Biomarker for diagnosing rheumatoid arthritis complicated with interstitial lung disease and related products thereof

Abstract

The invention discloses a biomarker for diagnosing rheumatoid arthritis combined with interstitial lung disease and a related product thereof, a serum biomarker anti-EXOSC4 autoantibody related to diagnosis of rheumatoid arthritis combined with interstitial lung disease is found for the first time, and the serum biomarker has relatively high diagnosis efficiency and can be applied to diagnosis of rheumatoid arthritis combined with interstitial lung disease. The invention provides a basis for clinical decision-making related to rheumatoid arthritis combined interstitial lung disease, lays a foundation for subsequent clinical research, and has good application prospects and important transformation significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a biomarker for diagnosing rheumatoid arthritis combined with interstitial lung disease and related products. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease. Approximately 50% of RA patients experience extra-articular manifestations, the most common of which is interstitial lung disease (ILD). Evidence suggests that the mortality rate of patients with rheumatoid arthritis and interstitial lung disease (RA-ILD) is 2-10 times higher than that of RA patients without ILD. The median survival of patients with RA-ILD is 2-14 years, and the 5-10 year survival rate is 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 RA-ILD. However, by the time RA-ILD is definitively diagnosed by HRCT, the patient may already have impaired lung function. In addition, due to factors such as ionizing radiation and cost, HRCT is subject to certain limitations in clinical use. Therefore, an increasing number of studies are exploring new diagnostic methods, including lung ultrasound, biomarkers, and novel autoantibodies. Autoantibodies play an important role in the diagnosis, classification, and monitoring of rheumatic and immune diseases. They can not only serve as biomarkers to help doctors determine the presence of the disease, but also reflect the activity and prognosis of the disease. Early diagnosis of RA-ILD through autoantibody detection is of great significance for clinical diagnosis and treatment.

[0004] EXOSC4 is a crucial component of the exosome complex and plays a crucial role in RNA processing and degradation. This complex, composed of multiple subunits, is crucial for maintaining stable RNA levels within cells. It also influences diverse biological processes, including gene expression, cell growth, and programmed cell death. Currently, EXOSC4 has been investigated as a therapeutic target for diseases such as pancreatic cancer, lung adenocarcinoma, and ovarian cancer. However, there have been no studies or reports on EXOSC4 as a diagnostic marker 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 combined with interstitial lung disease in the art.

[0006] The present invention is the first to discover anti-EXOSC4 autoantibodies, a serum biomarker associated with the diagnosis of rheumatoid arthritis combined with interstitial lung disease (RA-ILD). By detecting the expression level of the anti-EXOSC4 autoantibodies in subjects (for example, using EXOSC4 antigen for detection), rheumatoid arthritis combined with interstitial lung disease can be quickly and accurately diagnosed. Based on this groundbreaking research result, the present invention is specially proposed.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

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

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

[0010] Furthermore, the anti-EXOSC4 autoantibodies are autoantibodies in the serum, whole blood or plasma of the subject;

[0011] Optionally, the subject is a human.

[0012] In the present invention, the full name of EXOSC4 is exosome component 4 [Homo sapiens (human)], and the Gene ID of EXOSC4 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 combined with interstitial lung disease.

[0013] In a specific embodiment of the present invention, the present invention uses for the first time a reagent for detecting the anti-EXOSC4 autoantibody (e.g., EXOSC4 antigen) in the diagnosis of rheumatoid arthritis combined with interstitial lung disease. After verification in real clinical samples (training set and validation set) collected by the present invention, it was found that the serum biomarker has a 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 combined with interstitial lung disease.

[0014] In the present invention, the AUC refers to the area under the curve of the receiver operating characteristic (ROC) curve, which is well known in the art. The AUC measurement is useful for comparing the accuracy of classifiers across the entire data range. A classifiers with higher AUCs have a higher ability to correctly classify unknowns between two or more target groups. ROC curves are useful for describing the performance of specific features (e.g., any biomarker described herein and / or any entry of additional biomedical information) when distinguishing between two populations.

