Marker combination and its application in diagnosis and risk assessment of fever of unknown origin and pediatric still's disease

Abstract

The present application relates to the field of biotechnology, in particular to a marker combination and its application in fever of unknown origin and diagnosis and risk assessment of Still's disease in children. The present application provides a biomarker combination for sJIA diagnosis and risk assessment and a combined detection kit based on liquid chip technology, which realizes high-throughput, high sensitivity, high specificity and high accuracy of multi-index combined detection by combining multiple sJIA related cytokines with liquid chip high-throughput detection technology, and provides a new technical means and detection tool for early diagnosis, disease assessment, macrophage activation syndrome risk warning and individualized treatment of sJIA.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to biomarker combinations and their application in the diagnosis and risk assessment of fever of unknown origin and Still's disease in children. Background Technology

[0002] Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in childhood. Systemic juvenile idiopathic arthritis (sJIA) is one of the most severe subtypes of JIA, also known as childhood Still's disease (hereinafter referred to as sJIA), accounting for approximately 10%-20% of JIA cases. Typical clinical manifestations of sJIA include prolonged irregular fever, transient rash, arthritis, hepatosplenomegaly and lymphadenopathy, serositis, and other systemic inflammatory responses. Its pathogenesis involves abnormal activation of the innate immune system and is closely related to the overexpression of various pro-inflammatory cytokines.

[0003] sJIA is one of the more common febrile illnesses in children, often facing diagnostic challenges and inaccurate assessment of disease activity. Because the clinical manifestations of sJIA largely overlap with infectious diseases and other autoimmune diseases, a large number of differential diagnoses are often ruled out before a definitive diagnosis can be made, leading to diagnostic delays and missed opportunities for optimal treatment. Furthermore, children with sJIA are at high risk of developing macrophage activation syndrome (MAS), a life-threatening complication whose early identification and intervention are crucial for improving patient prognosis. However, MAS and sepsis share highly overlapping clinical manifestations, such as high fever, cytopenia (especially thrombocytopenia), hepatosplenomegaly, coagulation abnormalities (e.g., decreased fibrinogen, increased D-dimer), significantly elevated ferritin, and multiple organ dysfunction. This high degree of overlap makes differential diagnosis extremely difficult. Therefore, developing biomarker detection methods for early and accurate diagnosis of sJIA, assessment of disease activity, differential diagnosis from febrile diseases such as sepsis, and detection of MAS risk is of significant clinical importance.

[0004] Currently, clinical methods for detecting cytokines mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and electrochemiluminescence immunoassay. ELISA is cumbersome to perform, has low throughput, can only detect a single indicator at a time, and has limited sensitivity, making it difficult to meet the clinical needs for combined detection of multiple indicators. While chemiluminescence immunoassay has high sensitivity, the equipment is expensive, resulting in high detection costs, and it also suffers from limited throughput per test. Traditional methods require multiple separate tests when detecting multiple cytokines, increasing sample volume, prolonging detection time, and raising costs, making it difficult to achieve simultaneous and rapid detection of multiple indicators.

[0005] Liquid-chip technology (also known as flow cytometry or suspension array technology) is a novel high-throughput, multi-indicator detection platform. This technology uses microspheres with different fluorescent codes as reaction carriers, with specific antibodies coupled to the surface of each type of microsphere. Simultaneous detection of multiple indicators is achieved through flow cytometry. Compared to traditional detection methods, liquid-chip technology offers significant advantages such as high throughput, low sample volume, high sensitivity, good specificity, wide dynamic range, ease of operation, and short detection time. It can simultaneously quantify multiple cytokines in a single test, greatly improving detection efficiency and reducing costs, making it particularly suitable for clinical applications requiring multi-indicator combined diagnosis.

