Autoimmune disease-specific polypeptides, specific antibodies and uses

Abstract

The application discloses autoimmune disease specific polypeptides, specific antibodies and application, and polypeptides are at least selected from one polypeptide in the following amino acid sequences: CarSP-1: VFLCPWRAEGGQCcarPSLLFDLRDETRNV; CarSP-2: FLCPWRAEGGQCcarPS; CarSP-3: AEGGQCcarPSLLFDLR; CarSP-4: CcarPSLLFDLRDETRNV and CarSP-5: VFLCPWRAEGGQCcarPSLLFDLRDCTRNV, cys4&cys23bridge; in view of the threat of autoimmune diseases to human health and life treatment, the application will have broad application prospects for the diagnosis and treatment of autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to specific peptides, specific antibodies, and applications for autoimmune diseases. Background Technology

[0002] In recent years, the incidence of various autoimmune diseases has been rising, and the medical costs and disease status of autoimmune diseases place a heavy burden on society and families. There is an urgent need for technologies and methods for the diagnosis and treatment of autoimmune diseases. Currently, 3P medicine—predictive medicine, preventive medicine, and personalized medicine—has become an important direction for ensuring and improving human health. The discovery and application of autoimmune disease-specific autoantigens and autoantibodies are crucial for patients with autoimmune diseases to achieve 3P medicine.

[0003] Ankylosing spondylitis (AS) is an autoimmune disease. Its main manifestations are inflammation and structural destruction of the axial joints (sacroiliac joints, paravertebral soft tissues, vertebral processes, and peripheral joints), which may be accompanied by extra-articular manifestations. Clinically, it mainly presents as pain in the lower back, back, neck, buttocks, and hips, as well as joint swelling and pain. In severe cases, spinal deformities and joint ankylosis can occur. The prevalence of ankylosing spondylitis in my country is approximately 0.1%-0.3%, with a higher incidence in men than women. Ankylosing spondylitis presents in diverse ways depending on the organs and tissues involved, making diagnosis (especially early diagnosis) challenging. The main disease-specific molecule is HLA-B27; currently, other highly specific clinical indicators and therapeutic targets are lacking. Besides ankylosing spondylitis, other spinal joint diseases such as undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, and autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis also require targets for diagnosis and treatment related to autoantigens and autoantibodies.

[0004] With the rapid development of biotechnology, the application of mass spectrometry, peptide synthesis, antibody preparation, and ELISA technologies has greatly accelerated the discovery and application of novel autoantigens and antibodies. The technological basis for these methods largely involves screening autoantigens related to autoimmune diseases using mass spectrometry and biochemical techniques; verifying the antigenicity and specificity of these autoantigens using peptide synthesis, antibody preparation, and various immunological techniques, and preparing antigen-specific antibodies; and further detecting and confirming patient-associated autoantibodies using ELISA and other technologies. The discovery and application of modified antigenic peptides and specific antibodies for autoimmune diseases play a crucial role in establishing early warning screening, early diagnosis, qualitative diagnosis, and disease treatment for autoimmune diseases.

[0005] Similar to ankylosing spondylitis, for rheumatoid arthritis, existing literature reports that serum antibodies in rheumatoid arthritis can recognize citrullinated antigens, and autoantibodies against citrullinated modified peptides (ACPA) have been developed. Positive serum ACPA is included in the diagnostic criteria for rheumatoid arthritis. The emergence of anti-citrullinated modified peptide autoantibodies, as a serum heterogeneous marker for rheumatoid arthritis, has increased our understanding of the pathogenesis of rheumatoid arthritis. Currently, there are no known specific autoantigens for ankylosing spondylitis, and no corresponding autoantibody identification and detection methods exist.

[0006] Integrin (ITA2B)-carboxyethyl modified peptide is an autoantigen specifically produced by patients with ankylosing spondylitis (AS). This peptide is a novel modified polypeptide with a carboxyethyl cysteine ​​modification, obtained through mass spectrometry screening. Its modification form and antigenicity were confirmed using various in vitro and in vivo biochemical and immunological methods. The specific presence of integrin (ITA2B)-carboxyethyl modified peptide in the peripheral blood of AS patients helps to elucidate the pathogenesis of AS and to group the disease characteristics. Furthermore, the role of corresponding autoantibodies in AS cannot be ignored.

[0007] Anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibody is an autoantibody produced in peripheral blood plasma in patients with ankylosing spondylitis (AS) that can bind to the integrin-carboxyethyl modified peptide antigen. An anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibody detection kit is used for the diagnosis of AS. Clinical test results show that the positive rate of anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibody in AS patients is approximately higher than in healthy controls.

[0008] Currently, the diagnosis of ankylosing spondylitis in clinical practice mainly combines clinical manifestations, imaging, and laboratory tests. Laboratory diagnosis primarily includes immunological tests such as complete blood count, erythrocyte sedimentation rate (ESR), biochemistry, HLA-B27, and C-reactive protein. There are currently no specific antigen-antibody tests or treatments for ankylosing spondylitis. Disease-specific autoantigen-autoantibody screening is of great significance for revealing the pathogenesis of ankylosing spondylitis, grouping patients according to disease characteristics, and developing treatments based on specific autoantigen-autoantibodies. It also offers broad application prospects for the clinical diagnosis and treatment of spondyloarthritis (including ankylosing spondylitis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, etc.) and other autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis). Summary of the Invention

[0009] The purpose of this invention is to provide specific peptides, specific antibodies, and applications for autoimmune diseases. This invention screened and compared five autoimmune disease-specific modified antigen peptides. The anti-modified polyclonal antibodies prepared by immunizing animals with these anti-modified peptides have the characteristics of high specificity and high purity. Among them, the modified peptides can specifically detect anti-modified antibodies in the plasma of patients with ankylosing spondylitis.

[0010] Based on the above objectives, the solution of the present invention specifically includes:

[0011] Based on autoimmune disease-specific peptides, at least one peptide selected from the following amino acid sequences is used:

[0012] CarSP-1: VFLCPWRAEGGQCcarPSLLFDLRDETRNV;

[0013] CarSP-2: FLCPWRAEGGQCcarPS;

[0014] CarSP-3:AEGGQCcarPSLLFDLR;

[0015] CarSP-4: CcarPSLLFDLRDETRNV and

[0016] CarSP-5: VFLCPWRAEGGQCcarPSLLFDLRDCTRNV, cys4&cys23 bridge;

[0017] in:

[0018] Car represents carboxyl ethylation modification, Ccar represents carboxyl ethylated cysteine; Cys4 & Cys23 bridge refers to the disulfide bond formed between the cysteine ​​residues at positions 4 and 23 in the CarSP-5 peptide.

