Use of antigen peptide MG15 and antibodies thereof in the preparation of diagnostic products for rheumatoid arthritis

Abstract

The application discloses application of an antigen peptide MG15 and an antibody thereof in preparation of a rheumatoid arthritis diagnosis product, and belongs to the technical field of disease diagnosis. The application aims to solve the problem of lack of a serological diagnosis marker with high sensitivity and high specificity for RA (especially anti-CCP antibody negative RA), and relevant detection products show significant diagnostic value. The antigen peptide MG15 has an amino acid sequence of GNSSESESKTTHAYT or BSA-C-GNSSESESKTTHAYT, and is used for detecting the content of an epitope antibody capable of being specifically recognized by the antigen peptide MG15 in a biological sample of a patient. The epitope antibody has obvious differences between RA patients, healthy people and other easily-confused rheumatism and immunological disease patients, has a sensitivity of 47.47% and a specificity of 90.16% in serum RA diagnosis, can effectively identify and diagnose RA, and has unique diagnostic value in anti-CCP antibody negative RA patients.

Description

Technical Field

[0001] This invention relates to the field of rheumatoid arthritis diagnostic technology, specifically to the application of antigenic peptide MG15 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.

[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 sensitivity of anti-CCP antibodies is limited, with a positive rate of approximately 70%-80%, meaning that about 20%-30% of RA patients are serum negative. For this group of patients who are anti-CCP antibody negative, there is currently a lack of sensitive and specific diagnostic methods in clinical practice, which constitutes a significant shortcoming and unmet clinical need in the existing diagnostic system. Summary of the Invention

[0004] The purpose of this invention is to provide a novel antigenic peptide MG15, which can be used as a detection tool to detect the content of corresponding epitope antibodies in RA patients, and thus be used for the diagnosis of RA, which has important diagnostic value.

[0005] The technical solution of this invention is described in detail below: In a first aspect, the present invention provides an antigenic peptide MG15, the amino acid sequence of which is GNSSESESKTTHAYT (SEQ ID NO:1), or the amino acid sequence of which is BSA-C-GNSSESESKTTHAYT (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 MG15 in the preparation of a diagnostic product for rheumatoid arthritis. The product is used to detect the content of epitope antibodies in a patient's biological sample. The epitope antibodies can be recognized and bound by the above-mentioned antigenic peptide MG15, that is, the epitope antibodies are antibodies against the above-mentioned antigenic peptide MG15.

[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 mentioned include those who are negative for serum anti-CCP antibodies.

[0009] The biological sample is serum or plasma.

[0010] The diagnostic product is an ELISA test kit.

[0011] Thirdly, the present invention provides a diagnostic kit for rheumatoid arthritis, comprising: the above-mentioned antigen peptide MG15, 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.

[0012] 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 MG15, which consists of 15 amino acids. Epitope antibodies (anti-antigen peptide MG15) corresponding to this antigenic peptide MG15 are present in patient biological samples. The content of this epitope antibody in RA patients is significantly different from that in normal healthy individuals and other common rheumatic immune diseases that are easily confused with rheumatoid arthritis. The sensitivity in serum RA diagnosis is 47.47%, and the specificity is 90.16%, which can effectively distinguish RA from other rheumatic immune diseases that are easily confused with RA.

[0013] In addition, in RA patients with negative serum anti-CCP antibodies, the levels of the above-mentioned epitope antibodies (antibodies against the above-mentioned antigenic peptide MG15) were also significantly increased, with a positive rate of 36.59% and a specificity of 90.80%. This can effectively identify such patients who are easily missed, providing new key evidence for solving the clinical pain points in RA diagnosis and having important and unique diagnostic value. Attached Figure Description

[0014] Figure 1 This 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. A represents serum samples and B represents plasma samples. p<0.05, p<0.01, p<0.001, p < 0.0001 (Kruskal-Wallis test). [AU value = (OD value of test sample / OD value of positive standard serum)] 100, OD value is the value detected by the microplate reader at 450nm - 570nm.

[0015] Figure 2 The ROC curves for distinguishing RA patients, healthy individuals, and disease controls using epitope antibodies in Example 1 are shown, where A represents serum samples and B represents plasma samples.

[0016] Figure 3 This is a graph showing the relative abundance of epitope antibodies in anti-CCP negative RA patient samples detected by the ELISA method in Example 2, where A represents serum samples and B represents plasma samples. p<0.05, p<0.01, p<0.001, p < 0.0001 (Kruskal-Wallis test). [AU value = (OD value of test sample / OD value of positive standard serum)] 100, OD value is the value detected by the microplate reader at 450nm - 570nm.

[0017] Figure 4 The positive rates of epitope antibody detection in Example 2 are shown for anti-CCP positive RA patients, anti-CCP negative RA patients, and healthy individuals, where A is a serum sample and B is a plasma sample. Detailed Implementation

[0018] 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.

[0019] The amino acid sequence of the antigenic peptide MG15 is: BSA-C-GNSSESESKTTHAYT (SEQ ID NO:2), synthesized by Shanghai Nuoyou Biotechnology Co., Ltd.

[0020] This ELISA diagnostic kit includes: the antigenic peptide MG15 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.

[0021] 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.

[0022] Experimental steps for using ELISA diagnostic kits for diagnosis: (1) Plate coating: The antigenic peptide MG15 of SEQ ID NO:2 was diluted to 5 μg / mL with carbonate buffer at 4℃ and coated into a 96-well microplate at 100 μL / well. The plate was then incubated 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.

