Application of composition in preparation of liver fibrosis detection reagent for oligosaccharide chain chronic hepatitis B patient

Abstract

The invention provides a detection reagent for detecting hepatic fibrosis of chronic hepatitis B patients based on oligosaccharide chains and a preparation method and application of the detection reagent, the detection reagent is formed by mixing the following reagents: a reagent A is prepared by adding SDS with the mass concentration of 1-5% into an ammonium bicarbonate solution with the concentration of 10mM and the pH value of 8.3; the reagent B is prepared by mixing 0.05-10 units / 10 microliters of glycosamine acylase, NP-40 with the mass concentration of 10% and an ammonium bicarbonate solution with the concentration of 10 mM and the pH value of 8.3, and the pH value of the mixed solution is 5-9; the reagent C is an organic matter reducing agent with the concentration of 0.02 mM to 1M, which is prepared by dissolving 8-aminopyrene-1, 3, 6-trisulfonic acid in DMSO (Dimethylsulfoxide); and a reagent D: a stop solution. The invention provides a method for establishing a serum natural oligosaccharide N-glycome map model by determining a natural oligosaccharide N-glycome map in serum through the reagent and then quantizing a peak value and performing statistical analysis. The hepatic fibrosis of a chronic hepatitis B patient is detected by measuring the change of natural glycosylation modification of protein in a physiological and pathological state.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a method for detecting liver fibrosis in patients with chronic hepatitis B, and specifically to a method for detecting liver fibrosis in patients with chronic hepatitis B based on a serum oligosaccharide chain G-Test specific fingerprint. Background Art

[0002] Chronic hepatitis B (CHB) is a chronic liver inflammatory disease caused by persistent infection with hepatitis B virus (HBV) for more than 6 months.

[0003] Chronic and persistent inflammatory responses in the liver can lead to the development of liver fibrosis. Cirrhosis is the terminal stage of chronic inflammation and fibrosis, and severe cirrhosis will eventually lead to the development of liver cancer. Among patients with cirrhosis and liver cancer (HCC) in my country, 77% and 84% are caused by HBV, respectively, which has become an important public health issue. Although domestic and international guidelines have gradually relaxed the indications for antiviral treatment of chronic hepatitis B, there are still a considerable number of chronic hepatitis B patients who fail to receive antiviral treatment because they do not meet the existing standards, which may lead to further development of the disease. Therefore, it is currently a vital task to allow these chronically infected people to be tested and treated before more serious diseases develop.

[0004] The clinical diagnosis and treatment of chronic hepatitis B requires a comprehensive assessment based on the results of serological, virological, biochemical, imaging, pathological, and other auxiliary examinations in patients with chronic HBV infection. Changes in the liver function-related serum biochemical indicators ALT, AST, ALB, GLB, and total bilirubin can reflect liver function status. Virological indicators HBsAg, Anti-HBs, HBeAg, Anti-HBe, Anti-HBc, and HBV DNA can determine the level of HBV viral replication, predict disease progression, and be used to select indications for antiviral treatment and assess its efficacy. Imaging and pathological results can reflect the extent of liver lesions.

[0005] Due to the complexity of liver function, various technical means of evaluating liver fibrosis have certain limitations. For example, serological tests require the simultaneous detection of multiple indicators and a comprehensive judgment to draw conclusions, but they cannot accurately stage liver fibrosis, and there is a large discrepancy between them and pathological test results. Imaging examinations are relatively reliable in distinguishing between cirrhosis and mild liver fibrosis, but they cannot clearly stage liver fibrosis in the intermediate area, and are affected by the patient's transaminase level, bilirubin, ascites, obesity, and operator experience, resulting in deviations. The interpretation of the results needs to be combined with indicators such as the patient's ALT and bilirubin levels. Pathological examination Liver biopsy is the gold standard for assessing the degree of liver fibrosis, but due to its invasiveness, high risk of complications, and poor consistency, it is not suitable for routine use. Patient acceptance is poor and the cost is high. Therefore, new markers are needed to assist in the diagnosis of liver fibrosis in patients with chronic hepatitis B.

