Citrullinated SAA polypeptide and application of citrullinated SAA polypeptide in preparation of rheumatoid arthritis diagnosis product

Abstract

The invention discloses a citrullinated SAA polypeptide and application thereof in preparation of a rheumatoid arthritis diagnosis product, the amino acid sequence of the citrullinated SAA polypeptide is DKYFHA-Cit-GNYDAAK-Cit-GPGGA, through the polypeptide, the citrullinated autoantigen B cell epitope of SAA and a corresponding autoantibody, namely an anti-citrullinated SAA polypeptide antibody (anti-CAP), are identified. The expression level of anti-CAP in RA patients is obviously higher than that of other disease control groups and healthy groups, and the anti-CAP has better positive rate in early RA, ESR / CRP normal RA and anti-CCP antibody / RF negative RA, and provides a new strategy for diagnosis of RA.

Description

Technical Field

[0001] The present invention relates to the technical field of rheumatoid arthritis diagnosis, and in particular to citrullinated SAA polypeptides and applications thereof in the preparation of rheumatoid arthritis diagnosis products. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic systemic autoimmune disease characterized by overactivation of immune cells, progressive synovial inflammation, and erosive lesions of articular cartilage and bone. The prevalence of RA worldwide is about 1% (SMOLEN JS, ALETAHA D, MCINNES I B. Rheumatoid arthritis [J]. Lancet, 2016, 388(10055): 2023-38), and the prevalence in my country is about 0.24%~0.4% (LIR, SΜN J, REN LM, et al. Epidemiology of eight common rheumatic diseases in China: a large-scale cross-sectional survey in Beijing [J]. Rheumatology (Oxford), 2012, 51(4): 721-9). RA is mainly manifested by inflammatory reactions of the local synovium of multiple small joints throughout the body. The disease can gradually progress and eventually lead to joint deformities, seriously affecting the quality of life of patients. Early diagnosis and effective treatment are key to preventing long-term damage and disability in RA patients.

[0003] In addition to the patient's clinical manifestations and imaging changes, the diagnosis of RA largely relies on the detection of serum markers. Anti-cyclic citrullinated peptide (CCP) antibodies and rheumatoid factor (RF) are currently the classic serological markers for the diagnosis of RA and have been included in the classification criteria for the diagnosis of RA established by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EMLAR) in 2010. Among them, anti-CCP antibodies have high specificity but limited sensitivity (95% and 67%, respectively). About 30% of patients still have negative anti-CCP antibodies in their serum (NISHIMMURA K, SΜGIYAMA D, KOGATA Y, et al. Meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis [J]. Ann Intern Med, 2007, 146(11): 797-808). Therefore, there is an unmet need to identify potential diagnostic biomarkers for RA, especially for RA patients who are negative for anti-CCP antibodies, which still lacks specific biomarkers, limiting the early diagnosis and treatment of such patients in clinical practice.

[0004] In the previous study, our research group conducted a serum antigenomics study on rheumatoid arthritis using label-free proteomics technology. Based on the molecular characteristics of differentially expressed proteins, a random forest model algorithm was used to classify rheumatoid arthritis, anti-cyclic citrullinated peptide antibody (ACPA)-positive rheumatoid arthritis, and ACPA-negative rheumatoid arthritis. Among these proteins, serum amyloid A (SAA) may be one of the specific autoantigens for the diagnosis of CCP-negative rheumatoid arthritis.