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

[0016] An ROC curve can be generated for individual features or for other individual outputs. For example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single summed value, and this single summed value can be plotted on an ROC curve. In addition, any combination of multiple features where the combination is derived from individual output values ​​can be plotted on an ROC curve, which can be used to analyze diagnostic accuracy.

[0017] In the present invention, the sample refers to a composition obtained or derived from a 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 can be obtained from a blood sample, tissue sample, and other fluid samples of biological origin, such as a biopsy tissue sample or a tissue culture or cell derived therefrom, from a subject (the subject includes a human or non-human mammal, preferably, the subject is a human). The source of the tissue sample can be a solid tissue, such as an organ or tissue sample, biopsy tissue, or aspirate from fresh, frozen, and / or preserved; blood or any blood component; body fluid; cells from any time of pregnancy or development of an individual; or plasma. The term sample includes biological samples that have been processed in any way after their acquisition, such as 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 is not particularly limited to the specific type of the sample.

[0019] Exemplarily, the sample includes, but is not limited to, serum, whole blood, plasma, tissue, tissue-derived cells, blood-derived cells, lymph fluid, synovial fluid, exosomes, cell extracts, feces, urine, saliva, sputum, joint fluid, pleural effusion, serous effusion, lymph fluid, cerebrospinal fluid, uterine fluid, digestive fluid, bile, alveolar bronchial lavage fluid, or organ, or any combination thereof. In a specific embodiment of the present invention, the sample is a serum sample derived from a subject.

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

[0021] The second aspect of the present invention provides a reagent for detecting the biomarker according to the first aspect of the present invention.

[0022] Furthermore, the reagent is a reagent 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 through an antigen-antibody reaction;

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

[0025] Optionally, the reagent is 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 amino acid sequence of the anti-EXOSC4 autoantibody.

[0027] In the present invention, homology refers to sequence homology. Amino acid sequence homology 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 aligned using standard methods, homologs or variants of a polypeptide 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 shown in SEQ ID NO: 1;

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

[0031] (3) An amino acid sequence capable of recognizing the anti-EXOSC4 autoantibody obtained by modifying or mutating the amino acid sequence in (1) or (2) above.

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

[0033] The third aspect of the present invention provides use of a reagent for detecting the biomarker as described in the first aspect of the present invention in a sample in the preparation of a diagnostic product for diagnosing or screening rheumatoid arthritis combined with interstitial lung disease.

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

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

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

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

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

[0039] Optionally, the diagnostic kit comprises 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 amino acid sequence of the anti-EXOSC4 autoantibody.

[0041] Furthermore, the reagent is a reagent 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 through an antigen-antibody reaction;

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

[0044] Optionally, the reagent is 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 amino acid sequence of 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, and chemiluminescence immunoassay. Any method disclosed in the prior art 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] Illustratively, the method for detecting the biomarker as described in the first aspect of the present invention includes but is not limited to: enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, chemiluminescence immunoassay, radioimmunoassay (RIA), colloidal gold immunochromatography (GICA), immunoturbidimetry, latex agglutination test (Latex Agglutination Test, LAT), surface plasmon resonance (SPR), etc.

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

[0049] Furthermore, the diagnostic or screening product comprises the reagent according to the second aspect of the present invention;

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

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

[0052] In some embodiments, the diagnostic or screening product is used to diagnose or screen rheumatoid arthritis combined with interstitial lung disease by quantitatively detecting anti-EXOSC4 autoantibodies in a test sample derived from a subject.

[0053] In some embodiments, the diagnostic kit is an ELISA detection kit, which comprises a reagent for detecting the anti-EXOSC4 autoantibody. In a specific embodiment of the present invention, the reagent for detecting the anti-EXOSC4 autoantibody is an EXOSC4 antigen. In a specific embodiment of the present 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 amino acid sequence of the anti-EXOSC4 autoantibody.

[0054] In some embodiments, the ELISA detection kit may further comprise components necessary for performing ELISA. For example, the ELISA detection kit may further comprise: a solid phase carrier, an enzyme label, sample processing and reaction reagents, color development and termination reagents, calibration and quality control components, and auxiliary materials.

