Use of antigenic peptide eg16 and antibodies thereof in the preparation of diagnostic products for rheumatoid arthritis

Abstract

The application discloses application of an antigen peptide EG16 and an antibody thereof in preparation of a rheumatoid arthritis (RA) diagnostic product, and belongs to the technical field of disease diagnosis. The application aims to solve the problem of lack of a serological diagnostic marker with high sensitivity and high specificity for RA (especially early RA). The amino acid sequence of the antigen peptide EG16 is APKRIRLPHIREDDQP or BSA-C-APKRIRLPHIREDDQP. The RA diagnostic product is used for detecting the content of an epitope antibody capable of being specifically combined with the antigen peptide EG16 in a biological sample of a patient. The expression level of the epitope antibody in a patient with RA is significantly higher than that in a healthy population and other rheumatism and immunological disease patients, the diagnostic sensitivity is 43.43%, the specificity is 92.80%, and the sensitivity in a preclinical RA patient is 44.44%. A new auxiliary diagnostic index with high specificity is provided for RA (especially in the early stage).

Description

Technical Field

[0001] This invention relates to the field of rheumatoid arthritis diagnostic technology, specifically to the application of antigenic peptide EG16 and its antibody in the preparation of rheumatoid arthritis diagnostic products. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic and highly destructive autoimmune disease characterized by symmetrical joint pain and swelling. The disease typically progresses with intermittent periods of remission. Without proper treatment, a significant proportion of patients develop joint deformities, and some may even become disabled (Sun X, Li R, Cai Y, et al. Clinical remission of rheumatoid arthritis in a multicenter real-world study in the Asia-Pacific region. Lancet Reg Health West Pac. 2021;15:100240. Published 2021 Aug 12.). Among systemic rheumatic diseases, RA is the most common in China, causing not only physical damage and psychological stress but also imposing a significant economic burden on patients, their families, and society.

[0003] Early and accurate diagnosis of rheumatoid arthritis (RA) is crucial for differentiating between different types of inflammatory joint diseases, developing appropriate treatment plans, and preventing long-term disabling complications such as joint deformities (Zhou Y, Wang X, An Y, et al. Disability and health-related quality of life in Chinese patients withrheumatoid arthritis: A cross-sectional study. Int J Rheum Dis. 2018;21(9):1709-1715.). Currently, the clinical diagnosis of RA mainly relies on clinical symptom assessment, imaging examinations, and biomarker detection. Among these, biomarker detection, as a promising medical approach, plays an important role in the diagnosis, prognosis, and treatment guidance of RA. Among existing serological markers, anti-citrullinated protein antibody (ACPA), especially anti-cyclic citrullinated peptide (CCP) antibody detected by enzyme-linked immunosorbent assay (ELISA), is widely used in clinical practice due to its high specificity and has become a key indicator in the classification criteria for RA. However, the detection rate of anti-CCP antibodies remains significantly limited in the preclinical stage of rheumatoid arthritis, with a relatively low positive rate. This means that a considerable number of patients at this stage cannot be identified early using existing serological methods. Currently, there is a lack of sensitive and specific early diagnostic methods for this preclinical patient population, which constitutes a key weakness and an urgent clinical need in the early screening and intervention system for RA. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing rheumatoid arthritis (RA) diagnostic techniques, especially in early diagnostic indicators, by providing a novel antigenic peptide, EG16. Based on the detection of the corresponding epitope antibody levels in patients using this antigenic peptide EG16, the diagnostic efficacy of RA can be significantly improved, providing a novel auxiliary indicator with high sensitivity and specificity for clinical diagnosis.

[0005] The technical solution of this invention is described in detail below: In a first aspect, the present invention provides an antigenic peptide EG16 with the amino acid sequence APKRIRLPHIREDDQP (SEQ ID NO:1) or the amino acid sequence BSA-C-APKRIRLPHIREDDQP (SEQ ID NO:2), wherein BSA is bovine serum albumin, which serves as a carrier protein and is coupled to the N-terminus of the sequence shown in SEQ ID NO:1 via cysteine ​​residues, thereby further increasing immunogenicity.