[0006] Protein glycosylation is the most common post-translational modification of proteins. It involves the transfer of sugars to the amino groups of asparagine (ASN) on protein peptide chains under the action of glycosyltransferases to form N-linked glycans or to the hydroxyl oxygen atoms of threonine / serine to form O-linked glycans, which participate in regulating protein function. Most glycoproteins are secreted proteins and are widely present in cell membranes, extracellular matrix, plasma, and mucus. N-glycan chains on proteins regulate protein structure, stability, and activity through processing and modification. Therefore, sugar chains in glycoproteins play an important role in maintaining the body's biological functions, thereby endowing glycoproteins with multiple biological functions. Therefore, understanding changes in sugar chains can help elucidate the molecular mechanisms of abnormal biological behaviors such as inflammation, tumor cell invasion and metastasis to surrounding tissues.

[0007] Abnormal changes in protein N-glycans have been found in a variety of tumors and inflammatory diseases, and alterations in terminal sialic acid modification of N-glycans are one such finding. Sialic acid is a negatively charged, nine-carbon sugar compound that is widely present in organisms and often located at the termini of glycan chains. Sialic acid is linked to galactose or N-acetylgalactosamine on glycan chains via α-2,3 or α-2,6 glycosidic bonds by sialidases to form polysialic acid chains. Studies have shown that sialic acid at the termini of glycan chains plays an important role in regulating cell recognition, molecular interactions, viral infection, immune responses, and signal transduction. Furthermore, sialic acid modification of cell surface glycoproteins is tissue- and cell-specific. Numerous studies have shown that abnormal sialic acid modification contributes to the development and progression of various diseases, including infectious diseases and tumor infiltration and metastasis. Furthermore, N-glycoproteins in human serum are primarily synthesized by the liver and B lymphocytes. Abnormalities in the glycan structure and quantity of N-glycoproteins often reflect liver and B lymphocyte pathology. Therefore, detecting changes in sialic acid-related oligosaccharide chains has potential clinical value in assisting the diagnosis of chronic hepatitis B liver fibrosis. Summary of the Invention

[0008] There are problems with the current clinical testing for liver fibrosis in patients with chronic hepatitis B. For example, serological tests require the simultaneous detection of multiple indicators and a comprehensive judgment to draw conclusions, but they cannot accurately stage liver fibrosis and there is a large discrepancy between them and pathological test results. Imaging examinations are relatively reliable in distinguishing between cirrhosis and mild liver fibrosis, but cannot clearly stage liver fibrosis in the intermediate area and are subject to deviations caused by the patient's transaminase level, bilirubin, ascites, obesity, and operator experience. The interpretation of the results needs to be combined with indicators such as the patient's ALT and bilirubin levels. Pathological examination liver biopsy is the gold standard for assessing the degree of liver fibrosis, but due to its invasiveness, high risk of complications, and poor consistency, it is not suitable for routine use, has poor patient acceptance, and is expensive. The present invention provides a detection reagent for liver fibrosis in patients with chronic hepatitis B. The reagent is used to measure the natural oligosaccharide N-glycome profile in serum, and then the peak values are quantified and statistically analyzed, thereby providing a method for establishing a serum natural oligosaccharide N-glycome profile model for liver fibrosis in patients with chronic hepatitis B. Liver fibrosis in patients with chronic hepatitis B is detected by measuring changes in the natural glycosylation modification of proteins under physiological and pathological conditions.

[0009] The technical solution adopted in the present invention is as follows:

[0010] A detection reagent for detecting liver fibrosis in patients with chronic hepatitis B based on oligosaccharide chains, comprising the following reagents:

[0011] Reagent A: Prepared by adding 1-5% SDS to a 10 mM ammonium bicarbonate solution with a pH of 8.3.

