Citrullinated SAA Polypeptide and Its Application in the Preparation of Diagnostic Products for Rheumatoid Arthritis

Abstract

The present invention discloses citrullinated SAA polypeptides and their application in the preparation of diagnostic products for rheumatoid arthritis. The amino acid sequence of the citrullinated SAA polypeptide is: DKYFHA-Cit-GNYDAAK-Cit-GPGGA. Through this polypeptide, the present invention has identified the citrullinated autoantigen B cell epitope of SAA and the corresponding autoantibody, namely anti-citrullinated SAA polypeptide antibody (anti-CAP). The expression level of anti-CAP in RA patients is significantly higher than that in other disease control groups and healthy groups, and it has a good positive rate in early RA, ESR / CRP normal RA, and anti-CCP antibody / RF negative RA, providing a new strategy for the diagnosis of RA.

Description

Technical Field

[0001] The present invention relates to the technical field of rheumatoid arthritis diagnosis, and particularly relates to citrullinated SAA polypeptide and its application in the preparation of rheumatoid arthritis diagnostic products. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic systemic autoimmune disease, mainly characterized by overactivation of immune cells, progressive synovial inflammation, and erosive lesions of articular cartilage / bone. The prevalence of RA worldwide is approximately 1% (SMOLEN J S, ALETAHA D, MCINNES I B. Rheumatoid arthritis [J]. Lancet, 2016, 388(10055): 2023-38), and the prevalence in China is approximately 0.24% - 0.4% (LIR, SΜN J, REN L M, 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 mainly presents as inflammatory reactions in the local synovium of multiple small joints throughout the body, and the condition can gradually progress and ultimately lead to joint deformity, seriously affecting the quality of life of patients. Early diagnosis and effective treatment are the keys to preventing long-term damage and disability in RA patients.

[0003] In addition to relying on the clinical manifestations and imaging changes of patients, the diagnosis of RA largely depends on the detection of serum markers. Anti-cyclic citrullinated peptide (CCP) antibody and rheumatoid factor (RF) are classic serological markers for the diagnosis of RA and have been incorporated into the classification criteria for RA diagnosis developed by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EMLAR) in 2010. Among them, the anti-CCP antibody has high specificity but limited sensitivity (95% and 67% respectively), and there are still about 30% of patients with negative anti-CCP antibody in their sera (NISHIMΜRA 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, the need to identify potential diagnostic biomarkers for RA has not been met. In particular, there is still a lack of specific biomarkers for RA patients with negative anti-CCP antibody, which limits the early diagnosis and treatment of such patients in clinical practice.

[0004] In previous studies of our research group, serum antigenomics of rheumatoid arthritis was studied by 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] Serum amyloid A (SAA) is an acute-phase reactant and belongs to the apolipoprotein family. Under normal physiological conditions, its content in plasma is low. In the case of inflammatory stimuli such as bacterial and viral infections, hepatocytes will synthesize and secrete a large amount of SAA. As an inflammatory marker, in infectious diseases, its level can rapidly increase within hours after infection, which is more sensitive than traditional inflammatory indicators such as C-reactive protein (CRP), and can be used for the diagnosis of early infection, disease monitoring and efficacy evaluation; in cardiovascular diseases, its elevated level is associated with the risk of occurrence of cardiovascular diseases; it can also assist in the disease monitoring and activity assessment of autoimmune diseases such as rheumatoid arthritis. Research shows that the expression level of SAA increases in the serum, synovial fluid and synovial tissue of RA patients, and the concentration of SAA is closely related to the disease activity of RA. The level of SAA is an effective indicator for judging the activity degree of RA and the clinical treatment response, and has certain clinical significance for the diagnosis and prognosis of RA. Summary of the Invention

[0006] The object of the present invention is to provide a new detection antigen, citrullinated SAA polypeptide, through which the concentration of the corresponding epitope antibody, anti-citrullinated SAA polypeptide antibody (anti-CAP), in the body of RA patients can be detected, and thus it can be used for the diagnosis of RA.

