Markers for the diagnosis of sepsis and uses thereof

Abstract

The present application relates to the field of biotechnology, and in particular, discloses a marker for diagnosing sepsis and use thereof. The present application first discovers that serum METRNL can predict the survival of sepsis patients, and METRNL can be used as a marker for predicting the survival of sepsis patients. The AUC of METRNL combined with PCT for diagnosing sepsis can reach 1. Compared with the diagnosis effect of a single marker, the AUC value and specificity of METRNL combined with PCT are significantly improved, and sepsis patients can be completely distinguished, which is an ideal sepsis diagnostic index.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to biomarkers for diagnosing sepsis and their uses, including the use of reagents for detecting METRNL protein levels, and the combined diagnostic use of reagents for detecting METRNL protein levels and reagents for detecting PCT levels. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated infection response. Despite significant attention being paid to sepsis, its mortality rate remains high. One major reason is the lack of a rapid and accurate diagnostic method for sepsis, resulting in a lack of clinical means for early diagnosis and disease prediction. Currently, the diagnosis of sepsis still relies on the Sepsis 3.0 diagnostic criteria jointly published by the American College of Critical Care Medicine and the European Society of Critical Care Medicine in 2016: an infection with a Sequential Organ Failure Assessment (SOFA) score ≥2. Suspected infection requires confirmation through microbial culture, which is time-consuming and has a certain proportion of false negatives. Furthermore, the SOFA score involves 12 items across 6 systems, requiring not only laboratory test results but also specialist assessments of clinical medication dosages and neurological function. The current requirements for infection confirmation and SOFA scoring contribute to the diagnostic challenges of sepsis. Excellent biomarkers for disease diagnosis have a natural advantage. To provide accurate and timely diagnostic and prognostic data for clinical practice, biomarkers for sepsis have been a research hotspot, with the aim of finding a biomarker with good sensitivity and specificity that can independently diagnose sepsis and predict the condition of sepsis patients. Thousands of candidate biomarkers have been proposed and studied; unfortunately, to date, no biomarker has the ability to independently diagnose sepsis. Procalcitonin (PCT), which has been extensively studied, is the only biomarker included in clinical sepsis treatment guidelines and can guide antibiotic use, but it does not have independent diagnostic capabilities. C-reactive protein (CRP), as a traditional inflammation screening indicator, has poor diagnostic specificity.

[0003] Meteorin-like protein (METRNL) is an adipokine belonging to the same secretory protein family as the neurotrophic factor meteorin. Also known as Meteorin-β, Cometin, Subfatin, and interleukin-41, its amino acid sequence shows high similarity across different species. METRNL is widely distributed throughout the body's tissues and organs, with particularly high expression in white adipose tissue and barrier tissues such as skin, intestines, and respiratory epithelium. Since its discovery, METRNL has been reported to have multiple functions, involving lipid metabolism, atherosclerosis, neurotrophic factors, damage repair, and immune inflammation. The inventors' research group has also conducted several studies on METRNL in recent years. Among them, a patent application filed in 2016, "Application of METRNL protein or gene in the prevention and treatment of sepsis" (application number 2016101431102), discloses that in sepsis models, the survival rate of model mice is lower than that of WT mice, and the serum METRNL level in mice after modeling is significantly increased. Therefore, METRNL can be used as a diagnostic biomarker for sepsis. However, the patent does not disclose the diagnostic efficacy of METRNL, nor does it address whether METRNL can be used to predict the survival of sepsis patients.

[0004] In the prior art, there are no reports on the use of METRNL as a biomarker for predicting the survival of sepsis patients, or on the combined use of METRNL and PCT for the diagnosis of sepsis. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of the prior art by providing a reagent for detecting METRNL protein levels.

[0006] A second objective of this invention is to provide a kit for predicting the survival of patients with sepsis.

[0007] A third objective of this invention is to provide the use of reagents for detecting METRNL protein levels and reagents for detecting PCT levels.

[0008] A fourth objective of this invention is to provide a kit for diagnosing sepsis.

[0009] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows:

[0010] The purpose of reagents for detecting METRNL protein levels is to prepare kits or diagnostic reagents for predicting the survival of sepsis patients.

[0011] Preferably, the detection is a serum detection.

[0012] Preferably, the reagent for detecting METRNL protein levels is a specific antibody against METRNL protein.

[0013] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0014] A kit for predicting the survival of sepsis patients, the kit comprising reagents for detecting METRNL protein levels.

[0015] Preferably, the detection is a serum detection.

[0016] Preferably, the reagent for detecting METRNL protein levels is a specific antibody against METRNL protein.

[0017] To achieve the third objective mentioned above, the technical solution adopted by the present invention is as follows:

[0018] The uses of reagents for detecting METRNL protein levels and PCT levels are for the preparation of diagnostic kits or reagents for sepsis.