[0006] However, there is currently a lack of multi-cytokine combination assay kits specifically designed for the diagnosis and risk assessment of sJIA. Existing cytokine assay kits are mostly general-purpose products, not optimized for the characteristics of sJIA, and have shortcomings in areas such as the selection of biomarker combinations, setting of detection sensitivity, and establishment of clinical interpretation criteria, making it difficult to meet the clinical needs for accurate diagnosis and personalized treatment of sJIA. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a combination of biomarkers and their application in the diagnosis and risk assessment of fever of unknown origin and Still's disease in children. The present invention provides a combination of biomarkers that can achieve high-throughput, high-sensitivity and high-specificity joint detection of sJIA-related cytokines, providing reliable laboratory evidence for the early diagnosis of sJIA, monitoring of disease activity and risk assessment of MAS.

[0008] The present invention provides a combination of markers including at least two of IL-6, IL-10, IL-18, IFN-γ, IL-1β, IFN-α and IL-1α.

[0009] In some embodiments, IL-6, IL-10, IL-18, IFN-γ, and IL-1β are included.

[0010] This invention provides the application of the aforementioned biomarker combination in the preparation of products for diagnosing systemic juvenile idiopathic arthritis.

[0011] This invention provides the application of the aforementioned biomarker combination in the preparation of products for differentiating between systemic juvenile idiopathic arthritis and sepsis.

[0012] This invention provides the application of the aforementioned biomarker combination in the preparation of a product for detecting whether systemic juvenile idiopathic arthritis is complicated by macrophage activation syndrome.

[0013] This invention provides a product for diagnosing systemic juvenile idiopathic arthritis, including a reagent for detecting the expression levels of the said biomarker combination.

[0014] This invention provides a product for differentiating between systemic juvenile idiopathic arthritis and sepsis, including a reagent for detecting the expression levels of the said biomarker combination.

[0015] This invention provides a product for identifying whether systemic juvenile idiopathic arthritis is complicated by macrophage activation syndrome, including a reagent for detecting the expression level of the said biomarker combination.

[0016] In some embodiments, the product is selected from at least one of reagents, kits, chips, hybridization probes, and sequencing libraries.

[0017] In some embodiments, the product is a combined detection kit based on liquid chip technology, the kit comprising a solid-phase carrier and a capture antibody coated on the surface of the solid-phase carrier, the capture antibody being a specific antibody against the combination of biomarkers.

[0018] In some embodiments, the solid support is a microsphere containing a variety of different fluorescent codes.

[0019] In some embodiments, it further includes at least one of the following: antibody mixture, streptavidin-labeled phycoerythrin, standards, quality control products, sample diluent, and washing solution;

[0020] The antibody mixture contains a biotin-labeled detection antibody, which is a specific antibody against the combination of markers.

[0021] The standard product includes the aforementioned combination of markers.

[0022] In some embodiments, the microspheres are magnetic microspheres with a diameter of 5.0-6.0 μm, and are encoded using two fluorescent dyes, which can form up to 100 different coding combinations.

[0023] In some embodiments, the capturing antibody is covalently coupled to the carboxyl groups on the surface of the microspheres via carbodiimide (EDC)-mediated amide bonds.

[0024] In some embodiments, the detection sensitivities of the kit are as follows: the detection limit for IL-18 is 2.5 pg / mL, the detection limit for IL-6 is 2.5 pg / mL, the detection limit for IL-10 is 2.5 pg / mL, the detection limit for IFN-γ is 2.5 pg / mL, the detection limit for IL-1β is 2.5 pg / mL, the detection limit for IFN-α is 2.5 pg / mL, and the detection limit for IL-1α is 2.5 pg / mL.

[0025] In some embodiments, the detection dynamic range of the kit is 2.5-2000 pg / mL.

[0026] In some embodiments, the intra-batch precision CV of the kit is ≤8%, and the inter-batch precision CV is ≤15%.

[0027] This invention provides the use of the described biomarker combination and / or the described product in the preparation of a diagnostic product for assessing systemic activity in juvenile idiopathic arthritis.

[0028] This invention provides the use of the described biomarker combination and / or the described product in the preparation of a detection product for assessing the risk of systemic juvenile idiopathic arthritis complicated with macrophage activation syndrome.

