Application of ANGPTL3 in prognosis of hepatitis B related chronic and acute liver failure

Abstract

The invention discloses application of ANGPTL3 in prognosis of hepatitis B related chronic-acute liver failure, and relates to the technical field of disease prognosis and biological medicine. The invention discloses application of ANGPTL3 as a biomarker in preparation of a product for diagnosis and / or disease course diagnosis and / or prognosis evaluation of hepatitis B related chronic plus acute liver failure. The invention has the following beneficial effects: ANGPTL3 can be used as a biomarker for prognosis of hepatitis B-related chronic-acute liver failure, blood sample levels of patients in different groups of HBV-ACLF and dynamic ANGPTL3 levels are absolutely quantified by ELISA technology, and hepatitis B-related chronic-acute liver failure phenomena and disease courses can be timely found and detected with other related indexes; therefore, the ANGPTL3 is utilized to provide scientific guidance for management and decision-making of hepatitis B related chronic plus acute liver failure.

Description

Technical Field

[0001] The present invention belongs to the fields of disease prognosis and biomedicine, and particularly relates to the application of ANGPTL3 in the prognosis of hepatitis B-related acute-on-chronic liver failure. Background Art

[0002] Acute-on-chronic liver failure (ACLF) is a complex syndrome that occurs in patients with chronic liver disease (CLD) during acute liver injury. Its main features are a strong systemic inflammatory response, organ failure, and a high short-term mortality rate. Virus-associated acute-on-chronic liver failure (HBV-ACLF) presents multiple organ failure within 4 weeks, with a high mortality rate and poor prognosis. Timely clinical intervention can prevent the progression of HBV-ACLF and improve the survival rate of patients.

[0003] Currently, common prognostic methods for liver failure prognosis include the model for end-stage liver disease (MELD), the CLIF-C ACLF model introduced by the European Association for the Study of Chronic Liver Failure (CLIF), etc. However, they still have limitations. Many new prognostic markers and evaluation systems for liver failure are under research. Due to the complex pathogenesis, rapid disease progression, and numerous factors affecting prognosis of liver failure, accurate prediction of liver failure is difficult. Developing more effective and sensitive detection methods to predict the disease progression and grading of HBV-ACLF is the key to treating HBV-ACLF. Summary of the Invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide ANGPTL3 as a biomarker and its application in the prognosis of hepatitis B-related acute-on-chronic liver failure.

[0005] The applicant used allele-labeling-based quantitative proteomics to evaluate the proteomic changes related to HBV-ACLF disease, and found that angiopoietin-like 3 (ANGPTL3) can be used as a candidate biomarker for prognosis based on the differentially expressed proteins (DEPs) in HBV-ACLF patients.

[0006] ANGPTL3 is one of the members of the angiopoietin-like protein family involved in regulating lipid metabolism. The protein encoded by the ANGPTL3 gene is only produced in the liver and then secreted into the circulatory system. In the liver, ANGPTL3 is completely produced by hepatocytes, and its expression is regulated by the hydroxysterol-activated liver X receptor (LXR). Previous studies have shown that the biological effects of ANGPTL3 are manifested in two aspects: regulating angiogenesis and lipid metabolism. So far, there has been no relevant report on the association between ANGPTL3 and HBV-ACLF disease. This application discovers this correlation and finds that the ANGPTL3 level is closely related to the course changes of HBV-ACLF.

[0007] The present invention provides ANGPTL3 as a biomarker for predicting the prognosis of hepatitis B acute-on-chronic liver failure, which can be applied in an Enzyme-Linked Immunosorbent Assay (ELISA) product to detect the plasma of patients, and can efficiently, simply and accurately predict the disease progression, survival time and specific grading of HBV-ACLF patients, and better guide clinical medication.

[0008] The technical solution of the present invention is as follows: The first aspect of the present invention provides an application of ANGPTL3 as a biomarker in the preparation of products for the diagnosis and / or course diagnosis and / or prognosis assessment of hepatitis B-related acute-on-chronic liver failure.

[0009] Optionally, the hepatitis B-related acute-on-chronic liver failure includes hepatitis B-related acute-on-chronic liver failure with a basis of hepatitis B cirrhosis.

[0010] Optionally, the product includes a kit for detecting the content of the biomarker.