[0019] Preferably, the detection is a serum detection.

[0020] Preferably, the reagent for detecting METRNL protein level is a specific antibody against METRNL protein, and the reagent for detecting PCT level is a specific antibody against PCT.

[0021] To achieve the fourth objective mentioned above, the technical solution adopted by the present invention is as follows:

[0022] A kit for diagnosing sepsis includes reagents for detecting METRNL protein levels and reagents for detecting PCT levels.

[0023] Preferably, the reagent for detecting METRNL protein level is a specific antibody against METRNL protein, and the reagent for detecting PCT level is a specific antibody against PCT.

[0024] The advantages of this invention are:

[0025] 1. The combined AUC of METRNL and PCT in diagnosing sepsis can reach 1. Compared with the diagnostic efficacy of the individual markers, the combined AUC and specificity of METRNL and PCT are significantly improved, and they can completely distinguish sepsis patients. The combined use of these two markers can effectively improve the diagnostic efficacy of sepsis and is an ideal diagnostic indicator for sepsis.

[0026] 2. This is the first time that serum METRNL has been found to predict the survival of sepsis patients. Attached Figure Description

[0027] Appendix Figure 1 Comparison of serum METRNL, PCT, and CRP levels between sepsis patients and control groups.

[0028] Appendix Figure 2 ROC curves of METRNL, PCT, and CRP for diagnosing sepsis.

[0029] Appendix Figure 3 ROC curve of combined METRN and PCT for the diagnosis of sepsis.

[0030] Appendix Figure 4 Scatter plot showing the correlation between three biomarkers and SOFA score in sepsis patients.

[0031] Appendix Figure 5 ROC curve of METRNL predicting 28-day survival of sepsis patients. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0033] The inventors first verified the diagnostic efficacy of the classic sepsis markers PCT and CRP, as well as the marker METRNL previously explored by their research group, for sepsis. The results showed that METRNL, PCT, and CRP had high AUC values, but their diagnostic specificity was less than or equal to 0.9, failing to completely distinguish sepsis patients. In exploring the predictive ability of serum METRNL for the survival of sepsis patients, it was shown that serum PCT and CRP markers could not predict the survival of sepsis patients. However, during the exploration of the diagnostic and disease prediction efficacy of serum METRNL for various types of sepsis patients, the inventors unexpectedly discovered that serum METRNL has the potential to predict the survival of sepsis patients, with an AUC value of 0.934, a sensitivity of 1, and a specificity of 0.793. Based on this, the inventors proposed the use of a reagent for detecting METRNL protein levels, for the preparation of kits or diagnostic reagents for predicting the survival of sepsis patients. According to this invention, the survival of sepsis patients can be predicted by measuring the METRNL protein content in the initial serum of patients at diagnosis; any reagent capable of measuring the METRNL protein content in serum can be used as a diagnostic reagent. Methods for determining the level of METRNL protein in serum include commonly used methods such as ultraviolet spectrophotometry, Kjeldahl method, biuret method, Lowry method, BCA method, Coomassie brilliant blue staining method, silver staining method, and immunological methods. For example, antibodies against METRNL protein can be prepared using conventional methods, and qualitative or quantitative methods and corresponding reagents or kits for detecting METRNL protein can be established. Conventional methods may involve using exogenously expressed METRNL protein or chemically synthesized METRNL protein peptides as antigens to prepare antibodies for animal immunization experiments. The anti-METRNL protein antibodies include monoclonal antibodies or polyclonal antibodies. The qualitative or quantitative methods and corresponding reagents or kits for detecting METRNL protein specifically involve in vitro detection of whether the content of METRNL protein in sample tissue is abnormal. First, the content of METRNL protein in the test sample is detected; second, it is compared with the content of METRNL protein in a standard sample; and finally, it is determined whether there is upregulation in the test sample. The detection reagent or kit may be an enzyme-linked immunosorbent assay (ELISA) kit, a chemiluminescence assay kit, a solid or liquid chip kit, or other kits prepared according to the method of antibody-coated detection antigen.

[0034] Furthermore, it was found that combining the biomarker METRNL with PCT significantly improves diagnostic efficacy for sepsis. The combined use of METRNL and PCT for sepsis diagnosis achieved an AUC of 1, significantly improving both AUC and specificity compared to the individual biomarkers, effectively differentiating sepsis patients. This combination of biomarkers effectively enhances diagnostic efficacy for sepsis and represents an ideal diagnostic indicator for the disease. Based on this, the applications of reagents for detecting METRNL protein levels and PCT levels are proposed for the preparation of sepsis diagnostic kits or reagents.