[0029] This invention provides the application of the described biomarker combination and / or the described product in the preparation of a diagnostic product for the differential diagnosis of systemic juvenile idiopathic arthritis and other febrile diseases.

[0030] This invention provides the application of the aforementioned biomarker combination and / or the aforementioned product in the preparation of a diagnostic product for guiding the selection of treatment regimens and monitoring of efficacy in patients with systemic juvenile idiopathic arthritis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) High-throughput multi-index joint detection: Based on liquid chip technology, this invention can simultaneously quantify multiple sJIA-related cytokines in a single detection, overcoming the limitation of traditional ELISA and other methods that can only detect a single index in a single detection, greatly improving detection efficiency, reducing sample volume, and lowering detection costs.

[0033] (2) High sensitivity and wide dynamic range: The kit of this invention adopts the flow cytometry fluorescence detection principle. The detection limit of each index can reach 2.5 pg / mL, and the dynamic range is 3-4 orders of magnitude. It can accurately detect the changes in cytokine concentration under physiological and pathological conditions, and meet the needs of clinical diagnosis and efficacy monitoring.

[0034] (3) High specificity and accuracy: The present invention adopts the double antibody sandwich method principle of paired monoclonal antibodies, combined with the spatial separation of different fluorescently encoded microspheres, which effectively avoids cross-reaction and ensures high specificity and accuracy of each indicator detection.

[0035] (4) Optimization of biomarker combination: The combination of cytokines such as IL-18, IL-6, IL-10, IFN-γ, and IL-1β screened in this invention covers the key inflammatory pathways in the pathogenesis of sJIA. It includes both pro-inflammatory and anti-inflammatory factors, which can comprehensively reflect the patient's immune activation status and provide multi-dimensional information for the diagnosis, disease assessment and prognosis of sJIA.

[0036] (5) MAS risk warning function: This invention specifically incorporates MAS-related biomarkers such as IL-18 and IFN-γ, which can be used to identify sJIA children at risk of developing MAS in the early stage, providing early warning information for timely clinical intervention.

[0037] (6) Simple operation and fast detection: The detection process of the kit of the present invention is simple. It only takes 38 minutes from sample addition to obtaining results. Compared with the traditional ELISA method, the detection time is greatly shortened, which is convenient for clinical application.

[0038] (7) High clinical application value: This invention can be applied to multiple clinical scenarios such as auxiliary diagnosis of sJIA, disease activity assessment, MAS risk assessment, differential diagnosis, treatment plan selection and efficacy monitoring, and has important clinical application value.

[0039] In summary, this invention provides a combination of biomarkers for the diagnosis and risk assessment of sJIA and a combined detection kit based on liquid chip technology. By combining multiple sJIA-related cytokines with high-throughput detection technology using liquid chips, it achieves high throughput, high sensitivity, high specificity, and high accuracy in the combined detection of multiple indicators, providing new technical means and detection tools for the early diagnosis, disease assessment, MAS risk warning, and personalized treatment of sJIA. Attached Figure Description

[0040] Fig. 1 ROC curve of combined biomarker diagnosis of sJIA disease;

[0041] Fig. 2 ROC curves for the differential diagnosis of sepsis using a combination of biomarkers;

[0042] Fig. 3 ROC curves for diagnosing MAS using a combination of biomarkers. Detailed Implementation

[0043] This invention provides a combination of biomarkers and their application in the diagnosis and risk assessment of fever of unknown origin and Still's disease in children. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0044] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0045] Example 1

[0046] Based on the hospital's diagnosis, this example selected children aged ≤16 years with similar age and sex ratios. Ultimately, 18 patients were selected for the sJIA group (including 4 patients with MAS), 41 patients for the sepsis group, and 25 patients for the control group (HC). Specific information is shown in Table 1 below. Blood samples from these individuals were properly collected according to known procedures.

[0047] Table 1

[0048]

[0049] This invention detects the concentrations of various biomarkers in the blood samples of subjects, including: interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-18 (IL-18), interferon-gamma (IFN-γ), interleukin-1β (IL-1β), interferon-alpha (IFN-α), and interleukin-1α (IL-1α).