[0007] The technical solution of the present invention is described in detail as follows:

[0008] In the first aspect, the present invention provides a citrullinated SAA polypeptide, and its amino acid sequence is shown as follows:

[0009] DKYFHA-Cit-GNYDAAK-Cit-GPGGA (SEQ ID NO:1).

[0010] Optionally or preferably, the N-terminus of the amino acid sequence of the above citrullinated SAA polypeptide is coupled with a carrier protein BSA through a cysteine residue to increase immunogenicity, and the amino acid sequence after coupling is as follows:

[0011] BSA-C-DKYFHA-Cit-GNYDAAK-Cit-GPGGA (SEQ ID NO:2).

[0012] In the second aspect, the present invention provides the application of the above citrullinated SAA polypeptide in the preparation of a rheumatoid arthritis diagnostic product, and the product is used to detect the content of anti-citrullinated SAA polypeptide antibody in a biological sample of a patient.

[0013] Compared with normal healthy people, patients with Sjogren's syndrome, systemic lupus erythematosus, osteoarthritis and psoriatic arthritis, the content of anti-citrullinated SAA polypeptide antibody in the biological samples of rheumatoid arthritis patients increases significantly.

[0014] The rheumatoid arthritis patients include early rheumatoid arthritis patients with a disease course within 12 months, rheumatoid arthritis patients with normal ESR and / or CRP, and rheumatoid arthritis patients with negative serum anti-CCP antibody and / or negative serum RF.

[0015] The biological sample is serum.

[0016] The diagnostic product is an ELISA detection kit.

[0017] In a third aspect, the present invention provides a rheumatoid arthritis diagnostic kit, comprising: the above-mentioned citrullinated SAA polypeptide, an enzyme-labeled plate, an ELISA coating solution, a washing solution, a blocking solution, a sample diluent, an enzyme-labeled secondary antibody, a chromogenic substrate, and a termination solution.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This study provides a citrullinated SAA polypeptide, and through this polypeptide, the citrullinated autoantigen B cell epitope of SAA is identified, revealing the corresponding autoantibody, that is, the diagnostic value of anti-citrullinated SAA polypeptide antibody (anti-CAP) 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 it has a good positive rate in early RA, ESR / CRP normal RA, and anti-CCP antibody / RF negative RA, providing a new strategy for the diagnosis of RA. Description of the Drawings

[0020] Figure 1 It is the statistical result of detecting the expression level of anti-CAP in the sera of different groups of people using citrullinated SAA polypeptide as the detection antigen in the small sample study of Example 2. RA: Rheumatoid arthritis; SS: Sjogren's syndrome; OA: Osteoarthritis; SLE: Systemic lupus erythematosus; HC: Healthy control; anti-CAP: Anti-citrullinated SAA polypeptide antibody.

[0021] Figure 2 It is the statistical result (A) of detecting the expression level of anti-CAP in the sera of different groups of people using citrullinated SAA polypeptide as the detection antigen in the large sample study of Example 2 and the analysis result (B) of the large sample ROC curve and the area under the curve (AUC). RA: Rheumatoid arthritis; SS: Sjogren's syndrome; OA: Osteoarthritis; SLE: Systemic lupus erythematosus; PsA: Psoriatic arthritis; HC: Healthy control; anti-CAP: Anti-citrullinated SAA polypeptide antibody.

[0022] Figure 3For Example 3, citrullinated SAA polypeptide was used as a detection antigen to detect the statistical results of the expression level of anti-CAP in the sera of early RA patients at different disease courses. ERA: early rheumatoid arthritis; HC: healthy control; anti-CAP: anti-citrullinated SAA polypeptide antibody; n: the number of patients positive for anti-CAP antibody; N: the total number of RA patients.

[0023] Figure 4 For the statistical results of the expression level of anti-CAP in biological samples of RA patients with different ESR / CRP characteristics. HC: healthy control; anti-CAP: anti-citrullinated SAA polypeptide antibody; n: the number of patients positive for anti-CAP antibody; N: the total number of RA patients.

[0024] Figure 5 For the statistical results of the expression level of anti-CAP 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 negative for both anti-CCP antibody and RF; anti-CAP: anti-citrullinated SAA polypeptide antibody; n: the number of patients positive for anti-CAP antibody; N: the total number of RA patients.

