Use of citrullinated galectin-1 polypeptides for the preparation of diagnostic products for rheumatoid arthritis

Abstract

The application discloses application of citrullinated galectin-1 polypeptide in preparation of a rheumatoid arthritis diagnosis product, and belongs to the technical field of rheumatoid arthritis diagnosis. The amino acid sequence of the citrullinated galectin-1 polypeptide is GD-Cit-WSSQQGSKAVYPE; the product is used for detecting the content of anti-citrullinated galectin-1 polypeptide antibody in a biological sample of a patient, and the antibody level is significantly different between RA patients, normal healthy people and other common rheumatic immune disease patients who are easily confused with rheumatoid arthritis. The product has good sensitivity to common RA, RA with normal ESR and CRP, and anti-CCP antibody / RF negative RA patients, and has good supplementary diagnosis value for the diagnosis of RA.

Description

Technical Field

[0001] This invention relates to the field of rheumatoid arthritis diagnostic technology, specifically to the application of citrullinated lectin-1 polypeptide in the preparation of rheumatoid arthritis diagnostic products. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease characterized by symmetrical polyarthritis. Its pathological features include synovial inflammation and bone erosion, leading to joint deformities and loss of function. Statistics show that the global prevalence of RA is approximately 1%, and about 44% of patients lose their ability to work due to joint destruction within ten years of onset (Sokka, T., et al . Work disability in rheumatoid arthritis 10 years after the diagnosis. J. Rheumatol. 26, 1681–1685 (1999); Js, S. et al Rheumatoid arthritis. Lancet (Lond. Engl.) 388, 2023–2038 (2016) ). Studies have shown that early diagnosis and intervention can significantly delay disease progression, making the search for highly sensitive and specific biomarkers crucial. Currently, anti-cyclic citrullinated peptide antibody (CCP) and rheumatoid factor (RF) are commonly used biomarkers for the clinical diagnosis of RA; however, their diagnostic efficacy remains limited. The sensitivity and specificity of anti-CCP antibodies are 67% and 95%, respectively, while those of RF are 69% and 85% (Nishimura, K). et al. Meta-analysis: diagnostic accuracy of anti-cyclic citrμLlinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann. Intern. Med. 146, 797–808 (2007)). More than one-third of patients are anti-CCP antibody negative, known as seronegative RA, which leads to disability due to delayed treatment caused by diagnostic difficulties. Therefore, the need for potential diagnostic biomarkers for RA remains unmet, especially for anti-CCP antibody negative RA patients, where specific biomarkers are still lacking, limiting early diagnosis and treatment in clinical practice.

[0003] Intelectin-1 is a calcium-dependent lectin initially discovered in intestinal cells. It specifically recognizes glycans on the surface of microorganisms and participates in the innate immune response of the intestinal defense mechanism (Nonnecke, EB). et al. Human intelectin-1 (ITLN1) genetic variation and intestinal expression. Sci. Rep. 11, 12889 (2021) ). It is also expressed in visceral adipose tissue and acts as an adipokine to regulate glucose uptake and insulin signaling pathways, enhancing insulin-stimulated glucose uptake in human adipocytes (Czechowski, P.). et al. Expression of intelectin-1, also known as omentin-1, is related to clinical phenotypes such as overweight, obesity, insμLinresistance, and changes after bariatric surgery. Sci. Rep. 14, 22286 (2024). Previous studies have shown that Intelectin-1 is associated with the development and progression of Crohn's disease, obesity, asthma, and various cancers. Our team previously discovered differential expression of Intelectin-1 in the serum of RA patients (especially anti-CCP antibody-negative RA patients) through serum full-protein proteomic analysis (Wu Y, et al Increased expression of omentin-1 is associated with synovitis and bone destruction in rheumatoid arthritis. ClinExp Rheumatol. 2024 JμL;42(7):1442-1450.), suggesting its potential as a diagnostic marker. Summary of the Invention

[0004] The purpose of this invention is to address the current difficulties in diagnosing RA, especially for patients with anti-CCP antibody-negative / RF-negative RA, by providing the application of citrullinated endoglucosin-1 peptide in the preparation of diagnostic products for rheumatoid arthritis, which can improve the sensitivity of RA detection, especially for serum-negative RA.