[0035] The inventors also validated the results using sepsis data following major abdominal surgery in clinical settings, demonstrating the same findings. In diagnosing sepsis, the AUC of METRNL was slightly lower than that of PCT but higher than that of CRP. Combining METRNL with PCT for sepsis diagnosis significantly improved both the AUC and specificity, effectively differentiating sepsis patients. Regarding the prediction of sepsis patient survival, serum PCT and CRP failed to predict postoperative sepsis survival, while serum METRNL showed potential for prediction, with an AUC greater than 0.9, indicating good predictive efficacy.

[0036] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0037] Example: METRNL's potential for diagnosis and disease prediction in sepsis

[0038] I. Experimental Methods

[0039] (1) Study population

[0040] Patients with sepsis in the ICU and non-septic patients in the control group.

[0041] (2) Inclusion and exclusion criteria:

[0042] Inclusion criteria for the sepsis group: sepsis patients of all types in the ICU.

[0043] Exclusion criteria for the sepsis group: patients who have recently had a stroke or have ulcerative colitis or other diseases.

[0044] Inclusion criteria for the control group: non-septic patients in the ICU.

[0045] Exclusion criteria for the control group: patients who have recently experienced a stroke or have ulcerative colitis or other similar conditions.

[0046] (3) Observation indicators:

[0047] Basic information: age, gender.

[0048] Main observation indicator: serum METRNL level in patients in the sepsis group and the control group.

[0049] Secondary observational indicators: serum PCT and CRP levels, SOFA score, and 28-day survival in the sepsis group, and serum PCT and CRP levels in the control group.

[0050] (4) Implementation process:

[0051] Sample collection from patients in the sepsis group: 2 mL of blood was collected venously within 24 hours of the diagnosis of sepsis, either simultaneously with routine blood collection or from an existing central venous catheter to avoid additional punctures. SOFA scores were assessed on the day of blood collection, and 28-day survival was recorded.

[0052] Sample collection from control group patients: 2 mL of blood was collected from the vein within 24 hours of admission to the ICU, either simultaneously with routine blood collection or from an established deep vein catheter, to avoid patients undergoing additional punctures.

[0053] (5) Sample processing:

[0054] After blood collection, the blood was left to stand at room temperature for 2 hours, then centrifuged at 2000 rpm for 15 minutes to obtain serum. The serum was stored at -80℃. Serum METRNL levels were detected in the laboratory using an ELISA kit, while PCT and CRP levels were obtained directly from the hospital system.

[0055] (6) Enzyme-linked immunosorbent assay (ELISA)

[0056] Species-specific ELISA kits (Human-DY7867 / Mouse-DY6679) were used for detection, and the experimental procedures were performed according to the instructions. The standard curve must meet the R-value. 2≥0.99. A simplified operating procedure is as follows: Plate the capture antibody into a dedicated 96-well plate and incubate overnight (12-16 h). Wash the plate 3 times with wash buffer. Add reagent diluent and plate, incubate for 1 h. Wash the plate 3 times with wash buffer. Add standards and samples, and incubate for 2 h. Wash the plate 3 times with wash buffer. Add detection antibody and incubate for 2 h. Wash the plate 3 times with wash buffer. Add streptavidin-horseradish peroxidase and incubate in the dark for 20 min. Wash the plate 3 times with wash buffer. Add substrate solution and plate, and incubate in the dark for 20 min. Add stop solution to terminate the reaction. Detect the absorbance of the standards and samples using a microplate reader at a detection wavelength of 450 nm and a calibration wavelength of 540 nm. Plot a standard curve and calculate the sample concentration.

[0057] (7) Data Analysis and Processing

[0058] All data are expressed as mean ± standard error. SPSS 21 was used for statistical analysis. Two sets of data were compared: first, a normality test was performed. For normally distributed data, an independent samples t-test was used for two independent samples, and a paired samples t-test was used for two related samples. For non-normally distributed data, nonparametric tests were used. The diagnostic efficacy of the biomarker was assessed using the area under the ROC curve (AUC), and the cutoff value, sensitivity, and specificity were determined based on the Youden index. The correlation between the biomarker and the SOFA score was assessed using Kendall's test and Spearman's test. Logistic regression analysis was used to analyze the statistical significance of the biomarker's impact on 28-day survival. Significance criteria were used as follows: * p<0.05; ** p<0.01; *** p<0.001.

[0059] II. Experimental Results

[0060] (1) Serum METRNL levels are elevated in patients with sepsis.

[0061] Blood samples were collected from 35 patients in the sepsis group and 40 patients in the control group. The demographic characteristics of the patients in the sepsis group and the control group are shown in Table 1. To explore the potential of METRNL as a diagnostic biomarker for sepsis, we compared samples from the sepsis group and the control group, and also included the classic sepsis biomarkers PCT and CRP to evaluate their efficacy. The overall distribution of the three biomarkers is shown in Table 1. Results are as follows: Figure 1 As shown, serum METRNL, PCT, and CRP levels are elevated in patients with sepsis.