[0011] Optionally, the kit contains reagents for detecting the content of the biomarker, and the reagents include one of the reagents required for immunohistochemistry, Western-Blot, immunoblotting, and enzyme-linked immunosorbent assay.

[0012] Specifically, the kit is an ELISA kit, and the ELISA kit includes a capture antibody, a detection antibody, a recombinant human ANGPTL3 protein standard, a sample treatment solution, a chromogenic solution and a termination solution. More specifically, the detection antibody is a detection antibody labeled with horseradish peroxidase.

[0013] Optionally, the course diagnosis includes detecting the ANGPTL3 level of the patient before and after treatment using the product, and judging whether the course is improved, fluctuated or deteriorated according to the change of the ANGPTL3 level.

[0014] Specifically, the disease course diagnosis includes disease improvement, disease fluctuation, and disease deterioration. Among them, disease improvement means that the ANGPTL3 level of the patient increases during treatment, disease fluctuation means that the ANGPTL3 level of the patient does not change significantly during treatment, and disease deterioration means that the ANGPTL3 level of the patient decreases during treatment.

[0015] The second aspect of the present invention provides a kit containing reagents for detecting the content of the biomarker, the biomarker being ANGPTL3, and the reagents including one of the reagents required for immunohistochemistry, Western-Blot, protein immunoblotting, and enzyme-linked immunosorbent assay; When detecting the content of the biomarker in a test sample using the kit, when the detected concentration of the biomarker in the test sample is lower than 64.87 ng / mL, it is determined that the source of the test sample is a high-risk patient with HBV-ACLF.

[0016] Optionally, the kit is an ELISA kit, and the ELISA kit includes a capture antibody, a detection antibody, a recombinant human ANGPTL3 protein standard, a sample treatment solution, a chromogenic solution, and a termination solution. The detection antibody is a detection antibody labeled with horseradish peroxidase.

[0017] The third aspect of the present invention provides the application of ANGPTL3 in constructing a prognostic prediction model for hepatitis B-related acute-on-chronic liver failure.

[0018] Specifically, the molecular marker ANGPTL3 is made into a prognostic prediction model for prognostic prediction of hepatitis B-related acute-on-chronic liver failure.

[0019] The fourth aspect of the present invention provides the application of ANGPTL3 as a target in screening drugs for treating hepatitis B-related acute-on-chronic liver failure.

[0020] The fifth aspect of the present invention provides the application of ANGPTL3 in preparing an inhibitor for hepatitis B-related acute-on-chronic liver failure.

[0021] Specifically, targeted therapeutic drugs or inhibitors can be designed for ANGPTL3 to design therapeutic drugs or inhibitors targeting hepatitis B-related acute-on-chronic liver failure. By designing an mRNA vaccine against ANGPTL3, the immune system in the patient is stimulated to produce ANGPTL3, thereby increasing its content. Studying the expression regulation mechanism of ANGPTL3, small molecule substances that can induce the expression of ANGPTL3 are found, and drugs or inhibitors that can activate the expression of ANGPTL3 are screened and optimized through drugs or inhibitors.

[0022] The sixth aspect of the present invention provides a method for screening drugs, including the following steps: Detect the content of ANGPTL3 in the patient's sample before and after drug administration; If the content of ANGPTL3 increases, the drug to be screened has the effect of treating HBV-ACLF; If the content of ANGPTL3 remains unchanged or decreases, the drug to be screened does not have the effect of treating HBV-ACLF; The sample includes blood.

[0023] The present invention has at least one of the following beneficial effects: The applicant screened out a biomarker for the prognosis of hepatitis B-related acute-on-chronic liver failure through a large number of proteomics, and found that ANGPTL3 can be used as a biomarker for the diagnosis and / or course diagnosis and / or prognosis of hepatitis B-related acute-on-chronic liver failure. By using ELISA technology to absolutely quantify the level of ANGPTL3 in the blood specimens of patients in different groups of HBV-ACLF and the absolute quantitative dynamic ANGPTL3 level, the phenomenon and course of hepatitis B-related acute-on-chronic liver failure can be detected in a timely manner and other related indicators can be detected, so as to provide scientific guidance for the management and decision-making of hepatitis B-related acute-on-chronic liver failure by using ANGPTL3.

[0024] ANGPTL3 can also be used to construct a prognosis prediction model for hepatitis B-related acute-on-chronic liver failure for prognosis prediction of hepatitis B-related acute-on-chronic liver failure.