[0025] In each of the above figures, the Kruskal-Wallis H test was used to analyze the differences among multiple groups for the statistical results, and the Dunnet t test was used for multiple comparisons. **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed implementation manners

[0026] To enable those skilled in the art of this technology to better understand the solution of this application, the following will clearly and completely describe this application in combination with examples and the accompanying drawings. Obviously, the described examples are only a part of the examples of this application, rather than all of the examples. Based on the examples in this application, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application. The instruments and reagents used in the examples are from commercial channels unless otherwise specified.

[0027] Example 1 Synthesis of citrullinated SAA polypeptide and its application in ELISA detection technology

[0028] SAA polypeptide was synthesized at the 51-70 sites of SAA and citrullinated. The N-terminus was conjugated with the carrier protein BSA to increase immunogenicity; a cysteine residue was added at the N-terminus during synthesis to facilitate conjugation.

[0029] The amino acid sequence of the citrullinated SAA polypeptide conjugated to BSA is:

[0030] BSA-C-DKYFHA-Cit-GNYDAAK-Cit-GPGGA.

[0031] 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.

[0032] ELISA detection technology:

[0033] (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;

[0034] (2) Aspirate the components in the microplate and rinse with 300L of washing solution for 3 times;

[0035] (3) Pipette 200 μl of 3% BSA + PBST into each microwell and incubate at room temperature for 2 hours;

[0036] (4) Add the standard and diluted patient samples to the microwells, 100 μl per well, and incubate at room temperature for 1 hour;

[0037] (5) Aspirate the components in the microplate and rinse with 300L of washing solution for 3 times;

[0038] (6) Pipette 100 μL of enzyme conjugate (i.e., enzyme-labeled secondary antibody) into each microwell and incubate at room temperature for 30 minutes;

[0039] (7) Aspirate the components in the microplate and rinse with 300L of washing solution for 3 times;

[0040] (8) Pipette 100 μL of TMB substrate solution into each microwell and incubate at room temperature for 10 min.

[0041] (9) Add 100 μL of stop solution to each microwell and incubate at room temperature for 5 minutes;

[0042] (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.

[0043] Example 2: Diagnostic Value of Anti-CAP in RA

[0044] Small sample study: We used citrullinated SAA polypeptide conjugated with BSA as the detection antigen and detected the corresponding antibody epitope, namely anti-CAP, in serum samples of RA (83 cases), disease control Sjogren's syndrome (24 cases), osteoarthritis (24 cases), systemic lupus erythematosus (24 cases), and healthy controls (21 cases) by ELISA detection technology. The detection results are as Figure 1 shown. The results showed that the level of anti-CAP in RA serum was significantly higher than that in HC and disease control groups.

[0045] The above small sample study results have shown that there are epitope antibodies anti-CAP corresponding to citrullinated SAA polypeptide in the sera of different populations, and the expression level of this epitope antibody in RA patients is specifically increased, which suggests that anti-CAP in serum may be a potential diagnostic marker for RA.

[0046] To further study the clinical diagnostic value of anti-CAP in RA, we conducted a validation in a large sample cohort study. The detection cohort included 123 cases of RA, 48 cases of Sjogren's syndrome, 48 cases of osteoarthritis, 48 cases of systemic lupus erythematosus, 32 cases of psoriatic arthritis, and 68 healthy controls. The detection results are as Figure 2 shown. Among them, A is the statistical result of the expression level of anti-CAP in different populations, showing that the level of anti-CAP in RA patients is significantly higher than that in other disease controls and healthy controls. B is the ROC curve and the 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 is the area enclosed by the ROC curve and the coordinate axes, and its value ranges from 0 to 1. The larger the AUC value, the better the performance of the model.

[0047] To further evaluate the efficacy of anti-CAP in RA diagnosis, we set the mean of the anti-CAP AU value in the healthy control group plus 2 standard deviations 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.

[0048] Table 1 Sensitivity and Specificity of Anti-CAP Antibody in the Cohort

[0049]

[0050] The results showed that the diagnostic sensitivity of anti-CAP in serum for 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%.

