Application of ANXA3 protein in preparation of reagent for critical illness death early warning and sepsis diagnosis

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to application of annexin A3 (ANXA3) in detection of critical illness death early warning and sepsis diagnosis. It is found that the ANXA3 protein amount in plasma of a critically ill patient is remarkably higher than that in a healthy voluntary person, the ANXA3 protein amount in plasma of a critically ill sepsis patient is remarkably higher than that in plasma of a critically ill non-sepsis patient, and the ANXA3 protein amount in the plasma is further remarkably related to the death risk of the critically ill patient. By detecting the blood plasma ANXA3 protein amount, sepsis occurrence and death risks of critically ill patients can be evaluated, individualized treatment can be started as soon as possible, and the kit has important clinical application value for prognosis of critically ill patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to the use of ANXA3 protein in the preparation of a reagent for critical illness death early warning and sepsis diagnosis. BACKGROUND

[0002] Sepsis is a life-threatening multi-organ dysfunction caused by immune disorders in response to infection. Current research reports that sepsis accounts for about 40% of the total number of patients admitted to ICU, and the mortality rate can be as high as 25%-40%. It is estimated that there were 4890 million sepsis cases worldwide in 2017, resulting in 1100 million deaths. A large number of studies have confirmed that early and accurate treatment can significantly reduce the incidence of sepsis patients and improve the prognosis of sepsis patients. Among them, microbial culture is the gold standard for the diagnosis of sepsis, but microbial culture mostly requires 3-5 days and is accompanied by a high false negative result. Therefore, developing new sepsis treatment targets and biomarkers plays an important role in reducing the incidence and mortality of sepsis.

[0003] An ideal sepsis biomarker should be efficient, capable of quickly identifying potential pathogenic infection, and accurately reflecting the outcome of anti-infection therapy. The most widely used indicators for monitoring sepsis in clinical practice include C-reactive protein (CRP), procalcitonin (PCT), and interleukin 6 (IL-6), but their sensitivity and specificity have been controversial. PCT and CRP often increase to varying degrees in end-stage surgical major surgery, autoimmune diseases, and other underlying diseases, and are not highly specific. And PCT and CRP can only reflect the degree of systemic inflammatory response at the time of detection, and the results vary greatly with different detection time windows. IL-6 has a short residence time in the blood, making it difficult to accurately determine the appropriate detection time, and has limited impact on the outcome prediction of sepsis patients.

[0004] Annexin A3 (ANXA3) is one of the members of the annexin family, which belongs to the Ca 2+Dependent phospholipid and membrane-bound protein. Current studies have confirmed that ANXA3 is involved in the intracellular membrane fusion and granule aggregation of neutrophils, enhances the killing activity of neutrophils on microorganisms, and plays an important role in oxidative stress and immune inflammatory response. At the same time, ANXA3 may also play an important role in regulating the apoptosis process of neutrophils. Researchers have also found that increased expression of ANXA3 is associated with a higher neutrophil-lymphocyte ratio, and ANXA3 up-regulates the neutrophil-lymphocyte ratio by inducing the release of chemokines CXCL8 and CCL25. Genome-wide association analysis (GWAS) results show that ANXA3-rs2867461 is associated with the risk of rheumatoid arthritis and lupus erythematosus, and rs2867461 may be involved in regulating the expression of ANXA3, thereby affecting the susceptibility of inflammatory diseases such as rheumatoid arthritis. There is no research to confirm the clinical relevance of plasma ANXA3 to the risk of critical illness death and the occurrence of sepsis. Therefore, through a series of explorations, the inventors have found that ANXA3 can not only be used for early warning of the risk of critical illness death, but also can be used for assessment of the occurrence of sepsis. SUMMARY

[0005] In view of the above, the purpose of the present application is to use ANXA3 as a biomarker for early assessment of the risk of critical illness death and the occurrence of sepsis, to make up for the deficiencies in the prior art in the early warning of the risk of critical illness death and the diagnosis method of sepsis, and to reduce the mortality rate of critical illness patients.

[0006] The present application provides a use of ANXA3 protein as a biomarker for early warning of critical illness death or diagnosis of sepsis.