[0005] SAA is an acute phase response protein belonging to the apolipoprotein family. Under normal physiological conditions, its content in plasma is low. Under inflammatory stimulation, such as bacterial and viral infection, liver cells will synthesize and secrete SAA in large quantities. As an inflammatory marker, SAA levels can rise rapidly within hours after infection in infectious diseases. It is more sensitive than traditional inflammatory indicators such as C-reactive protein (CRP) and can be used for early diagnosis of infection, disease monitoring and efficacy evaluation; in cardiovascular diseases, its increased level is associated with the risk of cardiovascular disease; it can also assist in disease monitoring and activity evaluation of autoimmune diseases such as rheumatoid arthritis. Studies have shown that SAA expression levels are increased in the serum, joint fluid and synovial tissue of RA patients, and SAA concentration is closely related to RA disease activity. SAA level is an effective indicator for judging the activity of RA and clinical treatment response, and has certain clinical significance for the diagnosis and prognosis of RA. Summary of the invention

[0006] The purpose of the present invention is to provide a new detection antigen citrullinated SAA polypeptide, which can be used to detect the concentration of the corresponding epitope antibody, i.e., anti-citrullinated SAA polypeptide antibody (anti-CAP), in RA patients, and can be used to diagnose RA.

[0007] The technical solution of the present invention is described in detail as follows: In the first aspect, the present invention provides a citrullinated SAA polypeptide, the amino acid sequence of which is shown below: DKYFHA-Cit-GNYDAAK-Cit-GPGGA (SEQ ID NO: 1).

[0008] Optionally or preferably, the above-mentioned citrullinated SAA polypeptide has a carrier protein BSA coupled to the N-terminus of the amino acid sequence through a cysteine ​​residue to increase immunogenicity. The amino acid sequence after coupling is as follows: BSA-C-DKYFHA-Cit-GNYDAAK-Cit-GPGGA (SEQ ID NO: 2).

[0009] In a second aspect, the present invention provides the use of the above-mentioned citrullinated SAA polypeptide in the preparation of a diagnostic product for rheumatoid arthritis, wherein the product is used to detect the content of anti-citrullinated SAA polypeptide antibodies in a biological sample of a patient.

[0010] Compared with normal healthy people, patients with Sjögren's syndrome, systemic lupus erythematosus, osteoarthritis and psoriatic arthritis, the content of anti-citrullinated SAA peptide antibodies in biological samples of rheumatoid arthritis patients was significantly increased.

[0011] The rheumatoid arthritis patients include patients with early rheumatoid arthritis with a course of less than 12 months, patients with normal ESR and / or CRP, and patients with negative serum anti-CCP antibodies and / or negative serum RF.

[0012] The biological sample is serum.

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

[0014] In a third aspect, the present invention provides a rheumatoid arthritis diagnostic kit, comprising: the above-mentioned citrullinated SAA polypeptide, an ELISA plate, an ELISA coating solution, a washing solution, a blocking solution, a sample 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: This study provides a citrullinated SAA peptide, and through this peptide, the citrullinated autoantigen B cell epitope of SAA is identified, revealing the diagnostic value of the corresponding autoantibody, anti-citrullinated SAA peptide antibody (anti-CAP, anti-citrullinated SAA peptide antibody) for RA. The expression level of anti-CAP in RA patients is significantly higher than that in other disease control groups and healthy groups, and has a good positive rate in early RA, RA with normal ESR / CRP, and anti-CCP antibody / RF negative RA, providing a new strategy for the diagnosis of RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The statistical results of the small sample study in Example 2, using citrullinated SAA polypeptide as the detection antigen, to detect the expression level of anti-CAP in the serum of different groups of people. RA: rheumatoid arthritis; SS: Sjögren's syndrome; OA: osteoarthritis; SLE: systemic lupus erythematosus; HC: healthy control; anti-CAP: anti-citrullinated SAA polypeptide antibody.

[0017] Figure 2 In the large sample study of Example 2, the statistical results (A) and the large sample ROC curve and area under the curve (AUC) analysis results (B) of anti-CAP expression levels in the serum of different groups of people were detected using citrullinated SAA polypeptide as the detection antigen. RA: rheumatoid arthritis; SS: Sjögren's syndrome; OA: osteoarthritis; SLE: systemic lupus erythematosus; PsA: psoriatic arthritis; HC: healthy control; anti-CAP: anti-citrullinated SAA polypeptide antibody.