[0012] Reagent B: Prepared by mixing 0.05-10 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the mixed solution is 5-9.

[0013] Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent with a concentration of 0.02 mM to 1 M;

[0014] Reagent D: Stop solution.

[0015] Preferably, the volume ratio of reagent A, reagent B and reagent C is 1:1:1.

[0016] Preferably, the volumes of reagent A, reagent B, and reagent C are all 5 μl.

[0017] Preferably, the reagent D is ultrapure water.

[0018] A method for preparing a detection reagent for detecting liver fibrosis in chronic hepatitis B patients based on oligosaccharide chains, comprising the following steps:

[0019] Step 1 Preparation of oligosaccharide chains

[0020] Add 5 μl of reagent A to 5 μl of inactivated serum sample for denaturation. After cooling to room temperature, add 5 μl of reagent B and react at 37°C for 3 h, then dry.

[0021] Step 2: Labeling of oligosaccharide chains

[0022] Add 5 μl of reagent C to the sample obtained after drying in step 1, react at 60°C for 1 hour, and then perform fluorescent labeling. Then, add 100 μl of reagent D to terminate the labeling reaction.

[0023] Step 3: Oligosaccharide chain separation and analysis

[0024] Take 10 μl of the oligosaccharide chain labeled liquid and use an analyzer to separate and detect the N-oligosaccharide chains to obtain the natural oligosaccharide N-glycome profile;

[0025] Step 4: Data processing and analysis

[0026] Peak quantification of N-glycan profiles: The relative content of each peak was calculated by dividing the peak height of each peak by the sum of the heights of all peaks.

[0027] A composition is used in the preparation of an oligosaccharide chain reagent for detecting liver fibrosis in patients with chronic hepatitis B. The composition consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum. The composition is used to detect liver fibrosis in patients with chronic hepatitis B through the value of G2S2+G2S1+G2S2F.

[0028] The G1F is an isomer.

[0029] The present invention provides a new reagent and application method for detecting liver fibrosis in chronic hepatitis B patients using natural oligosaccharide chains, and statistical analysis is performed by measuring the G-Test specific fingerprint of serum natural oligosaccharide chains.

[0030] Materials and methods:

[0031] 1. Test samples: Serum from patients with chronic hepatitis B without fibrosis and patients with chronic hepatitis B with fibrosis.

[0032] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0033] 3. Reagent preparation:

[0034] 1. Reagent A: Prepared by adding 1-5% SDS to a 10 mM ammonium bicarbonate solution with a pH of 8.3.

[0035] 2. Reagent B: Prepared by mixing 0.05-10 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the mixed solution should be 5-9.

[0036] 3. Reagent C: Prepare an organic reducing agent with a concentration of 0.02 mM to 1 M by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO;

[0037] 4. Reagent D: stop solution.

[0038] 4. N-Glycan Profile Detection

[0039] 1. Preparation of oligosaccharide chains

[0040] 5 μl of reagent A was added to 5 μl of inactivated serum sample for denaturation; after cooling to room temperature, 5 μl of reagent B was added, reacted at 37°C for 3 h, and then dried.

[0041] 2. Labeling of oligosaccharide chains

[0042] 1) Add 5 μl of Reagent C to the dried sample and incubate at 60°C for 1 hour for fluorescent labeling.

[0043] 2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

[0044] 3. Oligosaccharide chain separation and analysis

[0045] 10 μl of the oligosaccharide chain labeled liquid was taken and N-oligosaccharide chain separation and detection was performed on the ABI3500dx instrument to obtain the natural oligosaccharide N-glycome map.

[0046] 4. Data processing and analysis

[0047] (1) Peak quantification of N-glycome profiles: The peak height of each peak was divided by the sum of the heights of all peaks to calculate the relative content of each peak.