[0025] ANGPTL3 can also be used as a target for screening drugs for treating hepatitis B-related acute-on-chronic liver failure and for preparing inhibitors for hepatitis B-related acute-on-chronic liver failure for screening drugs and inhibitors. Description of the Drawings

[0026] Figure 1 It is the correlation between the ANGPTL3 level at admission and HBV-ACLF patients. Among them, A in the figure is the distribution of ANGPTL3 in the survival group and non-survival group of HBV-ACLF patients. B in the figure is the receiver operating characteristic curve of ANGPTL3 predicting the 30-day mortality of HBV-ACLF patients. C in the figure is the survival rate of patients in the high ANGPTL3 group and low ANGPTL3 group after 30 days. D in the figure is the correlation between the ANGPTL3 level and the mortality rate. *P<0.001.

[0027] Figure 2 It is the change of the ANGPTL3 level among the improvement group, fluctuation group and deterioration group between the initial (at admission) and final (14 days after admission or at discharge) evaluations.

[0028] Figure 3 It is the standard curve equation in Example 1.

[0029] Figure 4 Sensitivity detection data in Example 3. Detailed implementation manners

[0030] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] Example 1 1) Collect blood specimens, including HBV-ACLF blood specimens; 2) Before detecting the ANGPTL3 level in the blood specimen by using ELISA technology, centrifuge the collected blood specimen at 4000 revolutions for 20 minutes to separate the serum. If it cannot be detected at one time, it needs to be aliquoted and stored frozen at a temperature below -20°C to avoid repeated freezing and thawing; thaw at room temperature before use to ensure that the sample is evenly and fully thawed.

[0032] 3) All components of the blood sample and the kit should be rewarmed for at least 120 min to ensure full rewarming to room temperature; the detection is carried out according to the following steps: Among them, step 3) specifically includes: (I) Set up standard product wells, 0-value wells, blank wells and sample wells. Add 50 μl of ANGPTL3 standard products with different concentrations (80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, 2.5 ng / ml) to each standard product well, add 50 μl of sample diluent to the 0-value well, add nothing to the blank well, and add 50 μl of the sample to be tested to the sample well.

[0033] (II) Except for the blank well, add 100 μl of the detection antibody labeled with horseradish peroxidase (HRP) to the standard product wells, 0-value wells and sample wells.

[0034] (III) Cover the reaction plate with a sealing plate film and incubate it in a 37°C water bath or incubator in the dark for 60 minutes.

[0035] (IV) Uncover the sealing plate film, discard the liquid, pat it dry on the absorbent paper, fill each well with washing solution, let it stand for 20 seconds, discard the washing solution, pat it dry on the absorbent paper, and repeat this 5 times. If an automatic plate washer is used, please wash the plate according to the operation procedure of the plate washer, and add a program of soaking for 30 seconds, which can improve the detection accuracy. After the plate washing is completed and before adding the substrate, pat the reaction plate dry on a clean paper that does not shed debris.

[0036] (V) Mix substrate A (hydrogen peroxide) and substrate B (TMB) in the ELISA kit in a volume ratio of 1:1 to obtain a substrate mixture; add 100 μl of the substrate mixture to all wells.

[0037] (vi) Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator in the dark for 15 minutes.

[0038] (vii) Add 50 μl of stop solution to all wells and read the absorbance (OD value) of each well on a microplate reader at a wavelength of 450 nm.

[0039] (VIII) Using the concentration of the standard as the horizontal axis (6 standard wells, plus 1 0 well, a total of 7 concentration points), and the corresponding absorbance (OD value) as the vertical axis, use computer software and four-parameter logistic curve fitting (4-pl) to create the standard curve equation, see Figure 3 , the standard curve is y=(3.08485-0.03122) / [1+(x / 57.01298) -1.57071 ]+0.03122, r 2 =0.99951, and the sample concentration is calculated using the equation through the sample absorbance (OD value). If the sample is diluted, the concentration value measured by the above method must be multiplied by the dilution factor to obtain the final concentration of the sample.

[0040] 4) Verify the prognostic value of ANGPTL3 in patients with HBV-ACLF; Among them, step 4) specifically includes: (I) Clinical data of HBV-ACLF patients were collected, including admission test and examination data, and complications. Patients were divided into the cirrhosis group and the non-cirrhosis group based on whether they had hepatitis B cirrhosis. Patients were divided into the survival group and the non-survival group based on whether they survived for 30 days.