[0023] (10) Calculation of results: 1) Selection of positive control standard serum: obtained by selecting serum from 10 RA cases with OD values ​​greater than 1.5 and mixing them in equal volumes; 2) AU value (Arbitrary units) is expressed by the formula: AU value = (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.

[0024] Example 1: Functional validation of antigenic peptide MG15 for the diagnosis of RA Serum samples included 158 patients diagnosed with rheumatoid arthritis (RA) (RA classification criteria: 2010 ACR / EULAR rheumatoid arthritis classification criteria), 51 patients with systemic lupus erythematosus (SLE), 48 patients with Sjögren's syndrome (SS), 47 patients with osteoarthritis (OA), and 98 healthy controls (HC).

[0025] Plasma samples included 105 patients diagnosed with rheumatoid arthritis (RA) (RA classification criteria: 2010 ACR / EULAR classification criteria for rheumatoid arthritis), 36 patients with systemic lupus erythematosus (SLE), 35 patients with Sjögren's syndrome (SS), 33 patients with osteoarthritis (OA), and 71 healthy controls (HC).

[0026] Peripheral blood was obtained from the above samples, and serum or plasma was separated and tested using the above ELISA kit.

[0027] The test results are shown in Table 1 and Figure 1 .

[0028] Table 1: Expression levels of epitope antibodies in RA, SLE, SS, OA and HC patients 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.

[0029] The results showed that the expression level of epitope antibodies that can be specifically recognized by the SEQ ID NO:2 antigenic peptide MG15 was significantly higher in the RA population than in the disease control group (osteoarthritis, systemic lupus erythematosus, Sjögren's syndrome) and the healthy control group.

[0030] The sensitivity and specificity of epitope antibodies were calculated based on the above test results.

[0031] Plot an ROC curve with the true positive rate (sensitivity) on the ordinate and the false positive rate (1-specificity) on the abscissa.

[0032] The area under the ROC curve (AUC) of epitope antibodies in serum samples was 0.7935. (See [link to relevant documentation]). Figure 2 In the study, the AU value of 20.46, 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 (47.47%) was significantly higher than that in patients with systemic lupus erythematosus (SLE) (9.80%), Sjogren's syndrome (SS) (10.42%), osteoarthritis (OA) (10.64%), and the healthy control group (HC) (9.18%). The sensitivity and specificity of epitope antibodies in diagnosing RA were 47.47% and 90.16%, respectively, demonstrating good diagnostic value.

[0033] The area under the ROC curve (AUC) of epitope antibodies in plasma samples was 0.7827. (See [reference needed]) Figure 2 In section B, the AU value of 29.80, the point of maximum Youden's index, was selected as the cut-off value. Based on this value, the positive rate of plasma epitope antibodies in RA patients (44.76%) was significantly higher than that in patients with systemic lupus erythematosus (SLE) (8.33%), Sjogren's syndrome (SS) (5.71%), osteoarthritis (OA) (9.09%), and the healthy control group (HC) (7.04%). The sensitivity and specificity of epitope antibodies in diagnosing RA were 44.76% and 92.57%, respectively, demonstrating good diagnostic value.

[0034] Table 2 Sensitivity and Specificity of Epitope Antibody ELISA Detection Example 2: Functional validation of antigenic peptide MG15 for the diagnosis of anti-CCP antibody-negative RA Some RA patients are negative for RA-specific antibodies (anti-CCP antibodies), which presents diagnostic challenges for these patients. Therefore, we explored the diagnostic value of the epitope antibody specifically recognized by the aforementioned antigen peptide MG15 in RA patients who are negative for anti-CCP antibodies.

[0035] Serum samples from 41 confirmed anti-CCP antibody-negative RA patients and plasma samples from 29 confirmed anti-CCP antibody-negative RA patients were selected. The above-mentioned ELISA kits were used for detection. Serum samples from 108 confirmed anti-CCP antibody-positive RA patients and plasma samples from 76 confirmed anti-CCP antibody-positive RA patients were also tested.

[0036] The expression level of epitope antibodies in anti-CCP antibody-negative RA populations is as follows: Figure 3 And Table 3.

[0037] Table 3: Diagnostic value analysis of epitope antibodies against CCP antibody-negative RA Table 3 shows that the sensitivity of epitope antibodies in serum samples of anti-CCP antibody-negative RA was 36.59%, and the specificity was 90.80%. In plasma samples of anti-CCP antibody-negative RA, the sensitivity was 34.48%, and the specificity was 91.07%. The positive rate of serum epitope antibodies in anti-CCP antibody-negative RA patients (36.59%) was significantly higher than that in the healthy control group (HC) (9.20%) (see Table 3). Figure 4 A) The positive rate of plasma epitope antibodies in anti-CCP antibody-negative RA patients (34.48%) was significantly higher than that in the healthy control group (HC) (8.93%) (see A). Figure 4 (B) This indicates that the epitope antibody specifically recognized by the aforementioned antigenic peptide MG15 has significant diagnostic value in anti-CCP antibody-negative RA patients, providing effective independent diagnostic evidence. This precisely fills the gap in existing serological testing, creating crucial conditions for early diagnosis and intervention, and has irreplaceable clinical value.

[0038] 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 MG15, characterized in that, The amino acid sequence is GNSSESESKTTHAYT, or the amino acid sequence is BSA-C-GNSSESESKTTHAYT, where BSA is bovine serum albumin.

2. The application of the antigenic peptide MG15 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 MG15 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 mentioned include those who are negative for serum anti-CCP antibodies.

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 MG15, 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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