[0050] The concentrations of the aforementioned biomarkers in the measured samples are substituted into one of the algorithm models, such as logistic regression or support vector machine (SVM), to calculate the probability of sJIA and MAS complications.

[0051] For Logistic regression, the regression equation is as follows:

[0052]

[0053] After calculating Logit(P) using the regression equation, substitute it into the following formula to calculate the P-value:

[0054]

[0055] After obtaining the P-value, ROC curve analysis is performed based on the P-value to obtain the Cut-off value. The Cut-off value can be used to determine the occurrence of sJIA and MAS complications in the sample.

[0056] This embodiment employs liquid chip technology to detect the concentration of the aforementioned biomarkers. The samples are added to the components of each kit, and the instrument automatically adds, incubates, washes, and analyzes the samples using flow cytometry to read the concentration results of each biomarker. The concentration results are then subjected to natural logarithmic transformation and logistic regression analysis (using R programming or SPSS software) to obtain the regression equation. ROC analysis is then performed to obtain the corresponding AUC value, as well as specificity and sensitivity values. This kit, based on flow cytometry and a double-antibody sandwich immunoreaction principle, enables the simultaneous quantitative detection of seven cytokines: human IFN-γ, IFN-α, IL-10, IL-18, IL-1α, IL-1β, and IL-6. Specifically, seven magnetic polystyrene microspheres with different fluorescent codes are cross-linked with monoclonal capture antibodies corresponding to the cytokines. During detection, the capture antibody on the microspheres specifically binds to the corresponding antigen in the serum. Subsequently, biotin-labeled paired detection antibody and streptavidin-labeled phycoerythrin (SA-PE) are added sequentially, forming the assay complex [microsphere-capture antibody-antigen-detection antibody-biotin-streptavidin-PE], which generates a specific fluorescent signal. Quantitative signal analysis uses median fluorescence intensity (MFI), calculated by collecting the fluorescence signals of 50-200 effective microspheres of each coded microsphere at 488 nm excitation and 575 nm emission wavelengths using a flow cytometer and taking the median value. This value is positively correlated with the target concentration. The detector distinguishes different targets by recognizing the fluorescent coding of the microspheres and achieves quantitative analysis using a dose-response standard curve established with calibrators. The standard curve is fitted using cubic splines, and the concentration of each cytokine in the test sample is calculated based on the fitted curve equation.

[0057] The specific components of this kit are as follows:

[0058] (1) Encoded microsphere mixture: Composed of seven different fluorescently encoded microspheres, each of which is made of magnetic polystyrene and has its surface activated by carboxyl groups, and then covalently cross-linked with monoclonal capture antibodies against IFN-γ, IFN-α, IL-10, IL-18, IL-1α, IL-1β, and IL-6 respectively; the seven encoded microspheres are mixed in a preset optimal ratio, and the final concentration of each microsphere is 1~5×10 5 The cells / mL were suspended in 20mM PBS buffer containing 1% BSA, 0.05% Tween-20, and pH 7.4.

[0059] (2) Antibody mixture: contains biotinylated antibodies against the above seven cytokines, with each antibody having a working concentration of 0.5~4 μg / mL, and is mixed and dissolved in 20 mM PBS buffer containing 1% BSA, 0.05% Tween-20, and pH 7.4.

[0060] (3) Fluorescent reporter reagent: SA-PE solution (phycoerythrin labeled with streptavidin), with a working concentration of 10~50 μg / mL, dissolved in 10 mM Tris-HCl buffer containing 1% BSA, 0.05% Tween-20, pH 7.4.

[0061] (4) Calibrators: a composite calibrator of seven cytokines, with six gradient concentrations of 0, 7.81, 31.25, 125, 500, and 2000 pg / mL.

[0062] (5) Quality control products: a composite quality control product for seven cytokines, with three concentration levels: high, medium and low, with concentrations of 10, 100 and 1000 pg / mL, respectively.