[0041] (II) SPSS 26.0 software was used to analyze whether ANGPTL3 could be used as an independent prognostic biomarker by univariate and multivariate Cox regression analysis combined with clinical data (Tables 1 and 2).

[0042] (III) Based on the results of step (II), ANGPTL3 was determined to be an independent prognostic biomarker. SPSS 26.0 software was used to divide HBV-ACLF into high ANGPTL3 group and low ANGPTL3 group using the receiver operating characteristic curve (ROC curve) and the area under the curve, and the cumulative survival rate was compared. Figure 1 .in, Figure 1 The correlation between ANGPTL3 level at admission and HBV-ACLF patients. Figure 1 A in the figure represents the distribution of ANGPTL3 in the survival and non-survival groups of HBV-ACLF patients. It can be observed that during the 30-day follow-up period, the ANGPTL3 level of the non-survival group at admission was significantly lower than that of the survival group.Figure 1 In which, B is the receiver operating characteristic curve of ANGPTL3 predicting the 30-day mortality of HBV-ACLF patients. Among them, the red line represents the reference line with no diagnostic value, and the blue line is the ROC curve. The more the ROC curve deviates from the red line, the more significant it is. As can be seen from Figure 1 B in Figure 1 , the area under the ROC curve (AUC) of the ANGPTL3 level for 30-day mortality is 0.77 (0.70 - 0.84), the sensitivity is 0.74, the specificity is 0.68, and the optimal cut-off value is 64.87 ng / mL. Figure 1 In C in Figure 1 , the survival rates of patients in the high ANGPTL3 group and the low ANGPTL3 group after 30 days are shown. It can be observed that the cumulative survival rate of the low ANGPTL3 group is significantly lower than that of the high ANGPTL3 group. Figure 1 In D in Figure 1 , the correlation between the ANGPTL3 level and the mortality rate is shown. Among them, 1 blue dot represents 1 sample, and the red line is the trend line. As can be seen from Figure 1 D in Figure 1 , it can be observed that the lower the ANGPTL3 level, the higher the mortality rate. As the ANGPTL3 concentration increases, the mortality rate shows a downward trend. *P < 0.001.

[0043] Step (i): The blood specimens include the blood of 219 HBV-ACLF patients. Among them, they are grouped according to survival and non-survival: 51 cases in the non-survival group and 168 cases in the survival group; they are grouped according to the presence or absence of hepatitis B cirrhosis: 88 cases of patients without hepatitis B cirrhosis and 131 cases of patients with hepatitis B cirrhosis (among them, 30 cases are non-surviving, accounting for 58.8% of the non-survival group; 101 cases are surviving, accounting for 60.1% of the survival group). See Tables 1 and 2 for details.

[0044] Step (ii): Data analysis indicates that for patients with a basis of hepatitis B cirrhosis, ANGPTL3 can still be used as an independent prognostic biomarker for HBV-ACLF. See Table 2.

[0045] Step (iii): The data analysis results indicate that the worse the outcome of HBV-ACLF patients, the higher the ANGPTL3 level.

[0046] Table 1 Table 2 5) Determine from the results described in step 4) that the ANGPTL3 level is related to the course of hepatitis B-related acute-on-chronic liver failure; Among them, step 5) specifically includes: (1) Collect the clinical data of HBV-ACLF patients during follow-up, including the test and examination results at admission and during regular reexaminations, and the complication status; divide the patients into a survival group and a non-survival group according to whether they survive within 30 days; divide the patients into an improvement group, a fluctuation group, and a deterioration group according to the CLIF-SOF score (Chronic Liver Failure-Sequential Organ Failure Assessment). Among them, The improvement group is defined as: the CLIF-SOFA score decreases by ≥2 points; The fluctuation group is defined as: the change (increase or decrease) in the CLIF-SOFA score is <2 points; The deterioration group is defined as: the CLIF-SOFA score increases by ≥2 points.

[0047] (2) Use SPSS 26.0 software and perform Mann-Whitney U test analysis, combined with clinical practice, to analyze whether there are significant differences in the ANGPTL3 levels among the groups.

[0048] The blood specimens in step (1) include 84 HBV-ACLF patients during follow-up; among them, grouped by disease course: the blood of 36 patients in the improvement group, 30 patients in the fluctuation group, and 18 patients in the deterioration group; grouped by non-survival and survival: the blood of 35 patients in the non-survival group and 49 patients in the survival group.

