Biomarker for diagnosing sepsis and application thereof

Abstract

The invention provides application of ARG2 as a biomarker in preparation of a sepsis early diagnosis product, and belongs to the field of medical diagnosis. Specifically, according to the technical scheme, the ARG2 level of serum is detected by adopting an ELISA method, an elisa kit is used, qualitative or quantitative analysis is carried out according to the color depth, and whether a patient is infected with sepsis or not can be effectively diagnosed. The serum ARG2 is applied to early diagnosis of sepsis for the first time, and the detection method has the advantages of high sensitivity, high specificity, simplicity, convenience and rapidness in operation and the like, is low in price and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis, and in particular, to a biomarker for diagnosing sepsis and applications thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection, often accompanied by organ dysfunction, and associated with high morbidity and mortality. Its rapid onset, complex course, and high mortality rate are significant. Despite significant advances in anti-infective therapy and intensive care, sepsis remains a leading cause of death worldwide.

[0003] Traditionally, the diagnosis of sepsis relies mainly on clinical manifestations and laboratory indicators, but their sensitivity and specificity are limited. Early diagnosis and timely intervention are key to improving the prognosis of patients with sepsis. Biomarkers can reflect the pathophysiological process of the disease through specific molecular changes in blood, urine or other body fluids. In the field of sepsis, researchers have discovered some potential biomarkers, such as C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), etc., but these markers still have limitations in specificity and sensitivity and cannot fully meet clinical needs. As a result, many patients are not diagnosed until their disease progresses to a serious stage, missing the best time for treatment.

[0004] In recent years, with the in-depth study of the pathogenesis of sepsis, immune metabolic disorders have gradually gained attention as one of its core pathophysiological foundations. In this context, enzymes related to arginine metabolism have gradually become a research hotspot. Among them, arginase 2 (ARG2), a mitochondrial-localized arginine degrading enzyme, plays a key role in regulating immune cell function, oxidative stress response, and tissue damage repair. Studies have shown that ARG2 expression changes dynamically in various inflammatory diseases, and it plays an important role in T cell immune regulation and endothelial dysfunction. However, current research has not yet addressed the role of ARG2 in the development and progression of sepsis. Therefore, research on the relationship between ARG2 and sepsis requires further exploration. Summary of the Invention

[0005] In response to the problem of insufficient specificity and sensitivity in the early diagnosis of sepsis in the prior art, the present invention provides a new early diagnostic biomarker ARG2 and its application to achieve efficient and accurate diagnosis of sepsis.

[0006] Specifically, the technical solution of the present invention includes: using ARG2 as a biomarker for sepsis, and applying it to the preparation of diagnostic products for detecting sepsis, especially ELISA kits, by detecting the ARG2 level in patient plasma, serum or blood samples to achieve the purpose of early diagnosis of sepsis.

[0007] The ELISA kit of the present invention comprises one or more components including an ARG2 standard, a sample dilution buffer, a biotin-labeled antibody working solution, an antibody dilution buffer, a SABC working solution, a SABC dilution buffer, a TMB substrate solution, and a stop solution.

[0008] In the present invention, the concentration of ARG2 is determined by the standard curve method. The specific steps include diluting the sample, adding a standard or sample, adding a labeled antibody working solution, incubating and washing, adding a substrate for a color reaction, adding a stop solution to terminate the reaction, and measuring the absorbance value at a wavelength of 450 nm, thereby achieving quantitative detection of the ARG2 concentration.

[0009] Furthermore, the present invention can effectively distinguish between sepsis patients and non-sepsis patients by setting the diagnostic threshold of ARG2 to ≥1.58 ng / mL, and is particularly suitable for the differential diagnosis of patients with lung infection combined with sepsis and patients with simple pneumonia.

[0010] Compared with the existing technology, the present invention clarifies for the first time that the expression level of ARG2 in the peripheral blood of patients with sepsis is significantly higher than that in healthy people and patients with simple pneumonia, establishes an accurate and reliable ARG2 threshold, has strong diagnostic specificity and high sensitivity, can effectively improve the accuracy of early diagnosis of sepsis, and is suitable for wide clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0012] Figure 1 To derive the AUC for the diagnosis of ARG2 in four cohorts based on public datasets;

[0013] Figure 2 The standard curve of ARG2 enzyme content obtained by enzyme-linked immunosorbent assay kit;

[0014] Figure 3 ARG2 levels in peripheral blood from healthy individuals, pneumonia patients, and sepsis patients;

[0015] Figure 4 This is the AUC graph of peripheral blood ARG2 content for diagnosing sepsis in actual clinical practice. DETAILED DESCRIPTION

[0016] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0017] Example 1 Screening of biomarkers (discovery set)

[0018] Four public datasets, E-MTAB-5273 (n=231), GSE28750 (n=30), GSE65682 (n=225), and GSE57065 (n=101), were obtained from the Array Express and Gene Expression Omnibus databases as discovery cohorts. They all contain peripheral blood transcriptional profile data from healthy controls and sepsis patients, as shown in Table 1:

[0019] Table 1 Human public datasets used to discover ARG2 as a diagnostic biomarker

[0020]

[0021]

[0022] After logarithmic transformation and normalization of the gene expression matrix, the ARG2 gene expression data of each sample and its corresponding group information (healthy control or sepsis) were extracted, and the binary ROC (Receiver Operating Characteristic) curve was drawn. The AUC (Area Under Curve) range for the diagnosis of ARG2 in the four cohorts was 0.755-0.905, as shown in Figure 2. Figure 1 As shown in the above multi-cohort public data, ARG2 is closely related to the occurrence of sepsis, and high expression of ARG2 in peripheral blood is a potential indicator for identifying and distinguishing sepsis.