[0006] Secondly, the present invention provides the application of the above-mentioned antigenic peptide EG16 in the preparation of a diagnostic product for rheumatoid arthritis, wherein the product is used to detect the content of epitope antibodies in patient biological samples and can bind to the above-mentioned antigenic peptide EG16.

[0007] Compared with normal healthy individuals, patients with systemic lupus erythematosus, patients with Sjögren's syndrome, and patients with osteoarthritis, the levels of epitope antibodies in biological samples from patients with rheumatoid arthritis were significantly increased.

[0008] The rheumatoid arthritis patients include those with preclinical rheumatoid arthritis, those with early-stage rheumatoid arthritis, and those with long-term rheumatoid arthritis.

[0009] Preclinical rheumatoid arthritis patients are defined as those who are older than 18 years, present with signs of hand joint pain, and have positive RF and / or CCP tests in the laboratory, but do not meet the diagnostic criteria for RA.

[0010] Early-stage rheumatoid arthritis patients are defined as those who meet the diagnostic criteria for RA and have a disease duration of ≤1 year.

[0011] Patients with long-term rheumatoid arthritis are defined as those who meet the diagnostic criteria for RA and whose disease duration is >1 year and ≤10 years.

[0012] The biological sample is serum or plasma.

[0013] The diagnostic product is an ELISA test kit.

[0014] Thirdly, the present invention provides a diagnostic kit for rheumatoid arthritis, comprising: the above-mentioned antigen peptide EG16, an enzyme-labeled plate, a coating solution, a washing solution, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a chromogenic substrate, and a stop solution.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Through extensive preliminary analysis and screening, this invention obtained the antigenic peptide EG16, composed of 16 amino acids. Epitope antibodies (anti-antigen peptide EG16) corresponding to this antigenic peptide EG16 were present in patient biosamples. These epitope antibodies showed significant differences in RA patients compared to healthy individuals and other common rheumatic immune diseases easily confused with rheumatoid arthritis. The sensitivity in diagnosing rheumatoid arthritis was 43.43%, and the specificity was 92.80%, particularly in preclinical rheumatoid arthritis patients, where the sensitivity was 44.44%. The detection of this antigenic peptide EG16 and its antibody provides a novel, highly specific auxiliary indicator for the diagnosis of rheumatoid arthritis (especially in its early stages), and is of significant value in improving the diagnostic accuracy and early detection capability of this disease. Attached Figure Description

[0016] Figure 1This is a graph showing the relative abundance of epitope antibodies detected by the ELISA method in Example 1 in samples from RA patients, healthy individuals, and other common rheumatic immune diseases that are easily confused with rheumatoid arthritis. p < 0.001 (Kruskal-Wallis test method). [AU value = (OD value of test serum / OD value of positive standard serum)] 100, OD value is the value detected by the ELISA reader at 450nm - 570nm. RA indicates rheumatoid arthritis patients, SLE indicates systemic lupus erythematosus patients, SS indicates Sjögren's syndrome patients, OA indicates osteoarthritis patients, and HC indicates healthy individuals.

[0017] Figure 2 The ROC curves for distinguishing RA patients, healthy individuals, and disease controls using epitope antibodies in serum in Example 1 are shown.

[0018] Figure 3 This is a graph showing the relative abundance of epitope antibodies in preclinical, early-stage, and long-term RA patient samples detected by the ELISA method in Example 2. p<0.01, p < 0.001 (Kruskal-Wallis test method). [AU value = (OD value of test serum / OD value of positive standard serum)] 100, OD value is the value detected by the ELISA reader at 450nm - 570nm. preRA indicates preclinical rheumatoid arthritis patients, earlyRA indicates early-stage rheumatoid arthritis patients, LsRA indicates long-term rheumatoid arthritis patients, and HC indicates healthy individuals.

[0019] Figure 4 This shows the positive rates of epitope antibodies in healthy individuals and preclinical, early-stage, and long-term RA patients in Example 2. preRA represents preclinical rheumatoid arthritis patients, earlyRA represents early-stage rheumatoid arthritis patients, LsRA represents long-term rheumatoid arthritis patients, and HC represents healthy individuals. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present application, the present application will be clearly and completely described below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application. Unless otherwise specified, the instruments and reagents used in the embodiments are all from commercial channels.

