4 autoantibody, closely related to the diagnosis of rheumatoid arthritis combined with interstitial lung disease (RA-ILD). RA-ILD can be rapidly and accurately diagnosed by detecting the expression level of this anti-EXOSC4 autoantibody (e.g., using the EXOSC4 antigen) in subjects. Furthermore, this invention provides a simple, rapid, and accurate RA-ILD diagnostic kit that can be used for the early diagnosis and screening of RA-ILD, providing a reliable basis for the early detection and treatment of RA-ILD.

[0078] (2) This invention is the first to use reagents for detecting the anti-EXOSC4 autoantibody (e.g., EXOSC4 antigen) in the diagnosis of RA-ILD. Validation in real clinical samples (training and validation sets) collected in this invention revealed that the serum biomarker anti-EXOSC4 autoantibody has high diagnostic efficacy, with high accuracy, sensitivity, and specificity. This invention provides a basis for clinical decision-making related to RA-ILD and lays the foundation for subsequent clinical research, demonstrating promising application prospects and significant translational value. Attached Figure Description

[0079] Figure 1 : Screening diagram of RA-ILD autoantigens in Example 1 of this invention;

[0080] Figure 2 Example 2 of this invention shows the fluorescence signal of anti-EXOSC4 autoantibodies in the serum of healthy donors, patients with RA-non-ILD (rheumatoid arthritis without interstitial lung disease), and patients with RA-ILD (rheumatoid arthritis with interstitial lung disease).

[0081] Figure 3 : The expression level of anti-EXOSC4 autoantibodies in serum samples from healthy donors, RA-non-ILD, and RA-ILD patients in the training set was detected by double-antibody sandwich ELISA in Example 3 of this invention;

[0082] Figure 4 ROC analysis statistics of EXOSC4 antigen in the training set for the diagnosis of rheumatoid arthritis complicated with interstitial lung disease in Example 3 of this invention;

[0083] Figure 5The expression level of anti-EXOSC4 autoantibodies in serum samples from healthy donors, RA-non-ILD, and RA-ILD patients was detected and verified by double-antibody sandwich ELISA in Example 4 of this invention.

[0084] Figure 6 ROC analysis statistics of EXOSC4 antigen in the validation set for the diagnosis of rheumatoid arthritis complicated with interstitial lung disease in Example 4 of this invention. Detailed Implementation

[0085] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. To facilitate understanding of the invention, the following terms used herein are explained:

[0086] As used herein, the terms “comprising” or “including” mean that any one or more of the stated elements or components are included, without excluding other elements or other components.

[0087] As used herein, the term "biomarker" refers to a molecular indicator possessing specific biological, biochemical, or other characteristics that can be used to determine the presence or absence of a particular disease or condition and / or the severity of a particular disease or condition. In a specific embodiment of the invention, the biomarker is an anti-EXOSC4 autoantibody.

[0088] As used herein, the term "expression level" is synonymous with "level," referring to the absolute or relative amount of the biomarker anti-EXOSC4 autoantibody described in this invention. The expression level of the biomarker anti-EXOSC4 autoantibody described in this invention can be determined by various techniques. In particular, the absolute or relative amount of the biomarker anti-EXOSC4 autoantibody described in this invention can be detected using methods well known to those skilled in the art.

[0089] As used herein, the term "diagnosis" refers to the discovery, judgment, or recognition of an individual's health status or condition based on one or more symptoms, data, or other information relevant to that individual. An individual's health status may be diagnosed as healthy / normal (i.e., without disease or ailment), or as unhealthy / abnormal (i.e., with disease or ailment), or as a specific degree of disease severity or progression (e.g., healthy individuals, RA-non-ILD patients (rheumatoid arthritis without interstitial lung disease), and RA-ILD patients (rheumatoid arthritis without interstitial lung disease)). The term diagnosis includes the early detection of a disease / symptom associated with a specific disease or ailment; the characteristics or classification of the disease; the discovery of disease progression, cure, or relapse; and the discovery of an individual's response to the disease after treatment or intervention. In a specific embodiment of the invention, the diagnosis includes the diagnosis or screening for rheumatoid arthritis with interstitial lung disease.