[0048] (2) Analysis of N-glycome data of chronic hepatitis B patients without fibrosis and chronic hepatitis B patients with fibrosis: The N-glycome data of chronic hepatitis B patients without fibrosis and chronic hepatitis B patients with fibrosis were compared and analyzed. The peak height of each peak was divided by the sum of the heights of all peaks to calculate the relative content of each peak, i.e., the peak quantification of the N-glycome spectrum. Then, the 9 N-oligosaccharide chain peaks in the quantified N-glycome spectrum of the chronic hepatitis B fibrosis group and the chronic hepatitis B non-fibrosis group were compared and statistically analyzed. The composition of the N-glycome spectrum consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2, of which G1F is an isomer; the liver fibrosis of chronic hepatitis B patients was detected by the composition G2S2+G2S1+G2S2F value.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The peaks of the G-Test fingerprint were quantified, and then the relative content results of each peak in patients with chronic hepatitis B with liver fibrosis (149 cases) and patients with chronic hepatitis B without liver fibrosis (136 cases) were compared and analyzed. It was found that the N-glycan compositions G2S2, G2S1 and G2S2F were significantly different between the two groups (p < 0.05); therefore, the model based on the composition G2S2+G2S1+G2S2F had an AUC value of 0.859 ( Figure 2 ), when the cutoff value of the G2S2+G2S1+G2S2F model detection was 0.405, the sensitivity of detecting liver fibrosis in chronic hepatitis B was 81.2% and the specificity was 78.5%, indicating that the N-glycan composition G2S2+G2S1+G2S2F in serum can be used as a marker for auxiliary diagnosis of liver fibrosis in patients with chronic hepatitis B.

[0051] (2) The G-Test method proposed in the present invention is based on the capillary microelectrophoresis technology (DSA-FACE) of a DNA sequencer. After fluorescently labeling the N-glycan chains of glycoproteins in serum samples, they are separated by capillary microelectrophoresis. The glycoprotein content obtained by measuring the fluorescence signal is the natural oligosaccharide N-glycome profile. By detecting the correlation between changes in sialic acid oligosaccharide chains under physiological and pathological conditions and the disease state, the present invention can establish a predictive model of N-glycan composition based on these changes to assist in the diagnosis of chronic hepatitis B with liver fibrosis.

[0052] (3) The G-Test natural N-glycome profile model constructed based on the method of the present invention can enable many patients to undergo routine, non-invasive testing, helping doctors and patients to timely monitor the occurrence and progression of liver fibrosis in hepatitis B patients. The detection technology of the present invention has the advantages of high sensitivity, simple operation, trace amount (5μl serum), high repeatability, good stability, and high throughput (96-well plate), and can be promoted and used in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is the serum natural oligosaccharide N-glycome profile of chronic hepatitis B patients without liver fibrosis and chronic hepatitis B patients with liver fibrosis;

[0054] Figure 2 This is a ROC curve diagram of a model for identifying chronic hepatitis B patients with liver fibrosis based on the N-sugar composition G2S2+G2S1+G2S2F; the total number of samples tested was 285, including 149 serum samples from chronic hepatitis B patients with liver fibrosis and 136 serum samples from chronic hepatitis B patients without liver fibrosis, and the area under the ROC curve AUC = 0.859. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below with reference to the examples and accompanying drawings. It should be noted that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or under conditions recommended by the manufacturer.

[0056] The liver fibrosis status of chronic hepatitis B patients was detected by measuring the serum natural oligosaccharide N-glycome profile and performing statistical analysis. The materials and methods used were as shown in the following examples.

[0057] Example 1

[0058] 1. Test samples: Serum from 149 patients with chronic hepatitis B and liver fibrosis and 136 patients with chronic hepatitis B and liver fibrosis.

[0059] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0060] 3. Reagent Preparation:

[0061] 1) Reagent A: Prepared by adding 3% SDS to a 10 mM ammonium bicarbonate solution.

[0062] 2) Reagent B: Prepared by mixing 5 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the mixed solution is 7.

[0063] 3) Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent at a concentration of 10 mM;

[0064] 4) Reagent D: ultrapure water.