[0019] Optionally, the amino acid sequence of the polypeptide may be all or part of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0020] Optionally, the amino acid sequence of the polypeptide is a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0021] Or the difference between the sequence and the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 shall not exceed 5, 4, 3, 2 or 1 amino acids;

[0022] Alternatively, it may be a variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, wherein the difference between the variant and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 includes substitution, deletion and / or insertion of one or more amino acid residues or at least one N- / C-terminal extension.

[0023] A specific antibody, wherein the antibody is obtained by immunization with an autoimmune disease-specific polypeptide as an antigen, as described in any of the present invention.

[0024] Optionally, the antibody is prepared by immunizing animals with any of the autoimmune disease-specific polypeptides described in this invention as immunogens; the antibody binds to the integrin-cysteine ​​carboxyethyl modified site and does not bind to the unmodified integrin site.

[0025] Optionally, the antibody is prepared using the following method:

[0026] Mice / rabbits are immunized with a mixture of the autoimmune disease-specific peptide and complete Freund's adjuvant as described in any one of the present invention. The antigen peptide is then mixed with incomplete Freund's adjuvant for booster immunization, and the booster immunization is performed four times. Blood is collected from mice 10 days after the last immunization, serum is obtained by centrifugation, and specific affinity purification is performed on the modified site to obtain specific antibodies.

[0027] The present invention relates to the application of autoimmune disease-specific peptides in the preparation of drugs for the diagnosis, prevention and / or treatment of autoimmune diseases.

[0028] Optional, autoimmune diseases are primarily spondyloarthritis / diseases, including ankylosing spondylitis, axial / peripheral spondyloarthritis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, and juvenile spondyloarthritis; as well as other autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis.

[0029] Optionally, the applications include, but are not limited to, one or a combination of peptide-based detection kits, diagnostic reagents, protein chips, and immunoassay strips.

[0030] The application of any of the specific antibodies described in this invention in the preparation of drugs for the diagnosis, prevention and / or treatment of autoimmune diseases includes, but is not limited to, one or a combination of specific antibody-based kits, diagnostic reagents, protein chips and immunoassay strips.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention utilizes mass spectrometry to screen peripheral blood-specific modified peptides for autoimmune diseases, obtaining highly reactive modified antigen sequences. Further HLA binding capacity analysis of the screened peptides yielded specific antibodies against these modified peptides in immunized animals. These antibodies exhibit high specificity and purity. The aforementioned modified antigen peptides and specific antibodies can be used for the clinical diagnosis and treatment of autoimmune diseases, primarily including spondyloarthritis (ankylosing spondylitis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, etc.), as well as other autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis). Given the threat posed by autoimmune diseases to human health and quality of life, this invention has broad application prospects. Attached Figure Description

[0033] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0034] Figure 1 The modified peptide CarSP-3: AEGGQC is specifically expressed in peripheral blood mononuclear cells from patients with ankylosing spondylitis. car PSLLFDLR secondary mass spectra;

[0035] Figure 2 Differences in the levels of peptide-specifically expressed modified peptides in peripheral blood mononuclear cells of patients with ankylosing spondylitis compared to those in healthy controls;

[0036] Figure 3 Fluorescence polarization technique was used to detect the optimal antigenic peptides involved in HLA-DR presentation.

[0037] Figure 4 Immunospot assay with specific antibodies. Specific antibodies bind to modified antigenic peptides, but do not react with unmodified peptides.

[0038] Figure 5 Schematic diagram of the structure of peptide CarSP-5. The modified peptide exhibits a secondary structure, specifically displaying the antigenic peptide of the modified epitope;

[0039] Figure 6The results of the specific binding detection of rabbit polyclonal antibodies and modified peptides; where A is a schematic diagram of the binding scheme of positive antibody and antigen peptide, that is, the modified ITA2B-carboxyethyl modified peptide CarSP-5 is coated on a 96-well plate, different concentrations of specific polyclonal antibody are added, and after reaction with HRP-labeled antibody, the ELISA reading is performed; B is the specific detection result of the binding of specific antibody and antigen peptide.

[0040] Figure 7 A comparison of the detection capabilities of an ankylosing spondylitis anti-integrin-carboxyethyl modified antibody detection kit with unmodified peptides and unmodified modified peptides as antigens. A compares the detection capabilities of three antigens—linear unmodified peptide (SP-1), linear modified peptide (CarSP-1), and modified cyclic peptide (CarSP-5)—to detect anti-modified antibodies; B compares the detection capabilities of three antigens—linear unmodified peptide (SP-1), linear modified peptide (CarSP-1), and modified cyclic peptide (CarSP-5)—to detect anti-unmodified antibodies.

[0041] Figure 8 Results of anti-ITA2B-carboxyethyl modified autoantibodies detected in plasma from patients with ankylosing spondylitis (AS) and healthy controls (HC); A is a schematic diagram of the plasma antibody-antigen peptide binding protocol, i.e., the modified ITA2B-carboxyethyl modified peptide (CarSP-5) is coated onto a 96-well plate, different plasma samples are added, and the reaction with HRP-labeled antibodies is performed before detection; B is the ELISA result of specific detection of plasma sample binding with the antigen peptide; * indicates... p <0.05;

[0042] Figure 9 The results are from the clinical ELISA test in Example 7;

[0043] Figure 10 The results are from the flow cytometry analysis of the immune response in Example 8. Detailed Implementation

[0044] The embodiments listed below are merely preferred examples of the present invention and are not intended to limit the invention. Those skilled in the art will recognize that the present invention can have various combinations, modifications, and variations. Any modifications and / or alterations made to the present invention within the requirements and principles of the invention will fall within the scope of protection of the present invention.

[0045] This invention is based on a polypeptide with a novel cysteine ​​carboxyl ethylation site discovered in integrins.

[0046] In this invention, "peptide" refers to a linear amino acid chain consisting of two or more amino acids linked by peptide bonds. Amino acids in a polypeptide can be modified, deleted, added, or substituted. Polypeptides can be synthesized using conventional techniques. For example, polypeptides can be obtained through artificial synthesis.

[0047] The term "carboxyethyl" here refers to the substitution of a hydrogen atom in a molecule (such as cysteine) by a carboxyethyl group. For example, cysteine ​​becomes carboxyethylated cysteine ​​after carboxylethylation.

[0048] This invention uses mass spectrometry to screen peripheral blood-specific modified peptides for autoimmune diseases, obtaining highly reactive modified antigen sequences for these diseases. Further HLA binding ability analysis of the screened peptides revealed that the selected modified antigen peptides can bind to HLA-DR molecules, participate in antigen presentation, and induce the production of related CD4+. + T-cell immune response. Specific antibodies are prepared by immunizing animals with the obtained modified peptides. These antibodies are highly specific, stable, simple, and universal, and can specifically bind to both the pre- and post-modification modified antigen peptide sequences. The aforementioned modified antigen peptides and the specific antibodies they produce can be used in the clinical diagnosis and treatment of autoimmune diseases, primarily including spondyloarthritis / arthritis (ankylosing spondylitis, axial / peripheral spondyloarthritis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, juvenile spondyloarthritis, etc.), as well as other autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis). Given the threat that autoimmune diseases pose to human health and quality of life, this invention has broad application prospects in the diagnosis and treatment of autoimmune diseases.