[0018] Figure 3The statistical results of Example 3 using citrullinated SAA polypeptide as the detection antigen to detect the anti-CAP expression level in the serum of early RA patients with different disease courses, ERA: early rheumatoid arthritis; HC: healthy control; anti-CAP: anti-citrullinated SAA polypeptide antibody; n: the number of patients with positive anti-CAP antibodies; N: the total number of RA patients.

[0019] Figure 4 The statistical results of anti-CAP expression levels in biological samples of RA patients with different ESR / CRP characterizations, HC: healthy control; anti-CAP: anti-citrullinated SAA peptide antibody; n: the number of anti-CAP antibody-positive patients; N: the total number of RA patients.

[0020] Figure 5 These are the statistical results of anti-CAP expression levels in biological samples of anti-CCP antibody / RF-negative RA patients; anti-CCP- RA: anti-CCP antibody-negative RA patients; RF- RA: RF-negative RA patients; anti-CCP- and RF- RA: seronegative RA patients with both anti-CCP antibody and RF negative; anti-CAP: anti-citrullinated SAA polypeptide antibody; n: number of anti-CAP antibody-positive patients; N: total number of RA patients.

[0021] In the above figures, the statistical results were analyzed by Kruskal-Wallis H test for differences among multiple groups, and Dunnet t test for multiple comparisons. **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0022] In order to make the technical personnel in this field better understand the present application scheme, the present application will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only embodiments of a part of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work should belong to the scope of protection of this application. The instruments and reagents used in the embodiments are all derived from commercial channels unless otherwise specified.

[0023] Example 1 Synthesis of citrullinated SAA polypeptide and its application in ELISA detection technology The SAA polypeptide was synthesized at the 51-70 position of SAA and citrullinated, and the N-terminus was coupled to the carrier protein BSA to increase immunogenicity; a cysteine ​​residue was added to the N-terminus during synthesis to facilitate coupling.

[0024] The amino acid sequence of the citrullinated SAA polypeptide conjugated to BSA is: BSA-C-DKYFHA-Cit-GNYDAAK-Cit-GPGGA.

[0025] ELISA diagnostic kit, including: citrullinated SAA peptide, ELISA plate (microplate), ELISA coating solution, washing solution, blocking solution (3% BSA + PBST), sample diluent, enzyme-labeled secondary antibody, color substrate (TMB substrate solution) and stop solution (sulfuric acid). The components in the kit are all volume percentages, the solvent for the enzyme-labeled secondary antibody is glycerol, and the solvents for the other reagents are water.

[0026] ELISA detection technology: (1) Dilute the citrullinated SAA peptide conjugated with BSA to 10 μg / ml with ELISA coating buffer, add 100 μl to each well of the microplate, seal the plate with sealing film, and place in a refrigerator at 4°C overnight; (2) Aspirate the components in the microplate and rinse with 300L of washing solution for 3 times; (3) Pipette 200 μl of 3% BSA + PBST into each microwell and incubate at room temperature for 2 hours; (4) Add the standard and diluted patient samples to the microwells, 100 μl per well, and incubate at room temperature for 1 hour; (5) Aspirate the components in the microplate and rinse with 300L of washing solution for 3 times; (6) Pipette 100 μL of enzyme conjugate (i.e., enzyme-labeled secondary antibody) into each microwell and incubate at room temperature for 30 minutes; (7) Aspirate the components in the microplate and rinse with 300L of washing solution for 3 times; (8) Pipette 100 μL of TMB substrate solution into each microwell and incubate at room temperature for 10 min. (9) Add 100 μL of stop solution to each microwell and incubate at room temperature for 5 minutes; (10) Read the optical density at 450 nm using a microplate reader and calculate the result. If dual wavelength measurement is used, the reference wavelength is 570 nm. The color produced should remain stable for at least 30 minutes, during which time the optical density is read.