[0065] 4. N-glycomic profile detection:

[0066] (1) Preparation of oligosaccharide chains

[0067] 5 μl of reagent A was added to 5 μl of serum sample (inactivated) for denaturation; after cooling to room temperature, 5 μl of reagent B was added, reacted at 37°C for 3 h, and then dried.

[0068] (2) Labeling of oligosaccharide chains

[0069] 1) Add 5 μl of Reagent C to the dried sample and incubate at 60°C for 1 hour for fluorescent labeling.

[0070] 2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

[0071] (3) Oligosaccharide chain separation and analysis

[0072] 10 μl of the oligosaccharide chain labeled liquid was taken and N-oligosaccharide chain separation and detection was performed on the ABI3500dx instrument to obtain the natural oligosaccharide N-glycome map.

[0073] (4) Data processing and analysis

[0074] 1) Peak quantification of N-glycome profiles: The relative content of each peak was calculated by dividing the peak height of each peak by the sum of the heights of all peaks.

[0075] 2) Analysis of N-glycome data between patients with chronic hepatitis B and those without liver fibrosis: Comparative analysis of N-glycome data between patients with chronic hepatitis B and those without liver fibrosis. Figure 1 As shown in the figure, the N-glycome map of human serum shows nearly 9 N-oligosaccharide chain peaks. Different oligosaccharide chains exhibit different mobility due to their different charges and molecular sizes. That is, different peaks on the N-glycome map represent different oligosaccharide chains, and the peak height represents the relative content of the oligosaccharide chains. Figure 1Figure A shows the serum N-glycan profile of a chronic hepatitis B patient without liver fibrosis, and Figure B shows the serum N-glycan profile of a chronic hepatitis B patient with liver fibrosis. The N-glycome profile consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, with G1F being an isomer. The G2S2+G2S1+G2S2F value is calculated to aid in determining the liver fibrosis status of chronic hepatitis B patients.

[0076] The model based on the combination of G2S2+G2S1+G2S2F achieved an AUC value of 0.859 under the ROC curve in differentiating patients with chronic hepatitis B and liver fibrosis ( Figure 2 ), when the cutoff value of the G2S2+G2S1+G2S2F model detection was 0.405, the sensitivity of detecting liver fibrosis in chronic hepatitis B was 81.2% and the specificity was 78.5%, indicating that the N-glycan composition G2S2+G2S1+G2S2F in serum can be used as a marker for auxiliary diagnosis of liver fibrosis in patients with chronic hepatitis B.

[0077] Example 2

[0078] 1. Test samples: Serum from 149 patients with chronic hepatitis B and liver fibrosis and 136 patients with chronic hepatitis B and liver fibrosis.

[0079] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0080] 3. Reagent Preparation:

[0081] 1) Reagent A: Prepared by adding 1% SDS to a 10 mM ammonium bicarbonate solution.

[0082] 2) Reagent B: Prepared by mixing 0.05 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the solution is 5.

[0083] 3) Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent at a concentration of 0.02 mM;

[0084] 4) Reagent D: ultrapure water.

[0085] 4. N-glycome profile detection was the same as in Example 1.

[0086] Example 3

[0087] 1. Test samples: Serum from 198 patients with chronic hepatitis B and liver fibrosis and 145 patients with chronic hepatitis B and liver fibrosis.

[0088] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0089] 3. Reagent Preparation: 1) Reagent A: Prepare by adding 5% SDS to a 10 mM ammonium bicarbonate solution;

[0090] 2) Reagent B: Prepared by mixing 10 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the solution is 9.

[0091] 3) Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent at a concentration of 1 M;

[0092] 4) Reagent D: ultrapure water.

[0093] 4. N-glycomic profile detection:

[0094] (1) Preparation of oligosaccharide chains

[0095] 5 μl of reagent A was added to 5 μl of serum sample (inactivated) for denaturation; after cooling to room temperature, 5 μl of reagent B was added, reacted at 37°C for 3 h, and then dried.