[0049] This invention provides specific peptides and antibodies for autoimmune diseases and their uses. This invention screened and compared five specific modified antigen peptides for autoimmune diseases. The anti-modified polyclonal antibodies prepared by immunizing animals with the anti-modified peptides have the characteristics of high specificity and high purity. Among them, the modified peptides can specifically detect anti-modified antibodies in the plasma of patients with ankylosing spondylitis.

[0050] Specifically, it includes: CarSP-1: VFLCPWRAEGGQC car PSLLFDLRDETRNV (SEQ ID No. 1); CarSP-2: FLCPWRAEGGQC car PS (SEQ ID No. 2);

[0051] CarSP-3: AEGGQC car PSLLFDLR (SEQ ID No. 3);

[0052] CarSP-4: C car PSLLFDLRDETRNV (SEQ ID No. 4);

[0053] CarSP-5: VFLCPWRAEGGQC carPSLLFDLRDCTRNV, cys4&cys23 bridge (SEQ IDNo.5);

[0054] Among them, C car It is a carboxyethylated cysteine, and the Cys4 & Cys23 bridge is a disulfide bond formed between the 4th and 23rd cysteine ​​residues in the CarSP-5 peptide.

[0055] Of the five peptides listed above, CarSP-3 is an in vitro synthesized peptide designed to mimic the in vivo peptide C72SP (SEQ ID NO: 6): AEGGQC +72.02 PSLLFDLR. C72SP is a specific polypeptide found in patients with ankylosing spondylitis, characterized by a 72.02-mass shift at position 6 of the polypeptide's cysteine ​​residue; the specific cause is unknown.

[0056] Polypeptides CarSP-1, CarSP-2, and CarSP-4: The CarSP-1 polypeptide sequence was obtained by extending both ends (i.e., -NH3, -COOH) of the in vitro synthesized polypeptide CarSP-3. To further compare key differential sites in the CarSP-1 sequence and in conjunction with the characteristic analysis of polypeptide immunogenicity, CarSP-1 was truncated into polypeptides CarSP-2 and CarSP-4, respectively.

[0057] Peptide CarSP-5: In order to optimize the above-mentioned synthetic peptides (CarSP-1, CarSP-2, CarSP-3, CarSP-4) and expose residues to enhance functional applications, CarSP-1 peptide was further subjected to site mutation (cysteine ​​at position 13 was replaced with carboxyethyl cysteine) and spatial structure modification (cysteine ​​at positions 4 and 23 formed a disulfide bond) to obtain CarSP-5. Experimental results showed that the autoantibody detection efficacy and specificity of the enhanced design peptide CarSP-5 were significantly improved.

[0058] The amino acid sequence of the polypeptide has an identity degree of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5;

[0059] Or the difference between the sequence and the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 shall not exceed 5, 4, 3, 2 or 1 amino acids;

[0060] Alternatively, it may be a variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, wherein the difference between the variant and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 includes substitution, deletion and / or insertion of one or more amino acid residues or at least one N- / C-terminal extension.

[0061] Generally, proteins / peptides with highly similar or identical amino acid sequences are considered to have similar functions. Furthermore, proteins and peptides from different species exhibit homology differences / similar amino acid sequences, but their basic structure and function remain relatively consistent. For the aforementioned peptides, sequence differences exceed 25% between different species, such as humans and mice, yet the basic functions of the proteins are similar. Therefore, the proposed site alterations or optimizations can maintain or even enhance the similar effects of the original peptides.

[0062] Of the five polypeptides listed above, the key structural functional site is C. car The carboxyethylated cysteine ​​residue represented by this site is highly conserved across different species, and specific antibodies targeting this site have verified a significant difference in antibody binding ability before and after modification. It is believed that in the aforementioned peptides, C... car The carboxyethylated cysteine ​​it represents is a key functional amino acid differential site. Therefore, provided that this functional residue site and its surrounding sequence are not altered, similar polypeptide sequences have the same or similar functions.

[0063] This invention relates to the application of autoimmune disease-specific peptides and antibodies in the preparation of drugs for the diagnosis, prevention, and / or treatment of autoimmune diseases. Since large-scale clinical studies are required to demonstrate in vivo preventive and therapeutic effects, these studies are not currently available. However, we believe that in vitro data suggests similar biological effects in vivo, and the in vitro results support the application of the peptides and antibodies in the preparation of drugs for the diagnosis, prevention, and / or treatment of autoimmune diseases. Because the diagnostic effects of the autoimmune disease-specific peptides and antibodies described in this invention will influence disease classification and corresponding treatment methods, they have diagnostic and therapeutic value.

[0064] Optionally, autoimmune diseases primarily include spondyloarthritis / arthritis, such as ankylosing spondylitis, axial / peripheral spondyloarthritis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, and juvenile spondyloarthritis; as well as other autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis. Spondyloarthritis comprises a group of interrelated diseases (ankylosing spondylitis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, and uveitis, etc.), with ankylosing spondylitis being the prototype. Its pathogenesis, diagnosis, and treatment are representative of this group of diseases, and therefore can be extrapolated to the currently listed types of autoimmune diseases.

[0065] Applications include, but are not limited to, one or a combination of peptide-based detection kits, diagnostic reagents, protein chips, and immunoassay strips.

[0066] The application of any of the specific antibodies described in this invention in the preparation of drugs for the diagnosis, prevention and / or treatment of autoimmune diseases includes, but is not limited to, one or a combination of specific antibody-based kits, diagnostic reagents, protein chips and immunoassay strips.

[0067] The modified polypeptide provided by this invention is specifically present in patients with autoimmune diseases. This polypeptide is immunogenic to patients with autoimmune diseases and can be presented to immune cells by the patient's HLA molecules, inducing an autoimmune response. This invention uses mass spectrometry screening to discover that peripheral blood mononuclear cells of patients with ankylosing spondylitis specifically express the modified polypeptide CarSP-3, wherein C... car The cysteine ​​residue was modified with a carboxyethyl group. Analysis revealed that the extended sequence CarSP-1 of the polypeptide possesses antigenicity. Furthermore, based on the binding preference of the antigenic peptide to HLA, CarSP-1 was divided into CarSP-2, CarSP-3, and CarSP-4, and their affinity for HLA was compared to identify potential antigenic epitopes.

[0068] This invention involves immunizing mice with an antigenic peptide represented by CarSP-2 mixed with complete Freund's adjuvant. Two weeks later, mice are boosted with a mixture of the same antigenic peptide and incomplete Freund's adjuvant. This booster immunization is repeated four times. Ten days after the final immunization, mouse blood is collected, serum is obtained by centrifugation, and then subjected to site-specific affinity purification to obtain a polyclonal antibody. The antibody specifically binds to the integrin-cysteine ​​carboxyethyl modified site and does not bind to the unmodified integrin site.