[0090] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0091] Example 1: Screening for potential autoantigens in RA-ILD using AT2 cells

[0092] 1. Immunoprecipitation

[0093] AT2 cells were washed twice with pre-chilled PBS and lysed on ice for 5 min using RIPA lysis buffer containing protease inhibitors. Cells were then scraped, and the cell suspension was transferred to EP tubes. The cells were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected, and all lysis buffer was mixed and aliquoted into new EP tubes. One tube of the lysis buffer served as a blank control, while the others were incubated with 5 μL of serum from different patients (RA-non-ILD patients and RA-ILD patients). RA-non-ILD patients refer to patients with rheumatoid arthritis without interstitial lung disease, while RA-ILD patients refer to patients with rheumatoid arthritis and interstitial lung disease. The cells were incubated on a shaker at 4°C for 1 h and then at room temperature for 30 min. Protein A / G beads were washed twice with PBS, and Protein A / G was mixed. 100 μL of Protein A / G was added to each tube, and the tubes were incubated at 4°C for 1 h and then at room temperature for 30 min. Then, centrifuge at 4000 rpm for 3 min at 4℃; slowly remove the supernatant and wash the precipitate 3 times with pre-cooled PBS, 1 mL each time.

[0094] 2. Pro-DIA Quantitative Proteomics

[0095] The sample was thawed at 4℃, washed 5 times with pre-cooled PBS, and the supernatant was discarded after short-term centrifugation. The centrifugation was repeated 5 times at 1000g for 30s. The remaining magnetic beads were redissolved in 500μL of 50mM NH4HCO3. DTT was added to a final concentration of 1mM, and the mixture was incubated at 65℃ for 30min, with repeated pipetting to mix. 0.5μg trypsin was added, and the mixture was incubated overnight at 37℃. 0.5μg trypsin was added, and the mixture was incubated at 37℃ for 3h. IAA was added to a final concentration of 2.5mM, and the mixture was incubated at room temperature in the dark for 30min. Formic acid was added to bring the pH to approximately 2-3, and the mixture was centrifuged at 12000rpm for 5min, and the supernatant was collected. The collected peptides were analyzed using SOLA. TM The SPE 96-well plate was desalted, and the desalted peptides were evaporated to dryness. The evaporated peptide samples were reconstituted using mass spectrometry A phase and the peptides were quantified. The separated peptide solution was injected into the mass spectrometer and detected by LC-MS / MS high-resolution mass spectrometry, with data acquisition performed in Pro-DIA mode.

[0096] 3. Experimental Results

[0097] The results of RA-ILD autoantigen screening are shown in the figure below. Figure 1As shown. The amino acid sequence of the EXOSC4 antigen is: MAGLELLSDQGYRVDGRRAGELRKIQARMGVFAQADGSAYIEQGNTKA LAVVYGPHEIRGSRARALPDRALVNCQYSSATFSTGERKRRPHGDRKSCEMGLQLRQTFEAAILTQLHPRSQIDIYVQVLQADGGTYAACVNAATLAVLDAGIPMRDFVCACSAGFVDGTALADLSHVEEAAGGPQLALALLPASGQIALLEMDARLHEDHLERVLEAAAQAARDVHTLLDRVVRQHVREASILLGD (SEQ ID NO:1).

[0098] Example 2: Comparison of serum EXOSC4 antibody levels in different patients using protein chips

[0099] 1. Experimental Methods

[0100] (1) Protein spotting: using a crystal-based Personal Arrayer TM 16. Spotting was performed using a spotting instrument. The Bio-polymer 3D substrate H was brought to room temperature. Three spotting replicates were set for each sample, and 90 pre-spottings were performed according to the experimentally designed spotting matrix. During and after the spotting process, the spotting results were observed, and any unspotted locations were recorded and replenished promptly. A barrier was affixed, and the sample was stored overnight at 4°C in the dark.

[0101] (2) Chip sealing: 5% BSA, room temperature, protected from light for 30 minutes.

[0102] (3) Incubation of serum: Dilute serum with 1% BSA (1:500), add 50 μL of diluted serum to each cell, and incubate at room temperature for 1 h.

[0103] (4) Washing: Wash with PBS three times, 2 minutes each time.

[0104] (5) Incubation of secondary antibody: Dilute anti-human Cy3 (1:500) with 1% BSA, add 50 μL of diluted secondary antibody to each cell, and incubate at room temperature for 1 h.

[0105] (6) Washing: Remove the chip enclosure and wash the chip with ultrapure water for 10 minutes.

[0106] (7) Imaging: After checking that the substrate is dry and clean, use a microarray scanner to scan the chip.

[0107] 2. Experimental Results

[0108] A microarray scanner was used to measure the signal intensity of all EXOSC4 antigen sites in each assay. Protein chip results showed that serum from RA-ILD patients exhibited the highest signal intensity upon binding to the EXOSC4 antigen. Figure 2 ).

[0109] Example 3: In the training set, ELISA was used to compare the serum EXOSC4 antibody levels and the diagnostic efficacy of EXOSC4 antigen for RA-ILD in different patients.