[0063] The core performance indicators of this kit are as follows: Detection sensitivity (limit of detection) is 2.5 pg / mL, calculated using the industry-standard method of combining the limit of blank (LoB) and limit of detection (LoD) (LoB = blank sample concentration + 1.645 × standard deviation of blank sample concentration, LoD = LoB + 1.645 × standard deviation of low concentration sample concentration); The linear range is 2.5~2000 pg / mL, covering the concentration range of target cytokines in common clinical samples. Verified by serial dilution of calibrators, the correlation coefficient R² of the fitted curve is ≥0.990, and the relative deviation at each concentration point is ≤±15%; Regarding precision, the intra-assay precision coefficient of variation (CV) is ≤8% (10-well replicates of each of the same batch of low, medium, and high quality control samples), and the inter-assay precision coefficient of variation (CV) is ≤15% (10-well replicates of each of three different batches of low, medium, and high quality control samples).

[0064] Sensitivity (also known as true positive rate, sensitivity, or accuracy) refers to the proportion of samples that are actually positive that are correctly identified as positive (the ability to correctly identify cases that are actually infected, i.e., the probability of a patient being diagnosed as positive). Higher sensitivity results in a lower false negative rate. Specificity (also known as true negative rate) refers to the proportion of samples that are actually negative that are correctly identified as negative (the ability to correctly identify cases that are actually not infected, i.e., the proportion of test results that are negative). Higher specificity results in a lower false positive rate.

[0065] The receiver operating characteristic (ROC) curve is a comprehensive indicator reflecting the sensitivity and specificity of continuous variables, used for binary classification. The area under the ROC curve (AUC) represents the accuracy of prediction; a higher AUC value corresponds to a larger area under the curve, indicating higher prediction accuracy.

[0066] 1. Seven cytokines were detected, including IL-6, IL-10, IL-18, IFN-γ, IL-1β, IFN-α, and IL-1α. Univariate logistic regression analysis of the seven cytokines yielded the results shown in Table 2 below.

[0067] Table 2

[0068]

[0069] Note: P<0.05

[0070] 2. The diagnostic specificity and sensitivity of the above biomarkers in the comparison between the sJIA group and the healthy control group (HC) were investigated. The results are shown in Table 3.

[0071] Table 3

[0072]

[0073] 3. The diagnostic specificity and sensitivity of the above biomarkers in the comparison between the sJIA group and the sepsis group were investigated, and the results are shown in Table 4.

[0074] Table 4

[0075]

[0076] 4. The diagnostic specificity and sensitivity of the above biomarkers in the comparison between the sJIA group with MAS and the group without MAS were investigated. The results are shown in Table 5.

[0077] Table 5

[0078]

[0079] The above results indicate that there were no statistically significant differences (P>0.05) between IL-1α and IFN-α in the sJIA group and the healthy control group, the sJIA group and the sepsis group, and the sJIA group with MAS and the group without MAS, thus lacking independent diagnostic value. The P-value for IFN-α was 0.804, and the P-value for IL-1α in the univariate logistic regression analysis was 0.244, both significantly greater than the 0.05 significance level. Therefore, IFN-α and IL-1α were not included in the multivariate regression model and were excluded from the final combination.

[0080] Example 2

[0081] Using the five cytokines selected in Example 1, in addition to the routine detection of single cytokine values, cytokine ratios were also introduced as derived variables. By performing a full combinatorial analysis of the five core factors—covering all linear and nonlinear combinations from single to five factors—and adhering to the principle of non-redundancy of single variables (e.g., IFN-γ combined with the IL-1β / IL-10 ratio), the area under the ROC curve (AUC), sensitivity, and specificity of each combination were systematically evaluated, thereby identifying the diagnostic model with the highest clinical diagnostic value. The results of combined diagnostic experiments with different factor combinations are shown in Table 6 below.