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

[0056] In some embodiments, the enzyme marker is used to bind to the antibody to be tested, and signal amplification is achieved by enzyme-catalyzed substrate color development (or luminescence). Common enzyme-substrate systems include: horseradish peroxidase (HRP)-tetramethylbenzidine (TMB, commonly used) / o-phenylenediamine (OPD), alkaline phosphatase (AP)-p-nitrophenyl phosphate (pNPP). The form of the marker includes: enzyme-labeled secondary antibody (such as goat anti-human IgG-HRP (for indirect detection of antibodies)), enzyme-labeled antigen (for competitive detection of antibodies (if the antibody in the sample is bound to the solid-phase antigen, the free antibody competes with the enzyme-labeled antigen for binding to limited sites)).

[0057] In some embodiments, the sample processing and reaction reagents include a sample diluent, a washing solution, and a blocking solution. The sample diluent is used to dilute the serum sample and reduce matrix interference (such as non-specific proteins in the serum). Common components include: phosphate buffered saline (PBS), Tris-HCl buffer, and bovine serum albumin (BSA) or gelatin to block non-specific binding sites. The washing solution is used to wash away unbound substances and reduce background signals. Common components include: PBS or TBS (Tris buffered saline) + 0.05% Tween-20 (surfactant, to enhance the washing effect). The blocking solution is used to block sites on the solid phase carrier that are not bound to the antigen and reduce non-specific adsorption. Common reagents include: 5% skim milk powder, 1% BSA (dissolved in PBS or TBS).

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

[0059] In some embodiments, the calibration and quality control elements include calibrators and quality control materials. The calibrators are used to establish a standard curve between antibody concentration and absorbance for further quantitative detection. The quality control materials are used to monitor the accuracy of the experimental process 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 embodiments, the auxiliary materials include an ELISA plate cover (to prevent evaporation and contamination), a sealing film (used to seal the microplate during incubation), and instructions (including operating steps, reagent composition, result interpretation method, etc.).

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

[0062] A fifth aspect of the present invention provides a system or device for diagnosing or screening rheumatoid arthritis combined 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 the computer-readable medium contains instructions, and the instructions, 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 combined with interstitial lung disease or the risk of suffering from rheumatoid arthritis combined with interstitial lung disease.

[0065] In the present invention, the system and / or device is a method for distinguishing different components, elements, parts, parts or assemblies at different levels. However, if other words can achieve the same purpose, the words can be replaced by other expressions. It is well known to those skilled in the art that the present invention can be implemented as an apparatus, method or computer program product. Therefore, the content disclosed in the present invention can be specifically implemented in the following forms, that is, it can be complete hardware, it can be complete software (including firmware, resident software, microcode, etc.), or it can be a combination of hardware and software. In addition, in some specific embodiments, the present invention can also be implemented in the form of a computer program product in one or more computer-readable media, and the computer-readable medium contains computer-readable program code.

[0066] The present invention also provides a computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, the system or device described in the fifth aspect of the present invention is implemented.

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

[0068] (1) Collect samples to be tested from subjects;

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

[0070] (3) Diagnosing or screening whether the subject suffers from rheumatoid arthritis combined with interstitial lung disease or is at risk of suffering from rheumatoid arthritis combined 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 combined with interstitial lung disease comprises the following steps:

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

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

[0074] (3) If the expression level of anti-EXOSC4 autoantibodies in the sample of the test subject is significantly increased compared with the expression level of anti-EXOSC4 autoantibodies in the control sample, it is judged that the test subject has rheumatoid arthritis combined with interstitial lung disease or has a higher risk of suffering from rheumatoid arthritis combined with interstitial lung disease.

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

[0076] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0077] (1) The present invention is the first to discover anti-EXOSC4 autoantibodies, a serum biomarker closely related to the diagnosis of rheumatoid arthritis combined with interstitial lung disease (RA-ILD). By detecting the expression level of the anti-EXOSC4 autoantibodies in a subject (for example, using EXOSC4 antigen for detection), RA-ILD can be quickly and accurately diagnosed. In addition, the present invention also provides a simple, rapid, and accurate RA-ILD diagnostic kit that can be used for the early diagnosis and screening of RA-ILD. The present invention provides a reliable basis for the early detection and treatment of RA-ILD.