[0049] The results in step (2) suggest that the ANGPTL3 level is significantly correlated with the disease condition, specifically as follows Figure 2 . Among them Figure 2 shows the changes in the ANGPTL3 levels in the improvement group, fluctuation group, and deterioration group between the initial (at admission) and final observation endpoints (14 days after admission or at discharge). It can be observed that there is no obvious change in the ANGPTL3 level in the fluctuation group between the initial and observation endpoints; the ANGPTL3 level in the deterioration group is significantly lower at the observation endpoint than at admission; the ANGPTL3 level in the improvement group is significantly higher at the observation endpoint than at admission.

[0050] Example 2 This example provides a detection kit for the prognosis assessment of acute-on-chronic liver failure.

[0051] (1) Composition of the kit: ANPTL3 specific antibody: including a capture antibody (coated on a microplate) and a human ANGPTL3 conjugate (coupled with horseradish peroxidase using a preservative), purchased from R&D Systems.

[0052] Standard: The concentration of recombinant human ANGPTL3 protein after reconstitution is 100 ng / mL, purchased from R&D Systems.

[0053] Standard diluent: A dilution buffer suitable for serum / plasma samples, which is animal serum containing preservatives and is purchased from R&D Systems.

[0054] Assay diluent: A buffered protein matrix containing preservatives, purchased from R&D Systems.

[0055] Chromogenic substrate: Chromogenic reagent A (HRP) and chromogenic reagent B (TMB) are uniformly mixed at a volume ratio of 1:1 and purchased from R&D Systems.

[0056] Stop solution: 2N sulfuric acid stop solution.

[0057] (2) Detection method Steps: (A) Sample collection: Collect venous blood from patients, centrifuge to separate serum / plasma (to be completed within 2 hours and stored at -80°C), collect the serum to obtain the sample to be tested.

[0058] (B) Detection process: Add the standard product and the sample to be tested to the microplate pre-coated with the capture antibody, and add 100 μl of assay diluent to each well, and incubate at 37°C for 2 hours.

[0059] After washing, add 200 μl of human ANGPTL3 conjugate to each well and incubate at room temperature for 1 hour.

[0060] After repeating the washing step, add 200 μl of chromogenic substrate to each well and incubate in the dark at room temperature for 30 minutes.

[0061] Add 50 μl of stop solution to each well. After termination, measure the absorbance at 450 nm within 30 minutes. At the same time, the absorbance at 540 nm or 570 nm can be set for wavelength correction.

[0062] (C) Result analysis: Calculate the ANGPTL3 concentration through the standard curve.

[0063] Using the above kit and detection method, 219 HBV-ACLF patients in a certain tertiary hospital were detected. According to the ANGPTL3 concentration of the patients, the patients were divided into a high-risk group and a low-risk group. Among them, the ANGPTL3 concentration in the high-risk group was lower than 64.87 ng / ml, and the low-risk group was higher than or equal to 64.87 ng / ml. The results showed that: The 28-day case fatality rate was highly matched with the ANGPTL3 level. Among them, there were 128 patients in the high-risk group, and 13 patients (10.1%) died within 30 days, while there were 91 patients in the low-risk group, and 37 (40.6%) died within 30 days.

[0064] Example 3 The kit in Example 2 was tested for specificity, sensitivity and precision.

[0065] Specificity of the kit in Example 2: The kit in Example 2 was used to detect natural and recombinant human angiopoietin-like protein 3 (ANGPTL3) and structural analogs (purchased from Jianglai Biotech, model name JL19947-48T). The results showed that this kit recognized natural and recombinant human angiopoietin-like protein 3 (ANGPTL3) and had no cross-reaction with structural analogs.

[0066] The sensitivity and precision of the kit in Example 2 were tested, as shown in Tables 3-5: Sensitivity: The kit and method in Example 2 were used to detect standard samples with ANGPTL3 concentrations of 0.156 ng / ml, 0.313 ng / ml, 0.625 ng / ml, 1.25 ng / ml, 2.5 ng / ml, 5 ng / ml and 10 ng / ml respectively, and the absorbance at 450 nm was measured. With the ANGPTL3 concentration as the abscissa and the absorbance at 450 nm as the ordinate, a standard curve was created. The results are shown in Figure 4 ; The kit in Example 2 was used to detect 10 zero standards (blank controls without the substance to be measured), and the absorbance at 450 nm was measured. The detection limit of the OD value (450 nm) was calculated by adding the average value of the OD of the 10 zero standard concentrations plus twice the SD (standard deviation), and the corresponding lowest detectable concentration was 0.006719 ng / ml. See Tables 3 and Figure 4 .