[0005] The technical solution of this invention is described in detail below:

[0006] In a first aspect, the present invention provides the application of citrullinated lectin-1 polypeptide (CIP) in the preparation of diagnostic products for rheumatoid arthritis, wherein the amino acid sequence of citrullinated lectin-1 polypeptide is: GD-Cit-WSSQQGSKAVYPE (SEQ ID NO:1); the product is used to detect the content of anti-citrullinated lectin-1 polypeptide antibody (anti-CIP) in patient biological samples.

[0007] Optionally or preferably, the N-terminus of the citrullinated lectin-1 polypeptide is also coupled with the carrier protein BSA via a cysteine ​​residue, and the amino acid sequence after coupling is as follows: BSA-C-GD-Cit-WSSQQGSKAVYPE (SEQ ID NO:2).

[0008] Compared with normal healthy individuals, patients with Sjögren's syndrome, patients with systemic lupus erythematosus, and patients with osteoarthritis, the levels of anti-citrullinated endoglucosin-1 polypeptide antibodies were significantly increased in biological samples from patients with rheumatoid arthritis.

[0009] The rheumatoid arthritis patients include those with normal ESR and / or CRP, and those with negative serum anti-CCP antibodies and / or negative serum RF.

[0010] The biological sample is serum or synovial fluid.

[0011] Optionally or preferably, the diagnostic product is an ELISA test kit.

[0012] Secondly, the present invention provides a diagnostic kit for rheumatoid arthritis, comprising: citrullinated lectin-1 polypeptide, an ELISA plate, ELISA coating solution, washing solution, blocking solution, sample dilution solution, enzyme-labeled secondary antibody, chromogenic substrate, and stop solution; the amino acid sequence of the citrullinated lectin-1 polypeptide is GD-Cit-WSSQQGSKAVYPE (SEQ ID NO:1) or BSA-C-GD-Cit-WSSQQGSKAVYPE (SEQ ID NO:2).

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention has discovered that citrullinated lectin-1 peptide can be used as a detection tool to detect anti-citrullinated lectin-1 peptide antibodies in patient biological samples. The antibody levels in RA patients are significantly different from those in normal healthy individuals and other common rheumatic immune diseases that are easily confused with rheumatoid arthritis.

[0015] The diagnostic sensitivity of serum anti-CIP in RA patients was 42.50%, the specificity was 92.75%, the positive predictive value was 85.86%, and the negative predictive value was 60.88%.

[0016] In RA patients with normal ESR or CRP, the positive rate of serum anti-CIP reached 32.67%; in RA patients with normal ESR and CRP, the positive rate reached 24.59%.

[0017] Serum anti-CIP showed a sensitivity of 40.00% in anti-CCP antibody-negative RA patients; 41.76% in RF-negative RA patients; and maintained a positive rate of 38.98% in double-negative (both anti-CCP antibody and RF negative) RA patients.

[0018] Therefore, CIP can be prepared into diagnostic products to detect the anti-CIP content in patient serum or arthritis samples, which has good supplementary diagnostic value for RA diagnosis. Attached Figure Description

[0019] Figure 1 This example presents the experimental results of large-sample detection of anti-CIP levels in different groups of individuals. A represents the anti-CIP antibody levels in the serum of patients with RA, SS, OA, SLE, and HC in a large-sample detection cohort. B represents the ROC curve and area under the curve analysis results for RA with negative or positive anti-CCP antibodies. RA: Rheumatoid Arthritis; SS: Sjögren's Syndrome; OA: Osteoarthritis; SLE: Systemic Lupus Erythematosus; HC: Healthy Control; anti-CIP: Anti-citrullinated lectin-1 polypeptide antibody; anti-CCP: Anti-cyclic citrullinated polypeptide antibody; AUC: Area under the curve; Std. error: Standard error; CI: Confidence interval. p<0.001, the Kruskal-Wallis H test was used to analyze the differences among multiple groups, and the Dunnet t test was used for multiple comparisons.

[0020] Figure 2 The above examples show the experimental results of detecting anti-CIP levels in the synovial fluid of RA and OA using the ELISA method. RA: rheumatoid arthritis; OA: osteoarthritis; anti-CIP: anti-citrullinated lectin-1 polypeptide antibody. p<0.01; Mann-Whitney U test.

[0021] Figure 3The above examples illustrate the experimental results of detecting anti-CIP levels in the serum of RA patients with normal ESR and CRP using an ELISA method. 28 RA patients had elevated ESR and CRP, 70 had normal ESR or CRP, and 57 had normal ESR and CRP. RA: Rheumatoid Arthritis; ESR: Erythrocyte Sedimentation Rate; CRP: C-Reactive Protein; HC: Healthy Controls; anti-CIP: Anti-citrullinated lectin-1 polypeptide antibody. p<0.001; Kruskal-Wallis H test was used to analyze differences among multiple groups, and Dunnet t test was used for multiple comparisons.