[0078] (2) The present invention is the first to use a reagent for detecting anti-EXOSC4 autoantibodies (e.g., EXOSC4 antigen) in the diagnosis of RA-ILD. Validation in real clinical samples (training and validation sets) collected by the present invention revealed that the serum biomarker anti-EXOSC4 autoantibodies 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, with promising application prospects and important translational significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 : Diagram of RA-ILD autoantigen screening in Example 1 of the present invention;

[0080] Figure 2 : The protein chip in Example 2 of the present invention compares the fluorescence signal of anti-EXOSC4 autoantibodies in the serum of healthy donors, RA-non-ILD (rheumatoid arthritis without interstitial lung disease) and RA-ILD (rheumatoid arthritis with interstitial lung disease) patients;

[0081] Figure 3 : Expression graph of anti-EXOSC4 autoantibodies in serum samples of healthy donors, RA-non-ILD and RA-ILD patients in the training set detected by the double antibody sandwich ELISA method in Example 3 of the present invention;

[0082] Figure 4 : ROC analysis statistics corresponding to the use of EXOSC4 antigen in the training set for diagnosing rheumatoid arthritis combined with interstitial lung disease in Example 3 of the present invention;

[0083] Figure 5: The expression graph of the content of anti-EXOSC4 autoantibodies in serum samples of healthy donors, RA-non-ILD and RA-ILD patients detected by the double antibody sandwich ELISA method in Example 4 of the present invention;

[0084] Figure 6 : ROC analysis statistical graph corresponding to the use of EXOSC4 antigen in the validation set for the diagnosis of rheumatoid arthritis combined with interstitial lung disease in Example 4 of the present invention. DETAILED DESCRIPTION

[0085] The present invention will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. In order to facilitate understanding of the present invention, the following terms involved in the present invention are explained here:

[0086] As used herein, the terms “include” or “comprising” mean including any one or more of the stated elements or components but not excluding other elements or components.

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

[0088] As used herein, the term "expression level" is the same as "level", and refers to the absolute amount or relative amount of the anti-EXOSC4 autoantibody, a biomarker, of the present invention. The expression level of the anti-EXOSC4 autoantibody, a biomarker, of the present invention can be determined by a variety of techniques. In particular, the absolute amount or relative amount of the anti-EXOSC4 autoantibody, a biomarker, of the present invention can be detected by methods well known to those skilled in the art.

[0089] As used herein, term " diagnosis " refers to the discovery, judgment or cognition of individual's health status or situation based on one or more symptoms, data or other information relevant to individual.Individual health status can be diagnosed as healthy / normal (i.e., there is no disease or illness), or can be diagnosed as unhealthy / abnormal (i.e., there is disease or illness), or can be diagnosed as specific disease severity or progression (such as: healthy person, RA-non-ILD patient (rheumatoid arthritis is not combined with interstitial lung disease patient) and RA-ILD patient (rheumatoid arthritis is not combined with interstitial lung disease patient)), term diagnosis includes the early discovery of the disease / illness related to specific disease or illness; The characteristic or classification of disease; The discovery of the progress of disease, cure or recurrence; The discovery of the reaction to disease after individual disposal or treatment. In a specific embodiment of the present invention, the diagnosis includes diagnosing or screening rheumatoid arthritis combined with interstitial lung disease.

[0090] The reagents, raw materials, and experimental consumables used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

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

[0092] 1. Co-immunoprecipitation

[0093] AT2 cells were rinsed twice with pre-chilled PBS and lysed on ice for 5 minutes using RIPA lysis buffer containing protease inhibitors. Cells were then scraped and the cell suspension transferred to an EP tube. Centrifuged at 12,000 rpm for 10 minutes at 4°C, the supernatant collected, and all lysates were mixed and aliquoted into new EP tubes with equal volumes. One tube of the lysate served as a blank control, and the others were supplemented 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 beads were incubated on a shaker at 4°C for 1 hour and then at room temperature for 30 minutes. Protein A / G beads were washed twice with PBS, mixed, and 100 μL of protein A / G was added to each tube. The beads were rotated at 4°C for 1 hour and then at room temperature for 30 minutes. Then, centrifuge at 4000 rpm for 3 min at 4°C; slowly remove the supernatant and wash the precipitate repeatedly with pre-cooled PBS three times, 1 mL each time.

