System for evaluating the activity status of vitiligo using serum cx3cl1 as a biomarker

Abstract

This invention belongs to the field of biomedical detection technology, specifically a vitiligo activity status assessment system using serum CX3CL1 as a biomarker. This invention employs ELISA detection technology to assess the activity status of vitiligo; it includes: a sample collection module for collecting serum samples from vitiligo patients; a detection kit for specifically and quantitatively detecting human serum CX3CL1, comprising anti-CX3CL1 antibody, standards, chromogenic substrate, and stop solution; and a data analysis unit for obtaining the vitiligo activity status assessment result based on a comparison of CX3CL1 concentration with a preset threshold. This invention's system is easy to operate, reduces human error, and the test results show high consistency and repeatability, filling the gap in current objective assessment of vitiligo activity status.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a vitiligo activity status assessment system. Background Technology

[0002] Vitiligo is an autoimmune disease characterized by the progressive destruction of epidermal melanocytes, with depigmented patches of varying sizes and numbers as typical skin lesions. Its core pathogenesis involves CD8... + Abnormal activation of cytotoxic T lymphocytes and targeted killing of melanocytes lead to depigmentation of the skin. Global epidemiological survey data show that vitiligo affects about 0.5%-2% of the global population, of which 30%-40% of patients have a rapidly progressive course, often accompanied by recurrent attacks and treatment resistance[1].

[0003] Current clinical assessments of vitiligo activity primarily rely on physicians' subjective visual scores (VASI / VIDA) and experience, making it difficult to capture dynamic changes in the immune microenvironment. The lack of effective disease biomarkers leads to missed early intervention windows and delayed individualized treatment.

[0004] Chemokines, as key factors influencing immune cell migration, are increasingly revealing the role of dysregulation in autoimmune diseases. In the skin microenvironment of vitiligo lesions, IFN-γ stimulates fibroblasts or keratinocytes to secrete CXCL10, recruiting CXCR3. + CD8 + T cells aggregate in the epidermis, forming a positive feedback loop of "inflammatory factors-chemokines-effect cells", which exacerbates disease progression [2]. At the level of immune cells, CXCR3 in the peripheral blood of vitiligo patients + CD8 + The proportion of T cell subsets was significantly higher than that of normal individuals[3]. In addition, the serum CCL20 concentration in patients with active disease was significantly higher than that of baseline, and the proportions of Th1 / 17 and Tc1 / 17 cells were significantly higher than those in patients with stable disease and healthy controls, and were positively correlated with the degree of disease activity[4]. This provides a new dimension for monitoring disease status. The pathogenesis of vitiligo is complex. Although CXCL10 showed good clinical relevance, it still has limitations such as insufficient sensitivity of a single marker, insufficient detection stability, and inability to accurately reflect the immune status of patients. Serum CX3CL1 has the potential to become a biomarker for the activity of inflammatory diseases. The serum CX3CL1 level in patients with psoriasis has been shown to be significantly higher than that in healthy individuals, and its concentration decreased significantly after treatment with anti-TNF-α biologics, suggesting that serum CX3CL1 may serve as a biomarker for assessing the inflammatory burden of the disease and the response to treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid, accurate, and reproducible system for assessing vitiligo activity using serum CX3CL1 as a biomarker.

[0006] This invention reveals a significant correlation between serum CX3CL1 concentration and the activity status of vitiligo. Quantitative detection of serum CX3CL1 levels using ELISA technology allows for an objective and accurate assessment of vitiligo activity. Serum CX3CL1, as a biomarker, exhibits high sensitivity and specificity, effectively distinguishing between active and stable phases of vitiligo, providing crucial information for developing clinical treatment strategies.

[0007] The vitiligo activity status assessment system provided by this invention, using serum CX3CL1 as a biomarker, includes:

[0008] Sample collection module: used to collect serum samples from vitiligo patients;

[0009] Test kit: This is an ELISA kit for the specific quantitative detection of CX3CL1 in human serum, containing anti-CX3CL1 antibody, standards, chromogenic substrate and stop solution;

[0010] Data analysis unit: Based on the comparison of CX3CL1 concentration with a preset threshold, the results of disease activity status assessment are obtained.

[0011] The assessment system is used to distinguish whether a vitiligo patient's disease is in an active or stable phase. Specifically:

[0012] A plasma CX3CL1 protein concentration < 441.2 pg / ml indicates that the vitiligo patient is in a stable phase.

[0013] A plasma CX3CL1 protein concentration ≥ 441.2 pg / ml indicates that the vitiligo patient is in the active phase, and the higher the concentration, the more severe the disease activity.

[0014] Furthermore, the serum CX3CL1 concentration is quantitatively detected using an ELISA method, and the concentration level is used to determine whether the disease is in an active state.