[0069] To specifically detect autoantibodies against the integrin 96-carboxyethylated cysteine-related peptide produced in patients with autoimmune diseases, this invention designs and modifies the integrin 96-carboxyethylated cysteine ​​peptide as a specific antigen CarSP-5 for antibody detection in samples. The modified antigen peptide CarSP-5 can specifically react with antibodies against the carboxyethylated modified peptide and does not cross-react with the corresponding unmodified antibodies.

[0070] The technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0071] Example 1: Mass spectrometry detection of specifically expressed modified peptides in peripheral blood mononuclear cells of patients with ankylosing spondylitis

[0072] 1. Experimental Materials

[0073] 1.1 Sample Source

[0074] Plasma samples (all clinical tests related to plasma sample collection were conducted by the Department of Rheumatology and Immunology, Xijing Hospital, Air Force Medical University of the Chinese People's Liberation Army)

[0075] This study used a total of 12 peripheral blood samples, including:

[0076] Seven peripheral blood samples were collected from patients diagnosed with ankylosing spondylitis. The median age (range) was 35 years (range 24-55).

[0077] Five peripheral blood samples from healthy individuals, with a median age (range) of 47 years (42-52).

[0078] All peripheral blood samples were collected from 2015 to 2016 by the Department of Rheumatology and Immunology, Xijing Hospital, Air Force Medical University of the Chinese People's Liberation Army. All peripheral blood samples were from patients diagnosed with ankylosing spondylitis.

[0079] 1.2 Reagents and Materials

[0080] BCA kit (Pierce, Thermo Scientific, Germany)

[0081] Protease inhibitors (Roche, Switzerland)

[0082] Phosphatase inhibitors (Roche, Switzerland)

[0083] DL-Dithiothreitol (DTT, Sigma-Aldrich, USA)

[0084] Trypsin (Sigma-Aldrich, USA)

[0085] ZipTip C18 centrifuge column (Millipore, USA)

[0086] 2. Experimental Methods

[0087] Mass spectrometry (LC-MS / MS) sample preparation: Human peripheral blood mononuclear cell samples were obtained from 7 patients with ankylosing spondylitis and 5 healthy controls. Total protein was isolated by RIPA lysis and incubated on ice for 1 hour with protease inhibitors and phosphatase inhibitors. Cell lysates were centrifuged, and the supernatant was collected and analyzed using a BCA kit. The samples were stored at -80°C until further use. To preserve the biochemical properties of native protein residues during protein preparation, reagents that might modify proteins, such as iodoacetamide (IAA) and urea, were omitted during protein isolation. 200 μg of cell lysate was digested with trypsin. The samples were dialyzed against ammonium bicarbonate and reduced with DL-dithiothreitol. Trypsin digestion was performed at 37°C for 24 hours, and peptide purification was achieved using a ZipTip C18 centrifuge column (Millipore, USA). Desalted peptides were dried in a SpeedVac and stored at -80°C until further use.

[0088] Mass spectrometry analysis using high-performance liquid chromatography (HPLC) to separate mixed peptides. Main steps: First, phase A was prepared with 2% CH3CN, 98% H2O, and NH3H2O ​​to adjust the pH to 10.0, and phase B was prepared with 98% CH3CN and 2% H2O. 200 µg of the mixed peptides were dissolved in 80 µL of phase A, and the sample injection volume was 80 μL. Based on their hydrophilicity, the mixed peptide solution was separated on a C18 column. The mobile phase gradient was set as follows: 0–3 min, 100% phase A, 3–5 min, 100%–70% A, 5–45 min, 70%–30% A, 45–55 min, 30%–5% A, 55–60 min, 5% A, always maintained at 0.7 mL / min. The UV detector was set to 214 nm. Based on the chromatogram of sample separation, 1.5 mL was collected per tube at approximately 5 minutes, for 1 minute. Finally, 55 components were collected in chronological order, numbered 1 to 55, and vacuum-dried in a SpeedVac. The 55 components were dissolved in an acetylene-water mixture (50% acetylene, 50% water) and combined into 10 fractions: 1, 11, 21, 31, 41, and 51 were combined into fraction 1, and so on, yielding fractions 1 to 10. The final 10 fractions were vacuum-dried in a SpeedVac and stored at -80°C for subsequent mass spectrometry analysis. Extracted and desalted peptides were separated by liquid chromatography on an EASY-nLC 1000 system (ThermoScientific) equipped with a long C18 column (300 mm × Ø0.075 mm, 3 μm particles). Samples were fractionated using a 90-minute linear gradient (5–35% acetonitrile containing 0.1% formic acid). MS and MS / MS spectra were obtained in a data-dependent manner using an LTQ-OrbitrapElite mass spectrometer (Thermo Scientific), where MS / MS fragments of the 20 most intense peaks were obtained for each complete MS scan.

[0089] Secondary mass spectra were searched against the human protein database using SEQUEST (see Yates, 2015) in Proteome Discover. Trypsin (complete cleavage) was specified as the lyase, allowing a maximum of two missing cleavages. Secondary mass spectra were searched with a maximum permissible deviation of 10 ppm for precursor mass and 0.6 Da for fragment mass. Oxidation of various amino acid residues, including methionine, was selected as dynamic modification, with a false detection rate (FDR) of 1%. Each sample was repeated three times using the same method.

[0090] 3. Results

[0091] The secondary mass spectrum of the modified peptide CarSP-3 (AEGGQCcarPSLLFDLR) (SEQ ID No. 2) was obtained from peripheral blood mononuclear cells of patients with ankylosing spondylitis. Figure 1 The modified peptides were only found in peripheral blood mononuclear cells from patients with ankylosing spondylitis and were not detected in peripheral blood mononuclear cells from healthy controls. Figure 2 ).

[0092] Example 2: Screening for the optimal antigenic peptide for HLA-DR presentation

[0093] 1. Experimental Materials

[0094] Gel filtration column (PD10; GE Healthcare)

[0095] Infinite F200 (Tecan) microplate reader

[0096] SP-1, CarSP-1, SP-2, CarSP-2, SP-3, CarSP-3, SP-4, CarSP-4 and FAM-conjugated peptide SP-6 (Hangzhou Zhongtai Biochemical Co., Ltd.)

[0097] 2. Experimental Methods

[0098] The modified peptide SP-6 (SEQ ID No. 6: KGGGAEGLRALLARSHVER), a FAM-labeled peptide derived from the EBNA1 protein of the Epstein-Barr virus, was conjugated with the fluorescent marker FAM and pre-bound with HLA-DR. FAM-SP-6 (1 μM) and HLA-DR (500 nM) were incubated at 37°C in sodium citrate buffer (150 mM sodium chloride, 50 mM sodium citrate buffer, pH 5.2) for 3 hours. Unbound peptides were then removed using a gel filtration column.