[0051] Example 3 Diagnostic value of Anti-CAP in early RA

[0052] By ELISA method, using the citrullinated SAA polypeptide of Example 1 as the detection antigen, the anti-CAP level in the serum of early RA (ERA) patients was detected. Among them, there were 85 RA patients with a disease course ≤ 12 months and 43 RA patients with a disease course ≤ 6 months, and 68 healthy controls were set up at the same time. The detection results are shown in Figure 3 , showing that anti-CAP was elevated in the serum of early RA patients, and with the prolongation of the disease course, the positive rate of anti-CAP antibody showed a gradually increasing trend.

[0053] The positive rates in patients with a disease course ≥ 12 months, ≤ 12 months, and ≤ 6 months were 57.89%, 52.94%, and 51.16% respectively.

[0054] Example 4 Supplementary diagnostic value of anti-CAP in RA with normal ESR / CRP

[0055] In the ACR / EULAR 2010 classification criteria, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are routinely used for the diagnosis and evaluation of RA disease activity. We further compared the anti-CAP level in the serum of RA patients with the two indicators ESR and CRP listed in the ACR / EULAR 2010 classification criteria. By ELISA method, using the citrullinated SAA polypeptide of Example 1 as the detection antigen, the anti-CAP level in RA patients with normal ESR / CRP was detected. There were 52 RA patients with both elevated ESR and CRP, 36 patients with elevated CRP and normal ESR or elevated ESR and normal CRP, and 36 RA patients with both normal ESR and CRP.

[0056] The positive rate of anti-CAP antibody in RA patients with elevated ESR and CRP was 53.85%, the positive rate of anti-CAP antibody in RA patients with elevated CRP and normal ESR or elevated ESR and normal CRP was 55.56%, and even the positive rate of anti-CAP antibody in RA patients with normal ESR and CRP could reach 55.56%, see Figure 4 and Table 2.

[0057] Table 2 Positive rate of anti-CAP in serum of different types of RA patients

[0058]

[0059] We evaluated the anti - cap levels and positive rates in RA patients with elevated ESR / CRP and normal ESR / CRP. As Figure 4 shown, the positive rate of anti - cap in RA patients with elevated ESR / CRP was higher. Even in RA patients with normal ESR or normal CRP, there was no significant difference in the positive rate, which was significantly higher than that in healthy people. This further indicates that it has certain supplementary diagnostic value for RA as a potential biomarker and supplementary disease activity index.

[0060] Example 5 Supplementary Diagnostic Value of Anti - CAP in RA with Normal Autoantibodies

[0061] Currently, the main diagnostic markers for RA are anti - CCP antibody and RF. However, there are still some patients with negative anti - CCP antibody and RF. Therefore, we further analyzed the supplementary diagnostic value of anti - CAP antibody in RA patients negative for anti - CCP antibody / RF.

[0062] By ELISA method, using the citrullinated SAA polypeptide of Example 1 as the detection antigen, the anti - cap levels in 34 RA patients negative for anti - CCP antibody, 32 RA patients negative for RF, and 20 seronegative RA patients negative for both anti - CCP antibody and RF were detected.

[0063] The results are shown in Figure 5 . The results showed that the positive rates of anti - CAP in RA patients negative for anti - CCP antibody and RA patients negative for RF were 44.12% and 43.75% respectively. More importantly, in seronegative RA patients negative for both anti - CCP antibody and RF, the positive rate of anti - CAP could reach 35.00%.

[0064] The experiment shows that the citrullinated SAA polypeptide provided by the present invention can effectively detect the corresponding epitope antibody anti - citrullinated SAA polypeptide antibody in the biological samples of patients, and the expression level of this antibody in RA patients is significantly higher than that in other disease control groups and healthy groups, and it has excellent diagnostic value in early RA, RA with normal ESR / CRP, and RA negative for anti - CCP antibody / RF.

[0065] Specific examples are used in this article to elaborate on the inventive concept in detail. The description of the above examples is only for helping to understand the core idea of the present invention. It should be noted that for those of ordinary skill in the art of this technology, any obvious modifications, equivalent replacements or other improvements made without departing from the inventive concept should be included within 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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