[0022] Figure 4 The above examples show the experimental results of detecting anti-CIP levels in serum of anti-CCP antibody / RF-negative RA patients using an ELISA method. RA: rheumatoid arthritis; erythrocyte sedimentation rate; CRP: C-reactive protein; anti-CCP: anti-cyclic citrullinated peptide antibody; RF: rheumatoid factor; anti-CIP: anti-citrullinated lectin-1 peptide antibody. p<0.001; Kruskal-Wallis H test was used to analyze differences among multiple groups, and Dunnet t test was used for multiple comparisons. Detailed Implementation

[0023] 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.

[0024] Example 1

[0025] I. Synthesis of Citrullinated lectin-1 polypeptide

[0026] Intelectin-1 polypeptide was synthesized using a solid-phase peptide synthesis method. The Intelectin-1 polypeptide was synthesized at sites 97-112, citrullinated, and a cysteine ​​residue was added to the N-terminus. The polypeptide was then coupled with the carrier protein BSA to increase immunogenicity. The sequence is BSA-C-GD-Cit-WSSQQGSKAVYPE (SEQ ID NO:2, abbreviated as CIP).

[0027] II. Verifying the specificity of CIP peptides as a detection tool

[0028] 1. Specific experimental steps

[0029] ELISA detection technology:

[0030] (1) Dilute the CIP peptide to 10 μg / ml with ELISA coating buffer, add 100 μL to each well of a microplate (i.e., microplate), seal the plate with sealing film, and store at 4°C overnight.

[0031] (2) Aspirate the components from the microplate and rinse with 300 μL of washing solution, repeating the rinsing 3 times;

[0032] (3) Add 200 μL of PBST containing 3% BSA (i.e., blocking solution) to each microwell and incubate at room temperature for 2 hours;

[0033] (4) Add the standard and diluted sample (both diluted with sample diluent) into the microwells, 100 μL per well, and incubate at room temperature for 1 hour;

[0034] (5) Aspirate the components from the microplate and rinse with 300 μL of washing solution, repeating the rinsing 3 times;

[0035] (6) Add 100 μL of enzyme conjugate (i.e., enzyme-labeled secondary antibody) to each microwell and incubate at room temperature for 30 minutes;

[0036] (7) Aspirate the components from the microplate and rinse with 300 μL of washing solution, repeating the rinsing 3 times;

[0037] (8) Add 100 μL of TMB substrate (i.e. chromogenic substrate) solution to each microwell and incubate at room temperature for 10 minutes;

[0038] (9) Add 100 μL of stop solution to each microwell and incubate at room temperature for 10 minutes;

[0039] (10) Use a microplate reader to read the optical density value at 450 nm and calculate the result. If using dual-wavelength assay, the reference wavelength is 570 nm. The resulting color should remain stable for at least 30 minutes, during which time the optical density value should be read.

[0040] (11) The serum of several RA patients with high OD values ​​was selected and mixed in the same volume to form the standard positive serum.

[0041] (12) Antibody levels are expressed as AU values ​​and are calculated using the following formula: AU = [(OD peptide - OD nonspecific background) test sample / (OD peptide - OD nonspecific background) positive serum] × 100.

[0042] 2. Clinical diagnostic value analysis

[0043] To investigate the clinical diagnostic value of anti-citrullinated lectin-1 polypeptide antibody (anti-CIP) in RA, we validated its efficacy using the aforementioned ELISA technique with CIP as the coating antibody in the serum of a large cohort. This cohort included 200 patients with RA, 45 with SS (Sjögren's syndrome), 45 with OA (osteoarthritis), 45 with SLE (systemic lupus erythematosus), and 59 healthy controls (HC), among whom 60 were anti-CCP antibody-negative RA patients.

[0044] The results are as follows Figure 1 As shown, the serum anti-CIP level in RA patients was significantly higher than that in healthy controls and other patients with rheumatic immune diseases, especially in RA patients who were negative for anti-CCP antibodies.

[0045] To further evaluate the efficacy of anti-CIP in the diagnosis of RA, we set the mean of the anti-CIP AU value plus two standard deviations as the cut-off value (AU value = 46.16) in the HC group, and calculated the sensitivity, specificity, positive predictive value and negative predictive value of anti-CIP in the cohort serum. The results are shown in Table 1 below.