[0067] Intra-assay precision: Three samples with known concentrations were randomly selected, and each sample was tested 20 times on the same plate to evaluate the precision of the internal analysis. The relevant data for the determination of internal precision are as follows: For the three samples, the number of tests n was 20 for all. The average value of sample one was 1.05 ng / ml, the standard deviation was 0.04, and the coefficient of variation was 3.8%; the average value of sample two was 3.36 ng / ml, the standard deviation was 0.09, and the coefficient of variation was 2.7%; the average value of sample three was 6.80 ng / ml, the standard deviation was 0.18, and the coefficient of variation was 2.6%.

[0068] Inter-batch precision: Three samples with known concentrations were taken and tested by at least three technicians using two batches of components in 20 independent analyses to evaluate the precision between analyses. The precision data between detection intervals showed that the number of tests n for all three samples was 20. The average value of sample one was 1.02 ng / mL, the standard deviation was 0.07, and the coefficient of variation was 6.9%; the average value of sample two was 3.18 ng / mL, the standard deviation was 0.20, and the coefficient of variation was 6.3%; the average value of sample three was 6.74 ng / mL, the standard deviation was 0.60, and the coefficient of variation was 8.9%.

[0069] Table 3 Table 4 Table 5 Therefore, it can be seen from Table 4 and Table 5 that the within-batch coefficient of variation < 10% and the inter-batch coefficient of variation < 15%. The above results indicate that the precision of the detection kit of the present invention is good and meets the requirements of clinical detection.

[0070] As described above, the above is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. Use of ANGPTL3 as a biomarker in the preparation of a product for the diagnosis and / or course diagnosis and / or prognosis assessment of hepatitis B-related acute-on-chronic liver failure.

2. The application according to claim 1, wherein The hepatitis B-related acute-on-chronic liver failure includes hepatitis B-related acute-on-chronic liver failure with a basis of hepatitis B cirrhosis.

3. The application according to claim 1, characterized in that, The product includes a kit for detecting the content of the biomarker, and the kit contains reagents for detecting the content of the biomarker. The reagents include one of the reagents required for immunohistochemistry, Western-Blot, immunoblotting, and enzyme-linked immunosorbent assay.

4. The application according to any one of claims 1 to 3, characterized in that, The course diagnosis includes using the product to detect the ANGPTL3 level in patients before and after treatment, and judging whether the course is improved, fluctuating, or deteriorated according to the change in the ANGPTL3 level.

5. A kit, characterized in that, Contains reagents for detecting the content of a biomarker, the biomarker is ANGPTL3, and the reagents include one of the reagents required for immunohistochemistry, Western-Blot, immunoblotting, and enzyme-linked immunosorbent assay; When using the kit to detect the content of the biomarker in a test sample, if the detected concentration of the biomarker in the test sample is lower than 64.87 ng / mL, then the source of the test sample is determined to be a high-risk patient with HBV-ACLF.

6. The kit according to claim 5, characterized in that, The kit is an ELISA kit, and the ELISA kit includes a capture antibody, a detection antibody, a recombinant human ANGPTL3 protein standard, a sample treatment solution, a chromogenic solution, and a stop solution.

7. Use of ANGPTL3 in the construction of a prognostic prediction model for hepatitis B-related acute-on-chronic liver failure.

8. Use of ANGPTL3 as a target in screening drugs for the treatment of hepatitis B-related acute-on-chronic liver failure.

9. Use of ANGPTL3 in the preparation of an inhibitor for hepatitis B-related acute-on-chronic liver failure.

10. A method for screening drugs, characterized in that, Including the following steps: Detect the content of ANGPTL3 in the patient's sample before and after administration; If the content of ANGPTL3 increases, the drug to be screened has a therapeutic effect on HBV-ACLF; If the content of ANGPTL3 remains unchanged or decreases, the drug to be screened does not have a therapeutic effect on HBV-ACLF; The sample includes blood.

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