[0099] Competitive peptides were added to the HLA-DR / FAM-SP-6 complex (100 μL in a 96-well plate). The competing peptides included: CarSP-1, CarSP-2, CarSP-3, CarSP-4, SP-1: VFLCPWRAEGGQCPSLLFDLRDETRNV (SEQ ID No. 7), SP-2: FLCPWRAEGGQCPS (SEQ ID No. 8), SP-3: AEGGQCPSLLFDLR (SEQ ID No. 9), SP-4: CPSLLFDLRDETRNV (SEQ ID No. 10), and the positive control peptide SP-PC: PKYVKQNTLKLAT (SEQ ID No. 11), which is known to bind strongly to HLA-DR. The fluorescence polarization (FP) values ​​of FAM were read at 25°C using an Infinite F200 (Tecan) microplate reader.

[0100] 3. Experimental Results

[0101] To determine the optimal peptide for HLA-DR presentation, autoimmune disease-specific antigen-associated peptides SP-1, CarSP-1, SP-2, CarSP-2, SP-3, CarSP-3, SP-4, and CarSP-4 were used to compete with the positive control peptide SP-PC. The modified peptides CarSP-1, CarSP-2, CarSP-3, and CarSP-4 all bound to HLA-DR and exhibited antigenicity. Among them, CarSP-2 showed the strongest binding affinity to HLA-DR, significantly stronger than the positive control group, suggesting that CarSP-2 was the most antigenic fragment in the experimental group. Figure 3 ).

[0102] Example 3: Preparation of rabbit polyclonal antibodies specific to modified peptides

[0103] 1. Experimental Materials

[0104] CarSP-2-KLH, CarSP-2-BSA, and SP-2-BSA (Hangzhou Zhongtai Biochemical Co., Ltd.)

[0105] CNBR Active Sepharose 4B Packing Material (GE)

[0106] Chelation buffer: 0.1M NaHCO3 pH 8.3 with 0.5M NaCl added (Tianjin Kemeio)

[0107] Blocking buffer: 0.1M Tris-HCl pH 8.0 (Tianjin Kemio)

[0108] Equilibration buffer: 0.01M pH 7.4 PBS (Shanghai Baisai Biotechnology Helix)

[0109] Elution buffer: 0.05 M Gly-HCl pH 2.5 (Tianjin Kemio)

[0110] 2. Experimental Methods

[0111] The ITA2B cysteine-2-carboxyethylated peptide CarSP-2 was conjugated with KLH. The antigenic peptide represented by CarSP-2 was mixed with complete Freund's adjuvant to immunize mice. The antigenic peptide was then mixed with incomplete Freund's adjuvant to boost the immunization of mice every other week. The booster immunization was performed four times. Blood was collected from mice 10 days after the last immunization. The serum was obtained by centrifugation and then purified by site-specific affinity purification to obtain polyclonal antibodies.

[0112] Weigh 4g of CNBr-4B dry powder and activate the agarose particles according to the CNBr-4B instruction manual. Dilute the modified peptide CarSP-2-BSA and the unmodified peptide SP-2-BSA to a concentration of 5mg / ml using chelation buffer. Add the protein dilution buffer to the activated sol at a ratio of 5mg / 10ml, incubate overnight at 4°C with shaking, rinse the sol 2-3 times with chelation buffer, add 3 volumes of blocking buffer, incubate at 4°C with shaking for 2 hours, and then pack the sol into columns.

[0113] Take 200 ml of rabbit serum ammonium sulfate precipitation mixture, centrifuge at 8000 rpm for 30 min and discard the supernatant. Dissolve the precipitated protein in 0.01 M pH 7.4 PBS buffer and filter through a 0.22 μm filter. Install an SP-2-BSA purification gel column and repeat the protein solution pre-purification steps of equilibration-loading-equilibration-elution-equilibration, collecting the protein solution transfusion until the elution peak is below 10 mAU. Install a CarSP-2-BSA purification gel column, equilibrate with 10 column volumes of equilibration buffer, load the sample at a rate of 3 ml / min, equilibrate with equilibration buffer to baseline after loading, elute with elution buffer at a rate of 1 ml / min, and quickly neutralize the eluent pH to 7.0 with 3 M Tris-HCl buffer. Concentrate with a 10 kDa ultrafiltration tube and replace the buffer with 0.01 M PBS pH 7.4.

[0114] 3. Experimental Results

[0115] Modified antigenic peptide CarSP-1 (see appendix) Figure 4 It can specifically react with antibodies against carboxyethyl modified peptides, and does not cross-react with the corresponding unmodified antibodies.

[0116] Example 4: Detection of binding of modified integrin ITA2B-carboxyethyl modified peptide CarSP-5 to specific antibody

[0117] 1. Experimental Materials

[0118] Anti-integrin-carboxyethyl modified antibody detection reagent, including:

[0119] 1) Coating with a modified antigen peptide; this modified peptide is derived from the modified integrin ITA2B-carboxyethyl modified peptide CarSP-5 ( Figure 5 );

[0120] 2) One 250 mL bottle of sample diluent, which is a phosphate buffer containing 10% bovine serum albumin;

[0121] 3) One 3 mL vial of antibody working solution contains horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin antibody (Thermo Scientific).

[0122] 4) Washing solution (20×) 1 bottle of 50 mL, which is a 20× phosphate buffer solution with pH 7.4 containing 1% Tween-20;

[0123] 5) 200 ng of rabbit polyclonal antibody against the modified antigen.

[0124] 2. Experimental Methods

[0125] Before use, mix all reagents thoroughly and equilibrate the buffer to room temperature. Coat a 96-well ELISA plate with 10 μg / mL of modified antigen peptide, seal with a sealing film, and incubate overnight at 4°C. Wash the pre-coated ELISA plate three times with washing buffer for 1 minute each time, and pat dry on absorbent filter paper. Dilute the positive antibody with sample diluent to a final concentration of 1 μg / mL, and perform six comparative dilutions: 1 μg / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 ng / mL. Add 100 μl of each concentration of positive antibody to the wells coated with the modified antigen (perform four replicates for each concentration), with 0 ng / mL as the negative control. Mix well, seal with a sealing film, and incubate at room temperature for 4 h. Add 300 μL of 1× washing buffer to each well and incubate for 1 min. Drain the liquid onto filter paper, and repeat three times. Add 100 μL of diluted horseradish peroxidase-labeled anti-rabbit immunoglobulin G antibody to each well, mix well, and then cover with a sealing film and incubate at room temperature for 1 h. Add 300 μL of 1× wash buffer to each well and incubate for 1 min, then drain the liquid onto filter paper. Repeat 3 times. Add 100 μL of TMB (Biolegend) to each well and incubate at room temperature in the dark for 5–30 min. After color development, quickly add 100 μL of stop solution (Biolegend) to each well to terminate the reaction. Read the value at 450 nm within 10 min after termination.