[0110] 1. Experimental materials

[0111] Human EXOCS4 pre-coated ELISA plate, 5% BSA, HRP goat anti-human IgG, PBST solution, chromogenic substrate TMB, stop solution, sealing film.

[0112] 2. Sample Source

[0113] Serum samples were collected from healthy donors (HC), patients with RA-non-ILD (rheumatoid arthritis without interstitial lung disease), and patients with RA-ILD (rheumatoid arthritis without interstitial lung disease) as the training set. The samples were obtained from the Department of Rheumatology, Guang'anmen Hospital, China Academy of Chinese Medical Sciences. All participants provided informed consent and signed informed consent forms.

[0114] The number of cases in each sample in the training set is as follows: healthy donor HC serum samples (n=30), RA-non-ILD patient serum samples (n=30), and RA-ILD patient serum samples (n=30).

[0115] 3. Experimental Methods

[0116] (1) Coating antigen: 1 μg of recombinant EXOSC4 protein (the amino acid sequence of the recombinant EXOSC4 protein is shown in SEQ ID NO:1) was thoroughly mixed with 10 mL of PBS, and 100 μL of the diluted protein antigen solution was added to each well. The ELISA plate was sealed with sealing film and incubated overnight at 4°C.

[0117] (2) Washing: After incubation, remove the microplate and discard the liquid in the wells. Add 200 μL of PBST washing buffer to each well, soak for 1-2 minutes, and then shake off the washing buffer. Repeat the washing process 3 times.

[0118] (3) Blocking: Add 100 μL of 5% BSA solution to each well, seal the microplate with the sealing membrane, and block at 37°C for 1 h.

[0119] (4) Washing: Same as step (2) above.

[0120] (5) Incubation of serum: Dilute the serum 100 times with PBS, add 100 μL of diluted serum to each well, seal the microplate with a sealing membrane, and incubate at 37°C for 1 h.

[0121] (6) Washing: Same as step (2) above.

[0122] (7) Incubation of secondary antibody: HRP goat anti-human IgG was diluted at a ratio of 1:10000. 100 μL of the diluted secondary antibody solution was added to each well. The plate was sealed with a sealing membrane and incubated at 37°C for 1 h.

[0123] (8) Washing: Same as step (2) above.

[0124] (9) Color development: Add 100 μL of substrate color development solution to each well and incubate at room temperature in the dark for 15 min to terminate the reaction.

[0125] (10) Measurement: The absorbance at 450 nm after color development was measured using an ELISA reader. This yielded the expression level of the EXOSC4 antibody.

[0126] 4. Experimental Results

[0127] In the training set, the levels of EXOSC4 antibodies in the serum of healthy donors (HC, n=30), RA-non-ILD, and RA-ILD, n=30, were detected by double-antibody sandwich ELISA. Figure 3 As shown in the figure, the results indicated that the level of EXOSC4 antibody in the serum of RA-ILD patients was significantly higher than that in the healthy group and the RA-non-ILD group; the results of ROC analysis of the data are shown in the figure below. Figure 4 As shown, the results indicate that in the training set, the EXOSC4 antigen has high diagnostic efficacy for RA-ILD, with an AUC value of 0.807, a sensitivity of 96.67%, and a specificity of 56.67%. This demonstrates that EXOSC4 can be used for the effective diagnosis of RA-ILD with high accuracy, sensitivity, and specificity.

[0128] Example 4: In the validation set, ELISA was used to compare the serum EXOSC4 antibody levels and the diagnostic efficacy of EXOSC4 antigen for RA-ILD in different patients.

[0129] 1. Experimental materials

[0130] Human EXOCS4 pre-coated ELISA plate, 5% BSA, HRP goat anti-human IgG, PBST solution, chromogenic substrate TMB, stop solution, sealing film.

[0131] 2. Sample Source

[0132] In addition, serum samples were collected from healthy donors (HC), RA-non-ILD (rheumatoid arthritis without interstitial lung disease), and RA-ILD patients as a validation set. These samples were obtained from the Department of Rheumatology, Guang'anmen Hospital, China Academy of Chinese Medical Sciences. All participants provided informed consent and signed informed consent forms.

[0133] The number of cases in each sample in the validation set is as follows: healthy donor HC serum samples (n=32), RA-non-ILD patient serum samples (n=32), and RA-ILD patient serum samples (n=32).

[0134] 3. Experimental Methods

[0135] (1) Coating antigen: 1 μg of recombinant EXOSC4 protein (the amino acid sequence of the recombinant EXOSC4 protein is shown in SEQ ID NO:1) was thoroughly mixed with 10 mL of PBS, and 100 μL of the diluted protein antigen solution was added to each well. The ELISA plate was sealed with sealing film and incubated overnight at 4°C.