[0015] As shown in Table 1, at the diagnostic threshold, the detection sensitivity of serum CX3CL1 is 60%, and the specificity is 88%. Sensitivity represents the detection rate in patients with active vitiligo, meaning that 60% of patients in the active phase can be correctly identified by detecting elevated serum CX3CL1 concentrations. Specificity represents the exclusion rate in patients without active vitiligo, meaning that 88% of patients without active vitiligo can be correctly excluded by detecting serum CX3CL1 concentrations below the threshold.

[0016] Table 1 shows the diagnostic threshold and efficacy of serum CX3CL1 as a biomarker for the active phase of vitiligo.

[0017] Table 1

[0018] .

[0019] Compared with the prior art, the present invention has the following beneficial effects.

[0020] This invention provides a new objective indicator and detection system for assessing the activity of vitiligo. By detecting the concentration of CX3CL1 in serum, it is possible to accurately and reliably assess whether vitiligo is in an active phase, thus compensating for the current lack of objective and quantitative assessment methods in clinical practice.

[0021] The ELISA detection technology used in this invention is mature, stable, and easy to operate. Detection results can be rapidly obtained using a microplate reader, exhibiting high repeatability and consistency, significantly reducing human error. Validation with multiple batches of samples demonstrates that the system's detection results have high reliability and reproducibility, making it easy to promote and use in clinical practice. Early and accurate assessment of vitiligo activity helps in the timely development and adjustment of treatment plans, improving patient prognosis and reducing the risk of disease progression. Attached Figure Description

[0022] Figure 1 The study investigated the serum CX3CL1 levels in vitiligo patients and the differences between groups.

[0023] Figure 2 Correlation analysis of serum CX3CL1 levels with disease activity and severity in vitiligo patients.

[0024] Figure 3 ROC curve analysis was performed to analyze the serum CX3CL1 level in vitiligo patients as a biomarker for the active phase of vitiligo. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0026] 1. Serum sample collection

[0027] Five mL of venous blood was collected from the elbows of vitiligo patients (50 in the active phase and 25 in the stable phase) and healthy controls (25 in total) into yellow procoagulant tubes. After standing at room temperature for 30 minutes, serum was separated (centrifuged at 3000×g for 10 minutes). The clear serum was aliquoted into cryovials, flash-frozen in liquid nitrogen, and then stored at -80°C for long-term storage.

[0028] 2. Assessment of disease activity and severity

[0029] All enrolled vitiligo patients underwent independent physical examinations, diagnoses, and disease assessments by two dermatologists. Based on the staging criteria for vitiligo, patients were divided into active and stable groups. Simultaneously, the percentage of body surface area affected by vitiligo (BSA%) and the Vitiligo European Task Force-assessment (VETFa) disease activity score were assessed for each patient.

[0030] The specific criteria for the VETFa Disease Activity Score are as follows: The patient's body surface is divided into five anatomical regions (head and neck, upper limbs, lower limbs, hands and feet, and trunk) for independent assessment. The scoring criteria for each region are based on a comparative analysis of the area of ​​vitiligo under Wood's lamp and natural light: when the area of ​​vitiligo under Wood's lamp is greater than the observation value under natural light, it is considered disease progression, and is scored +1 point; if the vitiligo area is equal under both light sources, it is considered stable, and is scored 0 points; when the area of ​​vitiligo under Wood's lamp is smaller than the observation value under natural light, it indicates repigmentation, and is scored -1 point. The final total score is the sum of the scores for each region.

[0031] The assessment criteria for Body Surface Area (BSA%) are shown in Table 2 below:

[0032] Table 2

[0033] .

[0034] 3. ELISA testing

[0035] (1) Reagent and sample pretreatment: Serum samples were transferred from -80℃ ultra-low temperature storage environment to room temperature for equilibration thawing; the test kit was left to stand at room temperature for 30 minutes; if the concentrated reagent showed crystallization, it was placed in a 37℃ water bath until the solution became clear.

[0036] (2) Buffer system preparation: Measure 50 mL of 20× concentrate and add 950 mL of deionized water to make up to 1 L, and mix with magnetic stirring; detection buffer: 5 mL of 10× concentrate and 45 mL of deionized water (store on ice); after vortexing the detection antibody stock solution, immediately dilute it with 1× buffer at a ratio of 1:100.

[0037] (3) Construction of standard curve: Take high concentration standard (1000 pg / mL) and perform serial dilution (final volume 230 μL / tube); use standard dilution as blank control.

[0038] (4) Pretreatment of reaction plate: plate well equilibration: inject 300 μL of 1× washing buffer into each well, let stand at room temperature for 30 seconds, and then aspirate and discard the liquid; invert the reaction plate and tap it three times on filter paper to remove liquid residue.

[0039] (5) Sample addition: Standard well group: double-duplicate wells, 100 μL gradient standard per well; Detection well group: double-duplicate wells, 100 μL serum to be tested; Blank control: 100 μL dilution buffer added to each well.

[0040] (6) Primary antibody incubation: Continuously and rapidly add 50 μL of detection antibody working solution (operation time ≤ 15 minutes); after sealing with sealing film, incubate on a horizontal shaker at 25°C for 120 minutes.