[0021] The amino acid sequence of the antigenic peptide EG16 is: BSA-C-APKRIRLPHIREDDQP (SEQ ID NO:2), synthesized by Shanghai Nuoyou Biotechnology Co., Ltd.

[0022] This ELISA diagnostic kit includes: the antigenic peptide EG16 of SEQ ID NO:2 above; a 96-well microplate; a coating buffer of carbonate buffer; an enzyme-labeled secondary antibody of goat anti-human IgM, which specifically recognizes human IgM and binds to horseradish peroxidase; a chromogenic substrate, namely TMB chromogenic solution, which develops color after being catalyzed by horseradish peroxidase, and the test results are presented according to the degree of substrate color development; and necessary diluents such as 1% BSA-PBST; washing buffer of 0.05% PBS-T; blocking buffer of 3% BSA-PBST; stop solution of 2 mol / L sulfuric acid; and positive standard serum. All percentage concentrations of the components in the kit are volume percentages.

[0023] Positive standard serum was used as a positive control. It was obtained by selecting serum from 10 cases of RA with an OD value greater than 1.5 and mixing them in equal volumes.

[0024] Experimental steps for using ELISA diagnostic kits for diagnosis: (1) Plate coating: The antigenic peptide EG16 (i.e., primary antibody) of SEQ ID NO:2 was diluted to 5 μg / mL with carbonate buffer at 4℃ and coated with 100 μL / well of 96-well microplates overnight at 4℃. (2) Washing: Wash the plate 3 times with 0.05% Tween 20 PBS (PBS-T, washing buffer), 200 μL / well. 2 minutes / session; (3) Blocking: Add 3% BSA-PBST (blocking solution), 200 μL / well, and block at 37℃ for 2 hours; (4) Washing: Wash the plate 3 times with 0.05% PBS-T (washing buffer), 200 μL / well. 2 minutes / session; (5) Primary antibody incubation: Dilute the sample serum or plasma with 1% BSA-PBST (diluent) at a ratio of 1:100 and add it to the 96-well microplate of step (4), 100 μL / well, and incubate at 37°C for 1 hour; (6) Washing: Wash the plate 3 times with 0.05% PBS-T (washing buffer), 200 μL / well 2 minutes / session; (7) Secondary antibody incubation: Add 100 μL of horseradish peroxidase-labeled goat anti-human IgM (enzyme-labeled secondary antibody) diluted 1:5000, incubate at 37℃ for 30 minutes, wash the plate 3 times, and add 200 μL / well. 2 minutes / session; (8) Color development: Add TMB color development solution (color development substrate), 100 μL / well, and react at 37℃ in the dark for 10-30 min; (9) Termination: The reaction was terminated with 2 mol / L sulfuric acid, 100 μL / well, and the OD values ​​were read at 450 nm and 570 nm respectively.

[0025] (10) Calculation of results: AU (Arbitrary Units) is a value calculated using the formula: AU = (OD value of the sample to be tested / OD value of the positive standard serum) 100, OD value is the value detected by the microplate reader at 450nm - 570nm.

[0026] Example 1: Functional validation of antigenic peptide EG16 for the diagnosis of RA Serum samples included 175 patients diagnosed with rheumatoid arthritis (RA) (RA classification criteria: 2010 ACR / EULAR classification criteria for rheumatoid arthritis), 52 patients with systemic lupus erythematosus (SLE), 51 patients with Sjögren's syndrome (SS), 53 patients with osteoarthritis (OA), and 125 healthy controls (HC).

[0027] Peripheral blood was obtained from the above samples, and serum or plasma was separated and tested using the above ELISA kit.

[0028] The test results are shown in Table 1 and below. Figure 1 .

[0029] Table 1: Expression levels of epitope antibodies in RA, SLE, SS, OA and HC populations Note: 'a' indicates that the AU value of epitope antibodies was significantly different between rheumatoid arthritis and the corresponding control group, i.e., p < 0.001.

[0030] The results showed that the expression level of epitope antibodies in the RA population was significantly higher than that in the disease control group (osteoarthritis, systemic lupus erythematosus, Sjögren's syndrome) and the healthy control group.

[0031] Based on the above results, the sensitivity and specificity of the epitope antibody were calculated.