[0007] In one embodiment according to the present application, the use comprises detecting the content of ANXA3 protein in the sample.

[0008] In one embodiment according to the present application, the sample in the use is a body fluid, plasma or serum.

[0009] In one embodiment according to the present application, the amount of ANXA3 protein in the sample in the use is positively correlated with the severity of organ dysfunction in critical illness patients.

[0010] In one embodiment according to the present application, the amount of ANXA3 protein in the sample in the use is related to the risk of critical illness death and the occurrence of sepsis.

[0011] In one embodiment according to the present application, the content of ANXA3 protein is detected by enzyme-linked immunoassay.

[0012] In one embodiment according to the present application, the enzyme-linked immunoassay is a double-antibody sandwich method.

[0013] The application also provides a reagent or kit for critical illness death early warning or sepsis diagnosis, comprising an antibody for targeting binding of ANXA3 protein.

[0014] The application further provides use of the above-mentioned reagent or kit for detecting ANXA3 protein in preparation of a composition for critical illness death early warning or sepsis diagnosis.

[0015] The above-mentioned technical solutions of the application have the following beneficial effects: The application takes ANXA3 protein as a biomarker, which is of great help to improving the early nature and accuracy of discovery and death risk assessment of critical illness sepsis. On one hand, the application provides that ANXA3 protein can be used as an early warning index of critical illness death risk; on the other hand, the application provides that ANXA3 protein can be used as an index for diagnosing critical illness sepsis. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a graph for detecting the amount of ANXA3 protein in the plasma of critical illness patients in Example 1 of the application, in which the amount of ANXA3 protein in the plasma of critical illness patients is obviously increased; Figure 2 Figure 2 is a graph for detecting the amount of ANXA3 protein in the plasma of critical illness sepsis patients in Example 1 of the application, in which the amount of ANXA3 protein in the plasma of critical illness sepsis patients is obviously increased (p<0.05) than that of non-sepsis patients; Figure 3 Figure 3 is a graph for detecting the correlation between the amount of ANXA3 protein in the plasma of critical illness patients and organ dysfunction score SOFA (a) and MODS (b) in Example 1 of the application; wherein, Figure 3 a is a graph for detecting the correlation between the amount of ANXA3 protein in the plasma of critical illness patients and organ dysfunction score SOFA in Example 1 of the application; Figure 3 b is a graph for detecting the correlation between the amount of ANXA3 protein in the plasma of critical illness patients and organ dysfunction score MODS in Example 1 of the application; Figure 4 Figure 4 is a graph for comparing and analyzing the diagnostic effect of ANXA3, PCT and IL-6 in critical illness sepsis by ROC curve in Example 2 of the application; Figure 5 Figure 5 is a graph for detecting the correlation between the amount of ANXA3 protein in the plasma and critical illness death risk (a) and sepsis death risk (b) in Example 3 of the application, wherein, Figure 5 a is a graph for detecting the correlation between the amount of ANXA3 protein in the plasma and critical illness death risk in Example 3 of the application; Figure 5b is a graph showing the correlation between the amount of ANXA3 protein in plasma and the risk of death from severe sepsis in Example 3 of this invention. Detailed Implementation

[0017] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0018] Example 1: Detection of ANXA3 protein levels in the plasma of critically ill patients and changes in its content in critically ill sepsis patients. Plasma samples were collected from 57 healthy individuals undergoing physical examinations and from 153 critically ill patients within 24 hours of admission. The protein content of ANXA3 was detected using an ELISA kit (purchased from Biorbyt, Germany), and the specific operating procedures were strictly followed according to the kit's instructions.

[0019] Furthermore, this invention conducted a differential analysis of ANXA3 protein levels in the plasma of critically ill patients and healthy volunteers, as well as critically ill sepsis patients and non-sepsis patients. It also conducted a correlation analysis of plasma ANXA3 protein levels with organ dysfunction scores (SOFA, MODS) in critically ill patients.