[0023] Example 2 Validation of ARG2 as a Sepsis Diagnostic Biomarker in Clinical Peripheral Blood Samples (Validation Set)

[0024] (1) Sample source and grouping:

[0025] Enrolled patients were recruited from the Department of Respiratory and Critical Care Medicine and Sleep Medicine at Xiang'an Hospital, Xiamen University. Pneumonia patients were included if they met the diagnostic criteria for community-acquired pneumonia based on symptoms, signs, chest radiographs, and laboratory tests. Sepsis patients were included if they had a diagnosis of infection and a Sequential Organ Failure Assessment (SOFA) score ≥ 2. Exclusion criteria included patients under 18 years of age, pregnant women, patients with hematologic malignancies, and those who had received immunosuppressive therapy within 15 days. All enrolled patients or healthy individuals provided written informed consent. Healthy individuals (n = 20) and pneumonia patients (n = 28) were classified as the non-sepsis group, and patients with sepsis (n = 20) were classified as the sepsis group.

[0026] (2) Detection of Arginase 2 (ARG2) content in samples:

[0027] From each patient, 3 mL of EDTA-2K anticoagulated peripheral blood was drawn and centrifuged horizontally at 1000 rpm for 10 min at room temperature, retaining the upper yellow plasma to avoid hemolysis;

[0028] The arginase 2 (ARG2) content in blood samples was detected by enzyme-linked immunosorbent assay kit (Shanghai Yansheng Industrial Co., Ltd.):

[0029] Take blank wells, standard wells and sample wells, add sample diluent to blank wells, add standard samples diluted in multiples (0.1-4 ng / mL) to standard wells, and add plasma samples diluted 2-4 times with sample diluent to sample wells; after adding the stop solution, the liquid in the wells turns from blue to yellow immediately, and the absorbance (OD value) of each well at a wavelength of 450 nm is measured within 10 minutes; zero the blank wells, use the concentration of the standard as the horizontal axis and the OD value as the vertical axis to draw a standard curve ( Figure 2 ) The corresponding concentration was obtained from the standard curve according to the OD value of the sample, and the actual concentration in plasma was obtained by multiplying it by the dilution factor.

[0030] Depend on Figure 3 It can be seen that the plasma ARG2 enzyme content in sepsis patients (n=20) was significantly higher than that in healthy people (n=20) and pneumonia patients (n=28). In the sepsis state, the ARG2 level increased nearly 3 times compared with the baseline level in healthy people and nearly 2 times compared with the baseline level in pneumonia patients.

[0031] (3) The diagnostic efficacy of ARG2 for sepsis was calculated using SPSS software;

[0032] Based on the ARG2 content levels in the non-sepsis group and the sepsis group, a binary ROC curve was drawn, and the AUC of ARG2 for diagnosing sepsis was 0.874 (95% CI: 0.794-0.955). Figure 4 As shown;

[0033] The maximum Youden Index (Table 2) was calculated based on sensitivity and specificity, indicating that the optimal threshold for diagnosing sepsis based on plasma ARG2 enzyme levels was 1.58 ng / mL. A level greater than the threshold was considered sepsis, while a level less than or equal to the threshold was considered non-sepsis. The ARG2 model for diagnosing sepsis had a sensitivity of 90% and a specificity of 77.1%.

[0034] Table 2 Correspondence between Youden Index and ARG2 thresholds for diagnosing sepsis

[0035]

[0036]

[0037] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of ARG2 in plasma, serum or blood as a biomarker in the preparation of a product for early diagnosis of sepsis, characterized in that: The product includes a kit.

2. The use according to claim 1, characterized in that The diagnostic product is an ELISA kit.

3. The use according to claim 2, characterized in that The ELISA kit includes one or more of an ARG2 standard, a sample dilution buffer, a biotin-labeled antibody working solution, an antibody dilution buffer, a SABC working solution, a SABC dilution buffer, a TMB substrate solution, and a stop solution.

4. The use according to claim 2 or 3, characterized in that The ELISA kit is used to detect the ARG2 expression level in a biological sample.

5. The use according to claim 4, characterized in that Compared with patients with lung infection without sepsis, patients with infection and sepsis had significantly elevated serum ARG2 levels.

6. The use according to claim 4, characterized in that The threshold value of ARG2 for diagnosing sepsis is ≥1.58 ng / mL.

7. The use according to claim 1, characterized in that The product can be used to differentiate patients with pneumonia from patients with sepsis.

8. The use according to claim 1, characterized in that The blood sample is a peripheral blood sample.

9. The use according to claim 4, characterized in that The detection method is to calculate the ARG2 concentration in the test sample by the standard curve method.

10. The use according to claim 2, characterized in that The detection steps of the ELISA method include diluting the sample, adding a standard or sample, adding a labeled antibody working solution, incubating and washing, adding a substrate for color development, and measuring the absorbance after adding a stop solution.

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