[0062] Table 1. Demographic information and overall distribution of three biomarkers in sepsis patients and control group.

[0063]

[0064] (2) Serum METRNL has the potential to diagnose sepsis.

[0065] We compared the sepsis group with the control group and plotted ROC curves for METRNL, PCT, and CRP. Figure 2 The AUC was calculated, and the cutoff value, corresponding sensitivity, and specificity were derived based on the Youden index. The results are shown in Table 2. The AUC of METRNL was slightly lower than that of PCT but higher than that of CRP (AUC = 0.973:0.99:0.926), indicating that the diagnostic efficacy of METRNL is slightly inferior to that of PCT but superior to that of CRP. Although METRNL, PCT, and CRP have high AUC values, their diagnostic specificity is less than or equal to 0.9, and they still cannot completely distinguish sepsis patients.

[0066] Table 2. AUC, cutoff value, sensitivity, and specificity of three biomarkers for diagnosing sepsis.

[0067]

[0068] Furthermore, the biomarker METRNL was combined with PCT for the diagnosis of sepsis, and ROC curves were plotted. Figure 3 The AUC was calculated and shown in Table 3. When METRNL and PCT were used together to diagnose sepsis, the AUC reached 1. Compared with the diagnostic efficacy of the individual markers, the combined AUC and specificity of METRNL and PCT were significantly improved, completely distinguishing sepsis patients. This indicates that the combined use of these two markers can effectively improve the diagnostic efficacy of sepsis and is an ideal diagnostic indicator for sepsis.

[0069] Table 3. AUC, sensitivity, and specificity of combined biomarkers for the diagnosis of sepsis.

[0070]

[0071] (3) Serum METRNL can predict the severity of sepsis.

[0072] To explore the predictive potential of METRNL for sepsis severity, we tested the correlation between clinical indicators from 35 sepsis patients and the SOFA score, which represents disease severity. The results of the normality test are shown in Table 4. SOFA score, METRNL, and PCT did not conform to a normal distribution, while CRP did, requiring nonparametric tests. The scatter plots of the correlations between the three markers and the SOFA score are shown below. Figure 4As shown in Table 5, the results of the two nonparametric tests are as follows: METRNL and PCT are correlated with SOFA scores, while CRP is not significantly correlated with SOFA scores. This suggests that in this part of the clinical study, METRNL and PCT have the potential to predict the severity of sepsis, while CRP cannot predict the severity of sepsis.

[0073] Table 4. Results of SOFA scores, serum METRNL, PCT, and CRP normality tests for sepsis samples.

[0074]

[0075] Table 5. Correlation test results of METRNL, PCT, CRP and SOFA score in sepsis samples.

[0076]

[0077] (4) Serum METRNL has the potential to predict 28-day survival in sepsis.

[0078] To explore the predictive ability of serum METRNL for 28-day survival in sepsis patients, we performed logistic regression analysis on the serum METRNL, PCT, and CRP levels and 28-day survival in 35 sepsis patients. The results are shown in Table 6. Serum METRNL had a statistically significant effect on the 28-day survival of sepsis patients, suggesting that serum METRNL has the potential to predict the 28-day survival of sepsis patients. Serum PCT and CRP did not have statistically significant effects on the 28-day survival of sepsis patients, suggesting that serum PCT and CRP cannot predict the 28-day survival of sepsis patients.

[0079] Table 6. Logistic regression analysis results of three biomarkers and 28-day survival of sepsis patients.

[0080]

[0081] Using METRNL as a predictor of 28-day survival in sepsis, an ROC curve for METRNL was plotted. Figure 5 The area under the ROC curve (AUC) was calculated, and the cutoff value, sensitivity, and specificity were determined based on the Youden index. The results are shown in Table 7. The AUC ranged from 0.5 to 1, with a higher AUC indicating better diagnostic efficacy. The results in the figure show that METRNL has an AUC of 0.934 for predicting 28-day survival in sepsis, indicating that METRNL has high predictive value for the survival (28-day clinical outcome) of sepsis patients.

[0082] Table 7. AUC, Sensitivity, and Specificity of METRNL in Predicting 28-Day Survival in Sepsis Patients

[0083]

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The use of reagents for detecting METRNL protein levels and reagents for detecting PCT levels in the preparation of sepsis diagnostic kits or diagnostic reagents, characterized in that, The diagnosis refers to distinguishing between patients with sepsis and those without sepsis.

2. The use according to claim 1, characterized in that, The test is a serum test.

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