[0032] ROC curves were plotted with the true positive rate (sensitivity%) on the ordinate and the false positive rate (100% - specificity%) on the x-axis. The area under the ROC curve (AUC) is a core indicator for evaluating the performance of binary diagnostic models, directly reflecting the model's overall ability to distinguish between "patients" and "non-patients." The closer the AUC value is to 1, the stronger the model's overall ability to differentiate between the two types of samples, and the higher its diagnostic value.

[0033] The AUC of epitope antibodies in serum samples was 0.7490. (See [reference needed]) Figure 2 The AU value of 69.81, the maximum value of the Youden index, was selected as the cut-off value. Based on this value, the positive rate of serum epitope antibodies in RA patients (43.43%) was significantly higher than that in patients with systemic lupus erythematosus (SLE) (11.53%), Sjogren's syndrome (SS) (9.8%), osteoarthritis (OA) (3.77%), and the healthy control (HC) (9.60%). The sensitivity and specificity of epitope antibodies in diagnosing RA were 43.43% and 92.8%, respectively, demonstrating good diagnostic value (see Table 2).

[0034] Table 2 Sensitivity and Specificity of Epitope Antibody ELISA Detection Example 2: Functional validation of antigenic peptide EG16 for the diagnosis of preclinical RA For preclinical rheumatoid arthritis patients, early clinical identification and diagnosis are challenging because their RA-specific antibodies (such as anti-CCP antibodies) are often negative or at low levels. To explore the supplementary diagnostic value of the epitope antibody recognized by the antigen peptide EG16 of this invention in such patients, we selected serum samples from 36 preclinical RA patients, 36 early RA patients, 36 long-term RA patients, and 36 healthy individuals (HC) for testing and analysis. The specific ELISA experimental procedures are the same as in Example 1.

[0035] Epitope antibody expression levels in RA populations at different stages, such as Figure 3 And Table 3.

[0036] Table 3. Diagnostic value of epitope antibody ELISA detection for different stages of RA. Note: a indicates that the AU value of epitope antibodies was significantly different from that of the healthy control group, i.e., p<0.001; b indicates that the AU value of epitope antibodies was significantly different from that of the healthy control group, i.e., p<0.01.

[0037] ROC curves were plotted with the true positive rate (sensitivity%) on the ordinate and the false positive rate (100% - specificity%) on the x-axis. The AU value of 69.87, where the Youden index was at its maximum, was selected as the cut-off value. Based on this value, the positive rates of serum epitope antibodies in preRA patients (44.44%), earlyRA patients (47.22%), and LsRA patients (41.67%) were all significantly higher than those in HC (8.83%). See [link to relevant documentation]. Figure 4 And Table 4.

[0038] Table 4. Sensitivity and specificity of epitope antibody ELISA detection for different stages of rheumatoid arthritis (RA). This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. Antigenic peptide EG16, characterized in that, The amino acid sequence is APKRIRLPHIREDDQP, or the amino acid sequence is BSA-C-APKRIRLPHIREDDQP, where BSA is bovine serum albumin and C linked to BSA is a cysteine ​​residue.

2. The application of the antigenic peptide EG16 according to claim 1 in the preparation of diagnostic products for rheumatoid arthritis, characterized in that, The product is used to detect the content of epitope antibodies in patient biological samples; the epitope antibodies can be recognized by the antigenic peptide EG16 of claim 1.

3. The application according to claim 2, characterized in that, Compared with normal healthy individuals, patients with systemic lupus erythematosus, patients with Sjögren's syndrome, and patients with osteoarthritis, the levels of epitope antibodies in biological samples from patients with rheumatoid arthritis were significantly increased.

4. The application according to claim 3, characterized in that, The rheumatoid arthritis patients include those with preclinical rheumatoid arthritis, those with early-stage rheumatoid arthritis, and those with long-term rheumatoid arthritis.

5. The application according to claim 2, characterized in that, The biological sample is serum or plasma.

6. The application according to claim 2, characterized in that, The diagnostic product is an ELISA test kit.

7. A diagnostic kit for rheumatoid arthritis, characterized in that, include: The antigen peptide EG16, enzyme-labeled plate, coating solution, washing solution, blocking solution, diluent, enzyme-labeled secondary antibody, chromogenic substrate and stop solution as described in claim 1.

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