[0126] 3. Experimental Results

[0127] The positive antibody specifically binds to the pre-coated modified antigen, resulting in color development after ELISA detection. This result indicates that the positive antibody exhibits high specificity and good reactivity in binding to the antigen. (See attached results.) Figure 6 .

[0128] Example 5: Comparison of binding affinity between linear unmodified peptide SP-1, linear modified peptide CarSP-1, and cyclic modified peptide CarSP-5 and anti-modification specific antibodies and anti-unmodified antibodies.

[0129] 1. Experimental Materials

[0130] Anti-integrin antibody detection reagents include:

[0131] 1) Coating with a modified antigen peptide; this modified peptide is derived from the modified integrin ITA2B-carboxyethyl modified peptide CarSP-5 ( Figure 5 );

[0132] 2) One 250 mL bottle of sample diluent, which is a phosphate buffer containing 10% bovine serum albumin;

[0133] 3) One 3 mL vial of antibody working solution contains horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin antibody (Thermo Scientific).

[0134] 4) Washing solution (20×) 1 bottle of 50 mL, which is a 20× phosphate buffer solution with pH 7.4 containing 1% Tween-20;

[0135] 5) Rabbit polyclonal antibody specific to the CarSP-2 peptide modified at 96 cysteine ​​position of integrin;

[0136] 6) Antibody against SP-2 peptide at the unmodified cysteine ​​residue 96 of integrin;

[0137] 7) Linear unmodified peptide SP-1 (VFLCPWRAEGGQCPSLLFDLRDETRNV)

[0138] 8) Linearly modified peptide CarSP-1 (VFLCPWRAEGGQCcarPSLLFDLRDETRNV)

[0139] 9) Modified cyclic peptide CarSP-5 (VFLCPWRAEGGQCcarPSLLFDLRDCTRNV, cys4 & cys23 disulfide bond linked)

[0140] 2. Experimental Methods

[0141] Before use, all reagents were thoroughly mixed and the buffer was equilibrated to room temperature. A 96-well microplate was coated with 10 μg / mL of linear unmodified peptide, linear modified peptide, and modified cyclic peptide, respectively, and then sealed with a sealing film and incubated overnight at 4°C.

[0142] Wash the pre-coated ELISA plate three times with washing buffer for one minute each time, and pat dry on absorbent filter paper. Dilute the anti-modified antibody (positive antibody) and the anti-unmodified antibody with sample diluent to a final concentration of 1 μg / mL, and perform six comparative dilutions: 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 ng / mL. Add 100 μL of each concentration of positive antibody to the wells coated with the modified antigen (perform four replicates for each concentration). 0 ng / mL serves as the negative control. After mixing, seal the plate and incubate at room temperature for 4 h.

[0143] Add 300 μL of 1× wash buffer to each well and incubate for 1 min. Remove the liquid from the filter paper and repeat 3 times. Add 100 μL of diluted horseradish peroxidase-labeled anti-rabbit immunoglobulin G antibody to each well, mix well, and cover with a sealing film. Incubate at room temperature for 1 h. Add 300 μL of 1× wash buffer to each well and incubate for 1 min. Remove the liquid from the filter paper and repeat 3 times. Add 100 μL of TMB (Biolegend) to each well and incubate at room temperature in the dark for 5–30 min. After color development, quickly add 100 μL of Stopsolution (Biolegend) to each well to terminate the reaction. Read the value at 450 nm within 10 min after termination.

[0144] 3. Experimental Results

[0145] The test results indicate that the modified cyclic peptide, as a pre-coated antigen, not only binds efficiently to anti-integrin-carboxyethylated antibodies compared to linearly modified and unmodified peptides, but also exhibits weaker cross-reactivity with unmodified anti-integrin antibodies (see...). Figure 7 Experiments have confirmed that the ankylosing spondylitis anti-integrin-carboxyethyl modified antibody detection kit of the present invention, used for the auxiliary diagnosis or differential diagnosis of ankylosing spondylitis, can efficiently and specifically detect in vitro and in vivo anti-integrin-carboxyethyl modified antibodies.

[0146] Example 6

[0147] The ITA2B-carboxyethyl modified antibody detection kit, used for the auxiliary diagnosis or differential diagnosis of ankylosing spondylitis, was expressed in the plasma of ankylosing spondylitis patients and healthy controls. The specific experimental procedure is as follows:

[0148] 1. Experimental Materials

[0149] 1.1 This study used a total of 166 plasma samples, including:

[0150] 126 plasma samples from patients diagnosed with ankylosing spondylitis, median age (range) years: 30 (14-68);

[0151] 40 plasma samples from healthy individuals, median age (range) years: 45 (25-68);

[0152] All plasma was collected by the Department of Rheumatology and Immunology, Xijing Hospital, Air Force Medical University of the Chinese People's Liberation Army, from 2015 to 2020. All disease plasma came from patients diagnosed with ankylosing spondylitis.

[0153] 1.2 Anti-integrin-carboxyethyl modified antibody detection reagent, including:

[0154] 1) The coating is made with a modified antigen peptide; the modified peptide is derived from the modified integrin ITA2B-carboxyethyl modified peptide CarSP-5;

[0155] 2) One 250 mL bottle of sample diluent, which is a phosphate buffer containing 10% bovine serum albumin;

[0156] 3) One 12 mL vial of antibody working solution contains horseradish peroxidase (HRP)-labeled anti-human immunoglobulin antibody (Thermo Scientific).

[0157] 4) One 3 mL vial of antibody working solution contains horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin antibody (Thermo Scientific).

[0158] 5) Washing solution (20×) 1 bottle of 50 mL, which is a 20× phosphate buffer solution with pH 7.4 containing 1% Tween-20;

[0159] 6) 200 ng of rabbit polyclonal antibody against the modified antigen.

[0160] 2. Experimental Methods

[0161] Before use, all reagents were thoroughly mixed, and the buffer was equilibrated to room temperature. A 96-well microplate was coated with 10 μg / mL of modified antigenic peptide, sealed, and incubated overnight at 4°C. Blank control, standard, and experimental groups were set up. 100 μL of diluted standard and sample dilution buffer (10 times the sample dilution) was added to each well, along with plasma from 126 patients with ankylosing spondylitis (AS) and 40 healthy controls (HC), in duplicate. After mixing, the wells were sealed and incubated at room temperature for 4 h. 300 μL of 1× wash buffer was added to each well, and after incubation for 1 min, the liquid was drained onto filter paper. This process was repeated 3 times. 100 μL of diluted horseradish peroxidase-labeled anti-human immunoglobulin G antibody (horseradish peroxidase-labeled anti-rabbit immunoglobulin G antibody was added to each well) was added to each well, mixed, sealed, and incubated at room temperature for 1 h. 300 μL of 1× wash buffer was added to each well, and after incubation for 1 min, the liquid was drained onto filter paper. This process was repeated 3 times. Add μL of TMB (Biolegend) to each well and incubate at room temperature in the dark for 5–30 min. After color development, quickly add 100 μL of stop solution (Biolegend) to each well to terminate the reaction. Within 10 min after termination, read the value using a detection wavelength of 450 nm.