[0094] 2. Pro-DIA quantitative proteomics

[0095] The sample was thawed at 4°C, washed 5 times with pre-cooled PBS, and the supernatant was discarded after a brief centrifugation. The beads were re-dissolved in 500 μL of 50 mM NH4HCO3 for 30 seconds at 1000 g. DTT was added to a final concentration of 1 mM, and the reaction was carried out at 65°C for 30 minutes, with repeated pipetting to mix. 0.5 μg of trypsin was added and the enzyme was hydrolyzed at 37°C overnight. 0.5 μg of trypsin was added and the reaction was carried out at 37°C for 3 hours. IAA was added to a final concentration of 2.5 mM and the reaction was carried out at room temperature in the dark for 30 minutes. Formic acid was added to a pH of about 2-3, and the supernatant was recovered. The collected peptides were purified by SOLA. TM The SPE 96-well plate was desalted and the desalted peptides were evaporated; the evaporated peptide samples were reconstituted using mass spectrometry phase A 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, using Pro-DIA mode for data acquisition.

[0096] 3. Experimental results

[0097] The results of RA-ILD autoantigen screening are shown in the figure Figure 1The 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 the Crystal Core Personal Arrayer TM Perform spotting using a 16-well spotter. Return BioAo Polymer 3D Substrate H to room temperature. Set up three replicate wells for each sample, and pre-spot 90 times according to the experimental design. Observe the spotting process and at the end of the spotting process, record any unspotted locations, and re-spot as needed. Attach a fence and store at 4°C in the dark overnight.

[0101] (2) Chip blocking: Block with 5% BSA for 30 min at room temperature in the dark.

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

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

[0104] (5) Incubation with secondary antibody: dilute anti-human Cy3 (1:500) in 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 fence and wash the chip with ultrapure water for 10 minutes.

[0106] (7) Imaging: After checking that the substrate is free of water 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 spots in each test. Protein chip results showed that the serum of RA-ILD patients showed the highest signal intensity when binding to the EXOSC4 antigen ( Figure 2 ).

[0109] Example 3: Comparison of serum EXOSC4 antibody levels and EXOSC4 antigen levels in different patients for the diagnosis of RA-ILD using ELISA in the training set

[0110] 1. Experimental Materials

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

[0112] 2. Sample Source

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

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

[0115] 3. Experimental methods

[0116] (1) Antigen coating: 1 μg of EXOSC4 recombinant protein (the amino acid sequence of the EXOSC4 recombinant 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 a sealing film and incubated in a 4°C refrigerator overnight.

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

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

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

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

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

[0122] (7) Incubation with 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 ELISA plate was sealed with a sealing film, and incubated at 37°C for 1 h.

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

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

[0125] (10) Measurement: Use a microplate reader to measure the absorbance at 450 nm after color development to obtain the expression level of the EXOSC4 antibody.

[0126] 4. Experimental results

[0127] In the training set, the double antibody sandwich ELISA method was used to detect the levels of EXOSC4 antibodies in the serum of healthy donors HC (n=30), RA-non-ILD (n=30), and RA-ILD (n=30). Figure 3 As shown in the figure, the EXOSC4 antibody content in the serum of RA-ILD patients was significantly higher than that in the healthy group and RA-non-ILD group; the results of ROC analysis of the data are shown in the figure Figure 4 As shown, the results showed that in the training set, EXOSC4 antigen had a high diagnostic efficacy for diagnosing RA-ILD, with an AUC value of up to 0.807, a sensitivity of 96.67%, and a specificity of 56.67%, indicating that EXOSC4 can be used for the effective diagnosis of RA-ILD with high accuracy, sensitivity, and specificity.

[0128] Example 4: Comparison of serum EXOSC4 antibody levels and EXOSC4 antigen levels in different patients for the diagnosis of RA-ILD using ELISA in the validation set

[0129] 1. Experimental Materials

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

[0131] 2. Sample Source

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

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

[0134] 3. Experimental methods

[0135] (1) Antigen coating: 1 μg of EXOSC4 recombinant protein (the amino acid sequence of the EXOSC4 recombinant 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 a sealing film and incubated in a 4°C refrigerator overnight.