[0046] Table 1. Sensitivity and specificity of anti-CIP in the cohort.

[0047]

[0048] The results showed that serum anti-CIP had a diagnostic sensitivity of 42.50%, a specificity of 92.75%, a positive predictive value of 85.86%, and a negative predictive value of 60.88% in RA patients (Table 1).

[0049] In addition, we measured the expression level of anti-CIP in the synovial fluid of 50 RA patients and 30 OA patients, such as Figure 2 As shown, the level of anti-CIP antibodies in the synovial fluid of RA patients was significantly higher than that of OA patients.

[0050] Table 2. Sensitivity and specificity of anti-CIP in synovial fluid cohorts

[0051]

[0052] III. Supplementary Diagnostic Value of Anti-CIP in RAP with Normal ESR / CRP

[0053] ESR (erythrocyte sedimentation rate) and CRP (C-reactive protein) are two inflammatory markers used in the ACR / EULAR 2010 classification criteria for the diagnosis of RA patients and the assessment of disease activity.

[0054] We examined the distribution of anti-CIP in the serum of RA patients under elevated ESR and CRP levels and under normal conditions. The results are as follows: Figure 3 As shown in Table 3.

[0055] In RA patients with elevated ESR and CRP, the positive rate of anti-CIP was 66.67% (26 / 39). In RA patients with normal ESR or CRP, the positive rate reached 32.67% (33 / 101), but in RA patients with both normal ESR and CRP, the positive rate was 24.59% (15 / 61). This suggests that anti-CIP has potential value in assessing RA disease activity.

[0056] Table 3. Supplemental diagnostic value of anti-CIP in RA with normal ESR / CRP.

[0057]

[0058] IV. Supplemental Diagnostic Value of Anti-CIP in Serone-Negative Rheumatoid Arthritis

[0059] Currently, the main diagnostic markers for RA are anti-CCP antibodies and RF, but some patients are still negative for both. Therefore, we further analyzed the expression level of anti-CIP in the serum of RA patients who were negative for both anti-CCP antibodies and RF. The results are as follows: Figure 4 .

[0060] We also analyzed the sensitivity and specificity of anti-CIP in diagnosing anti-CCP antibody / RF-negative RA, and the results are shown in Table 4.

[0061] Table 4. Diagnostic value of anti-CIP in seronegative rheumatoid arthritis

[0062]

[0063] The results showed that the anti-CIP antibody sensitivity reached 40.00% (24 / 60) in anti-CCP antibody-negative RA patients; 41.76% (38 / 91) in RF-negative RA patients; and maintained a positive rate of 38.98% (23 / 59) in double-negative (both anti-CCP antibody and RF negative) RA patients.

[0064] 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. The application of citrullinated lectin-1 polypeptide in the preparation of diagnostic products for rheumatoid arthritis, characterized in that, The amino acid sequence of the citrullinated lectin-1 polypeptide is: GD-Cit-WSSQQGSKAVYPE; the product is used to detect the content of anti-citrullinated lectin-1 polypeptide antibody in patient biological samples.

2. The application according to claim 1, characterized in that, The N-terminus of the citrullinated lectin-1 polypeptide is also coupled to the carrier protein BSA via a cysteine ​​residue. The amino acid sequence after coupling is as follows: BSA-C-GD-Cit-WSSQQGSKAVYPE.

3. The application according to claim 1 or 2, characterized in that, Compared with normal healthy individuals, patients with Sjögren's syndrome, patients with systemic lupus erythematosus, and patients with osteoarthritis, the levels of anti-citrullinated endoglucosin-1 polypeptide antibodies were significantly increased in biological samples from patients with rheumatoid arthritis.

4. The application according to claim 3, characterized in that, The rheumatoid arthritis patients include those with normal ESR and / or CRP, and those with negative serum anti-CCP antibodies and / or negative serum RF.

5. The application according to claim 1 or 2, characterized in that, The biological sample is serum or synovial fluid.

6. The application according to claim 1 or 2, characterized in that, The diagnostic product is an ELISA test kit.

7. A diagnostic kit for rheumatoid arthritis, characterized in that, include: Citrullinated lectin-1 peptide, ELISA plate, ELISA coating solution, washing solution, blocking solution, sample dilution solution, enzyme-labeled secondary antibody, chromogenic substrate and stop solution; the amino acid sequence of citrullinated lectin-1 peptide is GD-Cit-WSSQQGSKAVYPE or BSA-C-GD-Cit-WSSQQGSKAVYPE.

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