[0162] 3. Experimental Results

[0163] Clinical ELISA results showed that anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibodies were significantly higher in patients with ankylosing spondylitis than in the control group, with a positive rate of approximately 10%, while the positive rate in healthy controls was 0% (see Appendix). Figure 8 Among HLA-DR4 positive patients (HLA subtypes highly associated with autoimmune diseases), the positive rate of anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibodies was approximately 50%.

[0164] The detection of autoantibodies in patients with ankylosing spondylitis is influenced by factors such as HLA typing, disease treatment status, and disease activity. In the unscreened population, the overall positive rate was only 10%. Current evidence suggests that in HLA-DR4 positive patients (an HLA typing highly associated with autoimmune diseases), the positive rate of anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibodies is approximately 50%. This percentage is correlated with disease type and progression, and has clinical guiding significance and diagnostic value. In other words, when using the group of synthetic peptides and their derived sequences mentioned in the claims to detect or treat ankylosing spondylitis patients, a positive screening rate of 10% would be considered ideal.

[0165] Example 7:

[0166] The ITA2B-carboxyethyl modified antibody detection kit, used for the auxiliary diagnosis or differential diagnosis of rheumatoid arthritis and systemic lupus erythematosus, demonstrates the following experimental procedure for expression in plasma from rheumatoid arthritis and systemic lupus erythematosus:

[0167] 1. Experimental Materials

[0168] 1.1 This study used a total of 71 plasma samples, including:

[0169] 31 plasma samples from patients with rheumatoid arthritis (RA);

[0170] 25 plasma samples from patients with systemic lupus erythematosus (SLE)

[0171] 15 healthy control (HC) plasma samples;

[0172] All plasma was collected by the Department of Rheumatology and Immunology, Xijing Hospital, Air Force Medical University of the Chinese People's Liberation Army, from 2015 to 2020. All disease plasma came from patients diagnosed with rheumatoid arthritis and systemic lupus erythematosus.

[0173] 1.2 Anti-integrin-carboxyethyl modified antibody detection reagent, including:

[0174] 1) The coating is made with a modified antigen peptide; the modified peptide is derived from the modified integrin ITA2B-carboxyethyl modified peptide CarSP-5;

[0175] 2) One 250 mL bottle of sample diluent, which is a phosphate buffer containing 10% bovine serum albumin;

[0176] 3) One 12 mL vial of antibody working solution contains horseradish peroxidase (HRP)-labeled anti-human immunoglobulin antibody (Thermo Scientific).

[0177] 4) One 3 mL vial of antibody working solution contains horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin antibody (Thermo Scientific).

[0178] 5) Washing solution (20×) 1 bottle of 50 mL, which is a 20× phosphate buffer solution with pH 7.4 containing 1% Tween-20;

[0179] 6) 200 ng of rabbit polyclonal antibody against the modified antigen.

[0180] 2. Experimental Methods

[0181] Before use, all reagents were thoroughly mixed, and the buffer was equilibrated to room temperature. 10 μg / mL of the modified antigenic peptide was coated onto a 96-well ELISA plate, sealed, and incubated overnight at 4°C. Blank control, standard, and experimental groups were set up. 100 μL of diluted standard and 10-fold sample dilution buffer were added to each well, with replicates. After mixing, the plate was sealed and incubated at room temperature for 4 h. 300 μL of 1× washing buffer was added to each well, and after incubation for 1 min, the liquid was drained onto filter paper. This process was repeated 3 times. 100 μL / well of diluted horseradish peroxidase-labeled anti-human immunoglobulin G antibody (horseradish peroxidase-labeled anti-rabbit immunoglobulin G antibody was added to the positive control group) was added, mixed, sealed, and incubated at room temperature for 1 h. 300 μL of 1× washing buffer was added to each well, and after incubation for 1 min, the liquid was drained onto filter paper. This process was repeated 3 times. Add μL of TMB (Biolegend) to each well and incubate at room temperature in the dark for 5–30 min. After color development, quickly add 100 μL of stop solution (Biolegend) to each well to terminate the reaction. Within 10 min after termination, read the value using a detection wavelength of 450 nm.

[0182] 3. Experimental Results

[0183] Clinical ELISA results showed that anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibodies were significantly higher in patients with rheumatoid arthritis and systemic lupus erythematosus than in the control group, with a positive rate of approximately greater than 10%, while the positive rate in healthy controls was 0% (see Appendix). Figure 9 Among HLA-DR4 positive patients (HLA subtypes highly associated with autoimmune diseases), the positive rate of anti-integrin (ITA2B)-carboxyethyl modified peptide autoantibodies was approximately 50%.

[0184] Example 8: Flow cytometry detection of specific antibody-induced attenuation of CarSP-1 peptide-induced immune response

[0185] 1. Experimental Materials

[0186] AIM V® cell culture media (Gibco)

[0187] Recombinant human GM-CSF (Biolegend)

[0188] Recombinant human IL-4 (Biolegend)

[0189] Recombinant human TNF-α (Biolegend)

[0190] Recombinant human IL-6 (Biolegend)

[0191] Recombinant human IL-1β (Biolegend)

[0192] 2. Experimental Methods

[0193] After isolating PBMCs from peripheral blood using lymphocyte separation medium, the cells were resuspended in AIM-V medium. PBMCs (3 x 10⁶ / mL) were added to six-well plates, transferred to a 37°C, 5% CO₂ incubator, and allowed to adhere for 2 hours. After 2 hours, the six-well plates were gently shaken and tapped to resuspend non-adherent and semi-adherent cells. The suspension consisted primarily of lymphocytes (frozen non-adherent cells were reserved for later use). Immature DC (iDC) medium was added to the adherent cells (to a final concentration of 800 U / mL GM-CSF and 500 U / mL IL-4 in AIM-V medium) at 37°C and 5% CO₂ incubation. On day 6 of iDC culture, an equal volume of mature DC medium (AIM-V medium with a final concentration of 1600 U / mL GM-CSF, 1000 U / mL IL-4, and 10 ng / mL TNF-α, 10 ng / mL IL-1β, 320 ng / mL IL-6, and 2 μg / mL PGE2) was added to the peptide (CarSP-1), bringing the final concentration to 10 μg / mL. Control IgG or specific antibody was added to each group at a final concentration of 1 μg / mL. The ratio of the original conditioned medium to the mature DC medium was 1:1. The cell culture flasks were placed in a 37 ℃, 5% CO2 incubator for 16–18 h, allowing the iDCs to mature into mDCs.