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

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

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

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

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

[0141] (7) Incubation with 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 ELISA plate was sealed with a sealing film, and incubated at 37°C for 1 h.

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

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

[0144] (10) Measurement: Use a microplate reader to measure the absorbance at 450 nm after color development to obtain the expression level of the EXOSC4 antibody.

[0145] 4. Experimental results

[0146] In the validation set, the double-antibody sandwich ELISA method was used to detect the levels of EXOSC4 antibodies in the serum of healthy donors HC, RA-non-ILD, and RA-ILD. Figure 5 As shown in the figure, the EXOSC4 antibody content in the serum of RA-ILD patients was significantly higher than that in the healthy group and RA-non-ILD group; the results of ROC analysis of the data are shown in the figure Figure 6 As shown, the results showed that in the validation set, EXOSC4 antigen had a high diagnostic efficacy for diagnosing RA-ILD, with an AUC value of 0.883, a sensitivity of 84.38%, and a specificity of 87.50%. This result once again proved that EXOSC4 can be used for the effective diagnosis of RA-ILD with high accuracy, sensitivity, and specificity.

Claims

1. A biomarker for diagnosing or screening rheumatoid arthritis combined with interstitial lung disease, characterized in that: The biomarker is anti-EXOSC4 autoantibody.

2. The biomarker according to claim 1, characterized in that The anti-EXOSC4 autoantibodies are autoantibodies in the serum, whole blood or plasma of the subject; Optionally, the subject is a human.

3. A reagent for detecting the biomarker according to claim 1 or 2, characterized in that: The reagent is a reagent used for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay or microfluidic immunoassay of the biomarker.

4. The reagent according to claim 3, characterized in that The reagent is used to detect the biomarker through an antigen-antibody reaction; Optionally, the reagent is used to detect the biomarker by ELISA, fluorescent immunoassay or chemiluminescent immunoassay; Optionally, the reagent is EXOSC4 antigen; 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 amino acid sequence of the anti-EXOSC4 autoantibody.

5. Use of a reagent for detecting the biomarker according to claim 1 or 2 in a sample in the preparation of a diagnostic product for diagnosing or screening rheumatoid arthritis combined with interstitial lung disease.

6. The use according to claim 5, characterized in that The sample is serum, whole blood or plasma of the subject.

7. The use according to claim 5, characterized in that The diagnostic product is a diagnostic kit, a detection chip or a detection reagent strip; Optionally, the diagnostic kit is a diagnostic kit for detecting the biomarker by ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay or microfluidic immunoassay; Optionally, the diagnostic kit is used to detect the biomarker through an antigen-antibody reaction; Optionally, the diagnostic kit is an ELISA detection kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit; Optionally, the diagnostic kit comprises EXOSC4 antigen; 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 amino acid sequence of the anti-EXOSC4 autoantibody.

8. The use according to claim 5, characterized in that The reagent is a reagent for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay or microfluidic immunoassay of the biomarker; Optionally, the reagent is used to detect the biomarker through an antigen-antibody reaction; Optionally, the reagent is used to detect the biomarker by ELISA, fluorescent immunoassay or chemiluminescent immunoassay; Optionally, the reagent is EXOSC4 antigen; 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 amino acid sequence of the anti-EXOSC4 autoantibody.

9. A diagnostic or screening product, characterized in that The diagnostic or screening product comprises the reagent of claim 3 or 4; Optionally, the diagnostic or screening product is a diagnostic kit, a detection chip or a detection reagent strip; Optionally, the diagnostic kit is a diagnostic kit for detecting the biomarker by ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay or microfluidic immunoassay.

10. A system or device for diagnosing or screening rheumatoid arthritis combined 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 the biomarker described in claim 1 or 2 in the subject sample, and the computer-readable medium contains instructions, and the instructions, 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 combined with interstitial lung disease or the risk of suffering from rheumatoid arthritis combined with interstitial lung disease.

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