[0082] Table 6

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] The results showed that although existing literature reported the association between these factors and sJIA, the AUC values ​​of individual factors varied considerably when used for diagnosis. Some factors (such as IFN-γ alone for sJIA vs HC) had an AUC of only 0.2889, indicating almost no diagnostic value. While IL-1β showed no significant specificity or sensitivity in single-factor diagnosis, its diagnostic efficacy in combination modalities was significantly higher in differentiating sJIA from sepsis. The ROC curves for the combined diagnosis of sJIA, sepsis, and MAS using the five cytokine combinations are shown below. Figs. 1-3 As shown.

[0089] The concentrations (pg / mL) of five cytokines—IL-18, IL-6, IL-10, IFN-γ, and IL-1β—were quantitatively detected using flow cytometry. A joint diagnostic model was constructed using binary logistic regression analysis. The optimal predictive equation (Logit P) for distinguishing the two disease groups was obtained through iterative calculation using maximum likelihood estimation (MLE) as follows:

[0090] Logit(P) = 0.787 + 0.329 × C IL-18 -2.867×C IFN-γ -1.143×C IL-1β +0.097×C IL-10+0.004×C IL-6

[0091] Where: C represents the serum detection concentration (pg / mL) of each cytokine; P is the predicted probability of the sample having sJIA. Based on the Youden exponent maximization principle of the ROC curve, the optimal diagnostic cutoff value was determined to be P = 0.412. (That is: if the calculated P > 0.412, the sample result is considered sJIA; if the calculated P ≤ 0.412, the sample result is considered sepsis). Model validation: ROC curve analysis showed that the AUC of this five-factor combined model was as high as 0.997 (95% CI: 0.98-1.00). At the optimal cutoff value, the model's sensitivity in identifying the two groups of diseases was 100%, and its specificity was 97.6%.

[0092] In summary, the combination of five cytokines achieved optimal or near-optimal AUC values ​​in all three diagnostic scenarios. The synergistic detection of the five cytokines covered key inflammatory pathways in the pathogenesis of sJIA, enabling high-throughput, high-sensitivity, and high-specificity joint detection of sJIA-related cytokines. This provides reliable laboratory evidence for the early diagnosis of sJIA, monitoring of disease activity, and risk assessment of MAS.

[0093] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of markers, characterized in that, It includes at least two of IL-6, IL-10, IL-18, IFN-γ, IL-1β, IFN-α and IL-1α.

2. The marker combination according to claim 1, characterized in that, Including IL-6, IL-10, IL-18, IFN-γ and IL-1β.

3. The use of the biomarker combination of claim 1 or 2 in the preparation of a product for diagnosing systemic juvenile idiopathic arthritis.

4. The use of the biomarker combination of claim 1 or 2 in the preparation of products for differentiating between systemic juvenile idiopathic arthritis and sepsis.

5. The use of the biomarker combination of claim 1 or 2 in the preparation of a product for detecting whether systemic juvenile idiopathic arthritis is complicated by macrophage activation syndrome.

6. A product for diagnosing systemic juvenile idiopathic arthritis, characterized in that, Including reagents for detecting the expression levels of the biomarker combination as described in claim 1 or 2.

7. A product for differentiating between systemic juvenile idiopathic arthritis and sepsis, characterized in that, Including reagents for detecting the expression levels of the biomarker combination as described in claim 1 or 2.

8. A product for identifying whether systemic juvenile idiopathic arthritis is complicated by macrophage activation syndrome, characterized in that, Including reagents for detecting the expression levels of the biomarker combination as described in claim 1 or 2.

9. The product according to any one of claims 6 to 8, characterized in that, The product is selected from at least one of reagents, kits, chips, hybridization probes, and sequencing libraries.

10. The product according to claim 9, characterized in that, The product is a combined detection kit based on liquid chip technology. The kit includes a solid support and a capture antibody coated on the surface of the solid support. The capture antibody is a specific antibody against the biomarker combination as described in claim 1 or 2. Includes at least one of the following: antibody mixture, streptavidin-labeled phycoerythrin, standards, quality control products, sample diluent, and washing solution; The antibody mixture contains a biotin-labeled detection antibody, which is a specific antibody against the biomarker combination as described in claim 1 or 2. The standard includes the combination of markers as described in claim 1 or 2.

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