[0194] After 2 hours of culture, non-adherent cells (lymphocytes) were collected, and 1640 cell culture medium (containing 10% FBS, 10 ng / mL IL-7, and monoclonal antibody) was added. The cells were then cultured at 37 °C in a 5% CO2 incubator for 15 hours. After 15 hours of T cell culture, T cells and mDCs were counted for co-culture of mDCs and T cells. The mDC:T cell ratio was 1:10. The number of T cells in a 96-well plate was 1 x 10⁵ / well. 1640 medium was used. IL-7 was added to a final concentration of 10 ng / mL, 10% FBS, and 1% penicillin / streptomycin. The remaining mDCs were frozen for secondary co-culture. After 3 days of co-culture, the 1640 medium was replaced with IL-7 to a final concentration of 10 ng / mL, 10% FBS, 1% penicillin / streptomycin, and 50 U / mL IL-2. After co-culturing for 10 days, mDC cells were resuscitated. mDC and T cells were co-cultured based on T cell and mDC cell counts, with an mDC:T cell ratio of 1:10. 10% FBS was added to the 1640 medium. In these groups, the FACS assay group included blocking agents Brefeldin A solution (1000X) and Monensin solution (1000X).

[0195] After co-culturing T cells and mDC cells for 6 hours, cells were collected separately, centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were transferred to 1.5ml EP tubes. After washing once with PBS, the cells were centrifuged at 300g for 5 min, and 100ul PBS and 2ul flow cytometry antibodies (CD3, CD4, CD8) were added for extracellular staining for 30 min. After adding 1ml PBS, the cells were centrifuged at 500g for 5 min, the supernatant was discarded, 250ul of membrane-permeability agent was added, and after 20 min, 500ul of washing buffer was added for washing once, centrifuged at 300g for 5 min, the supernatant was discarded, and 100ul of washing buffer was added. The cells were incubated overnight at 4℃. The next day, 2ul of flow cytometry antibodies (APC-TNF-α, APC-IFN-γ) were added directly for intracellular staining. After 30 min, 500ul PBS was added for washing once, centrifuged at 300g for 5 min, the supernatant was discarded, and 200ul PBS was added. The cells were then transferred to flow cytometry tubes for flow cytometry analysis.

[0196] 3. Experimental Results

[0197] Combination Figure 10Flow cytometry analysis of the immune response indicated that the specific antibody could attenuate the antigen presentation response induced by CarSP-1 peptide and the autoimmune response associated with CD4 and CD8 T lymphocytes. After the addition of the specific antibody, the response markers—tumor necrosis factor (TNF) and interferon (IFN)—were significantly reduced, suggesting that the specific antibody attenuates the immune response induced by CarSP-1 peptide and has potential therapeutic effects.

[0198] In summary, this invention obtained specifically expressed modified antigenic peptide sequences through mass spectrometry screening of ankylosing spondylitis patients. Further immunogenicity identification of the screened modified antigenic peptides revealed that they can bind to HLA-DR molecules and participate in antigen presentation. Based on the specific antibodies generated after immunizing animals with the modified antigenic peptides, these antibodies exhibit high specificity and purity. This modified antigenic peptide and its specific antibody have broad application prospects in the early diagnosis, disease classification, and targeted drug development of spondyloarthritis (including ankylosing spondylitis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, etc.) and other autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis).

[0199] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. Nucleotide sequence listing electronic file <110> Air Force Medical University of the Chinese People's Liberation Army <120> Based on autoimmune disease-specific peptides, specific antibodies and their applications <160> 6 <210> 1 <211> 27 <212> PRT <213> SYNTHESIS <220> The cysteine ​​residue at position 13 of the synthesized polypeptide is carboxyethylated cysteine, CarSP-1 <400> Val Phe Leu Cys Pro Trp Arg Ala Glu Gly Gly Gln Cys Pro Ser 1 5 10 15 Leu Leu Phe Asp Leu Arg Asp Glu Thr Arg Asn Val 16 20 25 <210> 2 <211> 14 <212> PRT <213> SYNTHESIS <220> The cysteine ​​residue at position 12 of the synthesized polypeptide is carboxyethylated cysteine, CarSP-2 <400> Phe Leu Cys Pro Trp Arg Ala Glu Gly Gly Gln Cys Pro Ser 1 5 10 <210> 3 <211> 14 <212> PRT <213> SYNTHESIS <220> The cysteine ​​residue at position 6 of the synthesized polypeptide is carboxyethylated cysteine, CarSP-3. <400> Ala Glu Gly Gly Gln Cys Pro Ser Leu Leu Phe Asp Leu Arg 1 5 10 <210> 4 <211> 15 <212> PRT <213> SYNTHESIS <220> The cysteine ​​residue at position 1 of the synthesized polypeptide is carboxyethylated cysteine, CarSP-4. <400> Cys Pro Ser Leu Leu Phe Asp Leu Arg Asp Glu Thr Arg Asn Val 1 5 10 15 <210> 5 <211> 27 <212> PRT <213> SYNTHESIS <220> The cysteine ​​residue at position 13 of the synthetic polypeptide is a carboxyethylated cysteine, and the cysteine ​​residues at positions 4 and 23 form a disulfide bond; CarSP-5 <400> Val Phe Leu Cys Pro Trp Arg Ala Glu Gly Gly Gln Cys Pro Ser 1 5 10 15 Leu Leu Phe Asp Leu Arg Asp Cys Thr Arg Asn Val 16 20 25 <210> 6 <211> 14 <212> PRT <213> Human <220> The patient-derived peptide has a mass shift of 72.02 cysteine ​​residue at position 6, C72SP. <400> Ala Glu Gly Gly Gln Cys Pro Ser Leu Leu Phe Asp Leu Arg 1 5 10

Claims

1. A specific polypeptide for autoimmune diseases, characterized in that, A polypeptide having the following amino acid sequence: CarSP-1: VFLCPWRAEGGQCcarPSLLFDLRDETRNV; Car represents carboxyethylated modification, and Ccar represents carboxyethylated cysteine.

2. The use of the autoimmune disease-specific polypeptide of claim 1 in the preparation of drugs for the diagnosis, prevention and / or treatment of autoimmune diseases.

3. The application according to claim 2, characterized in that, Autoimmune diseases are mainly spondyloarthritis / arthritis, including ankylosing spondylitis, axial / peripheral spondyloarthritis, undifferentiated spondyloarthritis, psoriatic arthritis, enteropathic arthritis, uveitis, and juvenile spondyloarthritis; And other autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, myositis, and vasculitis.

4. The application according to claim 2, characterized in that, The applications include, but are not limited to, one or a combination of peptide-based detection kits, diagnostic reagents, protein chips, and immunoassay strips.

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