[0096] (2) Labeling of oligosaccharide chains

[0097] 1) Add 5 μl of Reagent C to the dried sample and incubate at 60°C for 1 hour for fluorescent labeling.

[0098] 2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

[0099] (3) Oligosaccharide chain separation and analysis

[0100] 10 μl of the oligosaccharide chain labeled liquid was taken and N-oligosaccharide chain separation and detection was performed on the ABI3500dx instrument to obtain the natural oligosaccharide N-glycome map.

[0101] (4) Data processing and analysis

[0102] 1) Peak quantification of N-glycome profiles: The relative content of each peak was calculated by dividing the peak height of each peak by the sum of the heights of all peaks.

[0103] 2) Analysis of N-glycome data between patients with chronic hepatitis B and those without liver fibrosis: Comparative analysis of N-glycome data between patients with chronic hepatitis B and those without liver fibrosis. Figure 1As shown in the figure, the N-glycome map of human serum shows nearly 9 N-oligosaccharide chain peaks. Different oligosaccharide chains exhibit different mobility due to their different charges and molecular sizes. That is, different peaks on the N-glycome map represent different oligosaccharide chains, and the peak height represents the relative content of the oligosaccharide chains. Figure 1 Figure A shows the serum N-glycan profile of a chronic hepatitis B patient without liver fibrosis, and Figure B shows the serum N-glycan profile of a chronic hepatitis B patient with liver fibrosis. The N-glycome profile consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, with G1F being an isomer. The G2S2+G2S1+G2S2F value is calculated to aid in determining the liver fibrosis status of chronic hepatitis B patients.

[0104] In order to further verify the accuracy of the model, serum normal control group sample data of 198 patients with chronic hepatitis B with liver fibrosis and 145 patients with chronic hepatitis B without liver fibrosis were used to verify the model established based on the composition G2S2+G2S1+G2S2F. Based on Example 1, when the cutoff value was 0.405, the discrimination was good. The sample model in this example was verified, and the sensitivity of chronic hepatitis B liver fibrosis detection was 80.81%, and the specificity was 79.80%, indicating that the N-glycan composition G2S2+G2S1+G2S2F in serum can be used as a marker for auxiliary diagnosis of liver fibrosis in patients with chronic hepatitis B. The verification results are shown in Table 1.

[0105] Table 1 Validation results of 198 patients with chronic hepatitis B and 145 patients without liver fibrosis

[0106] type Sensitivity Specificity Chronic hepatitis B with fibrosis 80.81(160 / 198) - Chronic hepatitis B without fibrosis - 79.80％(159 / 198)

[0107] Compared with existing technologies, this detection technology can detect the correlation between changes in sialic acid oligosaccharide chains under physiological and pathological conditions and disease status. Based on these changes, a predictive model of N-glycan composition can be established to assist in determining the liver fibrosis status of patients with chronic hepatitis B. The G-Test natural N-glycome profile model constructed based on the method of this invention can enable a large number of patients to undergo routine, non-invasive testing, helping doctors and patients to timely monitor the occurrence and progression of liver fibrosis in patients with chronic hepatitis B, and can be promoted for clinical use.

[0108] The specific embodiments described above in conjunction with the accompanying drawings further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above is only a specific embodiment of the present invention, but is not a limitation on the scope of protection of the present invention. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. that are made within the spirit and principles of the present invention without creative work should be included in the scope of protection of the present invention.

Claims

1. A composition for preparing an oligosaccharide chain for detecting liver fibrosis in chronic hepatitis B patients, characterized in that: The composition consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum, and the composition is used to detect liver fibrosis in chronic hepatitis B patients through the value of G2S2+G2S1+G2S2F.

2. Use of a composition according to claim 1 in the preparation of an oligosaccharide chain chronic hepatitis B patient liver fibrosis detection reagent, characterized in that: The G1F is an isomer.

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