[0136] (2) Washing: After incubation, remove the microplate and discard the liquid in the wells. Add 200 μL of PBST washing buffer to each well, soak for 1-2 minutes, and then shake off the washing buffer. Repeat the washing process 3 times.

[0137] (3) Blocking: Add 100 μL of 5% BSA solution to each well, seal the microplate with the sealing membrane, and block at 37°C for 1 h.

[0138] (4) Washing: Same as step (2) above.

[0139] (5) Incubation of serum: Dilute the serum 100 times with PBS, add 100 μL of diluted serum to each well, seal the microplate with a sealing membrane, and incubate at 37°C for 1 h.

[0140] (6) Washing: Same as step (2) above.

[0141] (7) Incubation of secondary antibody: HRP goat anti-human IgG was diluted at a ratio of 1:10000. 100 μL of the diluted secondary antibody solution was added to each well. The plate was sealed with a sealing membrane and incubated at 37°C for 1 h.

[0142] (8) Washing: Same as step (2) above.

[0143] (9) Color development: Add 100 μL of substrate color development solution to each well and incubate at room temperature in the dark for 15 min to terminate the reaction.

[0144] (10) Measurement: The absorbance at 450 nm after color development was measured using an ELISA reader. This yielded the expression level of the EXOSC4 antibody.

[0145] 4. Experimental Results

[0146] In the validation set, the levels of EXOSC4 antibodies in the serum of healthy donors with HC, RA-non-ILD, and RA-ILD were detected by double-antibody sandwich ELISA. Figure 5 As shown in the figure, the results indicated that the level of EXOSC4 antibody in the serum of RA-ILD patients was significantly higher than that in the healthy group and the RA-non-ILD group; the results of ROC analysis of the data are shown in the figure below. Figure 6 As shown, the results indicate that in the validation set, the EXOSC4 antigen has high diagnostic efficacy for RA-ILD, with an AUC value of 0.883, a sensitivity of 84.38%, and a specificity of 87.50%. This result further demonstrates that EXOSC4 can be used for the effective diagnosis of RA-ILD with high accuracy, sensitivity, and specificity.

Claims

1. The application of reagents for detecting biomarkers in samples in the preparation of diagnostic products for the diagnosis or screening of rheumatoid arthritis complicated with interstitial lung disease, characterized in that, The biomarker is an anti-EXOSC4 autoantibody.

2. The application according to claim 1, characterized in that, The anti-EXOSC4 autoantibody is an autoantibody found in the subject's serum, whole blood, or plasma.

3. The application according to claim 2, characterized in that, The subjects were humans.

4. The application according to claim 1, characterized in that, The sample is the subject's serum, whole blood, or plasma.

5. The application according to claim 1, characterized in that, The diagnostic products are diagnostic kits, detection chips, or detection strips.

6. The application according to claim 5, characterized in that, The diagnostic kit is a diagnostic kit for detecting the biomarkers using ELISA, protein / peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay.

7. The application according to claim 5, characterized in that, The diagnostic kit is used to detect the biomarkers via antigen-antibody reactions.

8. The application according to claim 5, characterized in that, The diagnostic kit is an ELISA detection kit, a fluorescence immunoassay kit, or a chemiluminescence immunoassay kit.

9. The application according to claim 5, characterized in that, The diagnostic kit contains the EXOSC4 antigen.

10. The application according to claim 9, characterized in that, The amino acid sequence of the EXOSC4 antigen is shown in SEQ ID NO:

1.

11. The application according to claim 1, characterized in that, The reagents are those used for ELISA, protein / peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay of the biomarkers.

12. The application according to claim 1, characterized in that, The reagent is used to detect the biomarker through an antigen-antibody reaction.

13. The application according to claim 1, characterized in that, The reagent is used to detect the biomarker by ELISA, fluorescence immunoassay, or chemiluminescence immunoassay.

14. The application according to claim 1, characterized in that, The reagent is the EXOSC4 antigen.

15. The application according to claim 14, characterized in that, The amino acid sequence of the EXOSC4 antigen is shown in SEQ ID NO:

1.

16. A system or device for diagnosing or screening rheumatoid arthritis complicated with interstitial lung disease, characterized in that, The system or device includes a processor, an input module, and an output module; The processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the expression level of anti-EXOSC4 autoantibody in the subject sample, and a computer-readable medium containing instructions that, when executed by the processor, execute an algorithm at the input expression level of the anti-EXOSC4 autoantibody; the output module is used to output whether the subject has rheumatoid arthritis with interstitial lung disease or the risk of having rheumatoid arthritis with interstitial lung disease.

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