[0041] (7) Secondary antibody labeling: After removing the liquid, add 100 μL of biotinylated secondary antibody and incubate at 37°C for 60 minutes.

[0042] (8) Avidin coupling: After washing the plate three times, add 100 μL of streptavidin-HRP complex and react at 37°C in the dark for 60 minutes.

[0043] (9) Substrate development: Inject 90 μL of TMB developer into each well, develop at room temperature for 15 minutes in the dark with tin foil; add 50 μL of 2M sulfuric acid stop solution.

[0044] (10) Detection: dual-wavelength detection using an ELISA reader (main wavelength 450 nm / reference wavelength 630 nm).

[0045] 4. Result Interpretation

[0046] Stable phase: Plasma CX3CL1 protein concentration < 441.2 pg / ml

[0047] Active phase: Plasma CX3CL1 protein concentration ≥ 441.2 pg / ml, and the degree of disease activity increases with increasing proportion.

[0048] The assessment results objectively quantify the activity level of vitiligo, providing key reference for clinicians to determine the disease stage, predict the risk of progression, develop individualized treatment plans (such as whether to initiate / enhance systemic immunotherapy), and evaluate treatment effectiveness.

[0049] Figure 1 This study presents the serum CX3CL1 levels and inter-group differences in vitiligo patients. The invention assessed the potential of CX3CL1 as a biomarker of disease activity by detecting serum CX3CL1 concentrations in healthy controls (25 cases), patients with stable vitiligo (25 cases), and patients with active vitiligo (50 cases). Results showed that serum CX3CL1 levels in patients with active vitiligo were significantly higher than those in patients with stable vitiligo (P < 0.001) and healthy controls (P < 0.0001). The increase in CX3CL1 levels in patients with stable vitiligo was not significant compared to healthy controls (P = 0.5741), indicating that the dynamic changes in serum CX3CL1 are closely related to the degree of vitiligo activity.

[0050] Figure 2 This study analyzed the correlation between serum CX3CL1 levels and disease activity and severity in vitiligo patients. The correlation between CX3CL1 and BSA (Bioassay Scale) and VETFa (Vitamin E5) disease activity scores was analyzed. Results showed that serum CX3CL1 levels were significantly positively correlated with BSA (R²=0.1932, P < 0.0001), while the correlation with VETFa scores was slightly weaker (R²=0.1440, P =0.0008).

[0051] Figure 3 ROC curve analysis was performed to evaluate the serum CX3CL1 level in vitiligo patients as a biomarker for the active phase of vitiligo. The ROC curve was used to assess the discriminant efficacy of serum CX3CL1 level in vitiligo patients for the active phase of the disease. The gray dashed line is the random reference line (AUC = 0.5).

[0052] References

[0053] [1] EZZEDINE K, ELEFTHERIADOU V, WHITTON M, et al. Vitiligo[J]. TheLancet, 2015, 386(9988): 74–84.

[0054] [2] LIU H, WANG Y, LE Q, et al. The IFN-γ-CXCL9 / CXCL10-CXCR3 axis invitiligo: pathological mechanism and treatment[J]. European Journal ofImmunology, 2024, 54(4): e2250281.

[0055] [3] WANG XX, WANG QQ, WU JQ, et al. Increased expression of CXCR 3and its ligands in patients with vitiligo and CXCL10 as a potential clinicalmarker for vitiligo[J]. British Journal of Dermatology, 2016, 174(6): 1318–1326.

[0056] [4] ZHANG L, KANG Y, CHEN S, et al. Circulating CCL20: A potentialbiomarker for active vitiligo together with the number of Th1 / 17 cells[J].Journal of Dermatological Science, 2019, 93(2): 92–100。

Claims

1. A system for evaluating the activity state of vitiligo using serum CX3CL1 as a biomarker, characterized in that, include: Sample collection module: used to collect serum samples from vitiligo patients; Test kit: This is an ELISA kit for the specific quantitative detection of CX3CL1 in human serum, containing anti-CX3CL1 antibody, standards, chromogenic substrate and stop solution; Data analysis unit: Based on the comparison of CX3CL1 concentration with preset threshold, the results of vitiligo activity status assessment are obtained.

2. The system for assessing the activity state of vitiligo according to claim 1, wherein, The serum CX3CL1 concentration was quantitatively detected using an ELISA method; the vitiligo activity status was divided into active phase or stable phase; specifically: A plasma CX3CL1 protein concentration < 441.2 pg / ml indicates that the vitiligo patient is in a stable phase. A plasma CX3CL1 protein concentration ≥ 441.2 pg / ml indicates that the vitiligo patient is in an active phase; and the higher the concentration, the more severe the disease activity.

3. The system for assessing the activity state of vitiligo according to claim 2, wherein, The diagnostic threshold and efficacy of serum CX3CL1 as a biomarker for active vitiligo are shown in the table below: 。

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