[0027] Example 2 Diagnostic value of Anti-CAP in RA Small sample study: We used citrullinated SAA peptide coupled to BSA as the detection antigen and detected the expression level of the corresponding antibody epitope, anti-CAP, in serum samples of RA (83 cases), disease control Sjögren's syndrome (24 cases), osteoarthritis (24 cases), systemic lupus erythematosus (24 cases) and healthy controls (21 cases) by ELISA detection technology. The test results are as follows Figure 1 As shown, the results showed that the anti-CAP level in RA serum was significantly increased compared with HC and disease control groups.

[0028] The results of the above small sample study have shown that the epitope antibody anti-CAP corresponding to the citrullinated SAA peptide exists in the sera of different populations, and the expression level of this epitope antibody is specifically increased in the serum of RA patients, which suggests that anti-CAP in serum may serve as a potential diagnostic marker for RA.

[0029] To further study the clinical diagnostic value of anti-CAP in RA, we conducted a validation study in a large sample cohort. The test cohort included 123 cases of RA, 48 cases of Sjögren's syndrome, 48 cases of osteoarthritis, 48 ​​cases of systemic lupus erythematosus, 32 cases of psoriatic arthritis, and 68 healthy controls. Figure 2 As shown in the figure, A is the statistical result of the expression level of anti-CAP in different populations, showing that compared with other disease controls and healthy controls, the anti-CAP level in RA patients is significantly increased, and B is the ROC curve and area under the curve (AUC) of the detection cohort. The ROC curve shows the classification performance of the model at different thresholds. The closer the curve is to the upper left corner, the better the performance of the model, that is, the higher the true positive rate and the lower the false positive rate. AUC refers to the area enclosed by the ROC curve and the coordinate axis, and the value range is between 0 and 1. The larger the AUC value, the better the performance of the model.

[0030] To further evaluate the efficacy of anti-CAP in the diagnosis of RA, we set the mean of the anti-CAP AU value of the healthy control group plus 2 times the standard deviation as the cut-off value (AU value = 76.24) and calculated the sensitivity, specificity, positive predictive value, and negative predictive value of anti-CAP in the cohort. The results are shown in Table 1.

[0031] Table 1 Sensitivity and specificity of anti-CAP antibodies in the cohort The results showed that the diagnostic sensitivity of serum anti-CAP in RA patients was 59.10%, the specificity was 94.10%, the positive predictive value was 83.69%, and the negative predictive value was 81.81%.

[0032] Example 3 Diagnostic value of Anti-CAP in early RA The anti-CAP level in the serum of patients with early RA (ERA) was detected by ELISA using the citrullinated SAA polypeptide of Example 1 as the detection antigen. Among them, 85 patients with RA with a course of ≤12 months, 43 patients with RA with a course of ≤6 months, and 68 healthy controls were also set up. The test results are shown in Figure 3 , showing that anti-CAP is elevated in the serum of early RA patients, and as the course of the disease increases, the positive rate of anti-CAP antibodies tends to gradually increase.

[0033] The positive rates in patients with disease duration ≥12 months, ≤12 months, and ≤6 months were 57.89%, 52.94%, and 51.16%, respectively.

[0034] Example 4 The supplementary diagnostic value of anti-CAP in RA with normal ESR / CRP In the ACR / EULAR 2010 classification criteria, erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) are routinely used for the diagnosis and assessment of RA disease activity. We further compared the anti-CAP levels in the serum of RA patients with the two indicators ESR and CRP listed in the ACR / EULAR2010 classification criteria. By ELISA, the citrullinated SAA polypeptide of Example 1 was used as the detection antigen to detect the anti-CAP levels in RA patients with normal ESR / CRP. There were 52 RA patients with elevated ESR and CRP, 36 patients with elevated CRP and normal ESR or elevated ESR and normal CRP, and 36 RA patients with normal ESR and CRP.