[0020] The results showed that compared with healthy volunteers (1.83 (1.35-2.54) ng / ml), the plasma ANXA3 level in critically ill patients was significantly higher (3.23 (2.34-4.37) ng / ml) (p<0.001). Figure 1 Furthermore, the plasma ANXA3 protein level in critically ill sepsis patients was significantly higher than that in non-sepsis patients (4.16 (3.58-5.14) ng / ml vs. 2.71 (1.82-3.37) ng / ml) (p<0.001). Figure 2 Furthermore, the level of ANXA3 protein in plasma was significantly positively correlated with the organ dysfunction severity scores SOFA and MODS in critically ill patients. Figure 3 a and Figure 3 b).

[0021] Example 2: Evaluation of the diagnostic efficacy of plasma ANXA3, PCT, and IL-6 in critically ill patients with sepsis Plasma samples were collected from 153 critically ill patients within 24 hours of admission. PCT and IL-6 antibodies were incubated with labeled luminescent detection antibodies and patient plasma using chemiluminescent immunoassay (CLIA). The levels of PCT and IL-6 in patient plasma were measured using a procalcitonin (PCT) assay kit and an interleukin-6 (IL-6) assay kit (purchased from Shenzhen Huamaixingwei, China) (see kit instructions for specific procedures). ROC curves for each biomarker were plotted, and the statistical differences in the area under the ROC curves were compared using MedCalc 15.8 software.

[0022] The results are as follows Figure 4 The results showed that the area under the ROC curve for ANXA3 was 0.815 (0.745-0.886), with a sensitivity of 83.33%, a specificity of 76.34%, and a cut-off value of 3.39 ng / ml. In contrast, the areas under the ROC curves for plasma PCT and IL-6 were 0.673 (0.584-0.761) and 0.672 (0.585-0.759), respectively. The area under the ROC curve for ANXA3 was significantly larger than that for PCT (p=0.006) and IL-6 (p=0.011). This indicates that ANXA3 has certain value in the detection of severe sepsis and is more effective than PCT and IL-6.

[0023] Example 3: The impact of ANXA3 on the risk of death in critically ill patients Plasma samples were collected from 153 critically ill ICU patients within 24 hours of admission. Patients were divided into survival and mortality groups based on their clinical outcomes within 28 days of admission. Survival curves were plotted using SPSS 19.0 software.

[0024] The results showed that among the 153 critically ill patients, 33 died. The survival rate of the ANXA3 high expression group was significantly lower than that of the ANXA3 low expression group (HR=2.16 (1.09-4.28)) (p=0.028). Figure 5 a); Among the 60 patients with sepsis, 19 died. The survival rate of the ANXA3 high expression group was trending lower than that of the ANXA3 low expression group (HR=1.63(0.65-4.06)) (p=0.276) Figure 5 (b) However, the difference did not reach statistical significance. This indicates that ANXA3 has a certain predictive effect on the risk of death in critically ill patients and those with sepsis.

[0025] The above description represents the 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 principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of ANXA3 protein as a biomarker for early warning of death in critically ill patients or for the diagnosis of sepsis.

2. The use as described in claim 1, comprising detecting the ANXA3 protein content in a sample.

3. The use as described in claim 1 or 2, wherein, The sample is body fluid, plasma, or serum.

4. The use as described in any one of claims 1-3, wherein, The amount of ANXA3 protein in the samples was positively correlated with the severity of organ dysfunction in critically ill patients.

5. The use as described in any one of claims 1-3, wherein, The amount of ANXA3 protein in the samples was associated with the risk of critical illness and death, as well as the occurrence of sepsis.

6. The use as described in any one of claims 1-5, wherein, The ANXA3 protein content in the sample was determined by enzyme-linked immunosorbent assay (ELISA).

7. The use as described in claim 6, wherein, The enzyme-linked immunosorbent assay (ELISA) is a double-antibody sandwich method.

8. A reagent or kit for early warning of death in critically ill patients or for the diagnosis of sepsis, comprising an antibody for targeting and binding to the ANXA3 protein.

9. Use of reagents or kits for detecting ANXA3 protein in the preparation of compositions for early warning of critical illness or diagnosis of sepsis.

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