[0035] The positive rate of anti-CAP antibodies in RA patients with elevated ESR and CRP was 53.85%, and the positive rate of anti-CAP antibodies in RA patients with elevated CRP and normal ESR or elevated ESR and normal CRP was 55.56%. Even in RA patients with normal ESR and CRP, the positive rate of anti-CAP antibodies could reach 55.56%, see below Figure 4 and Table 2.

[0036] Table 2 Anti-CAP positive rate in serum of patients with different types of RA We evaluated the anti-cap levels and positive rates in RA patients with elevated ESR / CRP and normal ESR / CRP. Figure 4As shown in the figure, the positive rate of anti-cap is higher in RA patients with elevated ESR / CRP. Even in RA patients with normal ESR or normal CRP, there is no significant difference in the positive rate, which is significantly higher than that of healthy people. This further shows that it has a certain supplementary diagnostic value for RA as a potential biomarker and supplementary disease activity indicator.

[0037] Example 5 The supplementary diagnostic value of Anti-CAP in RA with normal autoantibodies Currently, the main diagnostic markers for RA are anti-CCP antibodies and RF, but some patients are still negative for anti-CCP antibodies and RF. Therefore, we further analyzed the supplementary diagnostic value of anti-CAP antibodies in anti-CCP antibody / RF-negative RA patients.

[0038] The anti-CAP levels in anti-CCP antibody-negative RA patients (34 cases), RF-negative RA patients (32 cases), and seronegative RA patients with both anti-CCP antibody and RF negative (20 cases) were detected by ELISA using the citrullinated SAA polypeptide of Example 1 as the detection antigen.

[0039] Results Figure 5 The results showed that the positive rates of anti-CAP in anti-CCP antibody-negative RA patients and RF-negative RA patients were 44.12% and 43.75%, respectively. More importantly, in seronegative RA patients with both anti-CCP antibody and RF negative, the positive rate of anti-CAP could reach 35.00%.

[0040] Experiments show that the citrullinated SAA polypeptide provided by the present invention can effectively detect the corresponding epitope antibody anti-citrullinated SAA polypeptide antibody in the patient's biological sample, and the expression level of the antibody in RA patients is significantly higher than that in other disease control groups and healthy groups, and has excellent diagnostic value in early RA, ESR / CRP normal RA, and anti-CCP antibody / RF negative RA.

[0041] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. A citrullinated SAA polypeptide, characterized in that The amino acid sequence is shown below: DKYFHA-Cit-GNYDAAK-Cit-GPGGA.

2. The citrullinated SAA polypeptide according to claim 1, characterized in that The N-terminus of the amino acid sequence is also coupled with a carrier protein BSA through a cysteine ​​residue. The amino acid sequence after coupling is as follows: BSA-C-DKYFHA-Cit-GNYDAAK-Cit-GPGGA.

3. Use of the citrullinated SAA polypeptide according to claim 1 or 2 in the preparation of a diagnostic product for rheumatoid arthritis, characterized in that: The product is used to detect the content of anti-citrullinated SAA polypeptide antibodies in patient biological samples.

4. The use according to claim 3, characterized in that: Compared with normal healthy people, patients with Sjögren's syndrome, systemic lupus erythematosus, osteoarthritis and psoriatic arthritis, the content of anti-citrullinated SAA peptide antibodies in biological samples of rheumatoid arthritis patients was significantly increased.

5. The use according to claim 4, characterized in that: The rheumatoid arthritis patients include patients with early rheumatoid arthritis with a course of less than 12 months, patients with normal ESR and / or CRP, and patients with negative serum anti-CCP antibodies and / or negative serum RF.

6. The use according to claim 3, characterized in that: The biological sample is serum.

7. The use according to claim 3, characterized in that: The diagnostic product is an ELISA detection kit.

8. A rheumatoid arthritis diagnostic kit, characterized in that: include: The citrullinated SAA polypeptide according to claim 1 or 2, an ELISA plate, an ELISA coating solution, a washing solution, a blocking solution, a sample diluent, an enzyme-labeled secondary antibody, a chromogenic substrate and a stop solution.

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