CN115825202A - Detection reagent for detecting hepatic fibrosis of chronic hepatitis B patient based on oligosaccharide chain, preparation method and application - Google Patents

Abstract

The invention provides a detection reagent for detecting hepatic fibrosis of a chronic hepatitis B patient based on oligosaccharide chains, a preparation method and application thereof, wherein the detection reagent is formed by mixing the following reagents, namely a reagent A: adding SDS with the mass concentration of 1-5% into ammonium bicarbonate solution with the concentration of 10mM and the pH value of 8.3 to prepare the ammonium bicarbonate solution; and (3) reagent B: is prepared by mixing 0.05-10 units/10 mul of glycosaminoglycan-acyl enzyme, NP-40 with the mass concentration of 10 percent and ammonium bicarbonate solution with the concentration of 10mM and the pH value of 8.3, and the pH value of the mixed solution is 5-9; and (3) reagent C: 8-aminopyrene-1, 3, 6-trisulfonic acid is dissolved in DMSO to prepare an organic reducing agent with the concentration of 0.02mM to 1M; and (3) reagent D: and (4) stopping the 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 by the reagent, quantifying a peak value and carrying out statistical analysis, and the hepatic fibrosis of a chronic hepatitis B patient is detected by determining the change of natural glycosylation modification of protein under a physiological and pathological state.

Description

A test for detecting liver fibrosis in patients with chronic hepatitis B based on oligosaccharide chains Agents, preparation methods and applications

technical field

The invention belongs to the technical field of biomedicine and relates to a method for detecting liver fibrosis in patients with chronic hepatitis B, in particular to a method for detecting liver fibrosis in patients with chronic hepatitis B based on the specific fingerprint of serum oligosaccharide chain G-Test.

Background technique

Chronic hepatitis B (CHB) is a chronic liver inflammatory disease caused by persistent infection of hepatitis B virus (HBV) for more than 6 months. The global prevalence of HBV infection in 2019 was 4.1%, suggesting that there are still 316 million HBV-infected people worldwide. At present, the prevalence rate of HBsAg in the whole population in my country is 5%-6%, and there are about 70 million cases of chronic HBV infection, including 20-30 million cases of CHB patients.

Chronic and persistent inflammatory response in the liver can lead to liver fibrosis. Cirrhosis is the end stage of chronic inflammation and fibrosis, and severe cirrhosis will eventually lead to liver cancer. HBV causes 77% and 84% of patients with liver cirrhosis and liver cancer (HCC) in my country, which has become an important public health problem. Although domestic and foreign guidelines have gradually relaxed the indications for antiviral treatment of chronic hepatitis B, there are still a considerable number of patients with chronic hepatitis B who do not meet the existing standards and do not receive antiviral treatment, which may lead to further development of the disease , so it is a crucial task for these chronically infected people to detect and treat them before more serious diseases appear.

The clinical diagnosis and treatment of chronic hepatitis B need to be judged based on the results of serology, virology, biochemistry, imaging, pathology and other auxiliary examinations of patients with chronic HBV infection. Among them, changes in liver function-related serum biochemical indexes ALT, AST, ALB, GLB and total bilirubin can reflect the state of liver function; virological indexes HBsAg, Anti-HBs, HBeAg, Anti-HBe, Anti-HBc and HBV DNA can judge The virus replication level of HBV infection can predict the progress of the disease and be used in the selection of indications for antiviral therapy and judgment of curative effect, etc. Imaging and pathological results can reflect the degree of liver lesions.

Due to the complexity of liver function, various technical means for evaluating liver fibrosis have certain limitations. For example, serological examination needs to detect multiple indicators at the same time, and a conclusion can be drawn through comprehensive judgment, but it cannot accurately stage liver fibrosis. And there is a large difference between pathological examination results; imaging examinations are relatively reliable in distinguishing liver cirrhosis or mild liver fibrosis, but liver fibrosis in the middle area cannot be clearly staged, and it is affected by the patient's transaminase level, bilirubin, ascites, Obesity and operator experience can cause deviations, and 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 evaluating the degree of liver fibrosis, but due to invasiveness, complications, etc. High risk and poor consistency, not suitable for routine application, poor patient acceptance and high cost. Therefore, new markers are needed for auxiliary diagnosis of liver fibrosis in patients with chronic hepatitis B.

Protein glycosylation (Glycosylation) is one of the most common post-translational modifications of proteins. It is the transfer of sugars to the amine groups of asparagine (ASN) on the protein peptide chain under the action of glycosyltransferases to form N-linked polymers. The hydroxyl oxygen atoms of sugars or threonine/serine form O-linked glycans, which are involved in the regulation of protein functions. Most glycoproteins are secreted proteins, widely present in cell membranes, interstitial cells, plasma, and mucus. N-sugar chains on proteins regulate the structure, stability and activity of proteins through processing and modification, so the sugar chains in glycoproteins play an important role in maintaining the biological functions of the body, thus endowing glycoproteins with various biological functions. Therefore, understanding the changes in sugar chains is helpful to elucidate the molecular mechanisms of abnormal biological behaviors such as inflammation, tumor cell invasion and metastasis of surrounding tissues.

At present, abnormal changes of protein N-sugar chains have been found in various tumors and inflammatory diseases, and the modification of the terminal sialic acid of N-sugar chains is one of them. Sialic acid is a kind of negatively charged 9-carbon sugar compound, which widely exists in organisms and is often located at the end of the glycan chain; under the action of sialidase, it can be combined with Galactose or N-acetylgalactosamine on the sugar chains are linked to form polysialic acid chains. Studies have found that the sialic acid at the end of the glycan chain plays an important role in regulating cell mutual recognition, molecular interaction, virus infection, immune response and signal transduction, and the sialic acid modification of cell surface glycoproteins has tissue and cell specificity. Many studies have shown that abnormal sialic acid modification leads to the occurrence and development of various diseases, such as the occurrence of infectious diseases, and the infiltration and metastasis of tumors are closely related. In addition, N-glycoprotein in human serum is mainly synthesized by liver and B lymphocytes, and abnormal glycan structure and quantity of N-glycoprotein can often reflect liver and B lymphocyte lesions. Therefore, detection of changes in sialic acid-related oligosaccharide chains has potential clinical value in the auxiliary diagnosis of liver fibrosis in chronic hepatitis B.

Contents of the invention

Aiming at the problems existing in the detection of liver fibrosis in patients with chronic hepatitis B currently used clinically, such as serological examination needs to detect multiple indicators at the same time, and a conclusion can be drawn from a comprehensive judgment, but it cannot accurately stage liver fibrosis, and and The differences between pathological test results are large; imaging examinations are relatively reliable in distinguishing liver cirrhosis or mild liver fibrosis, but liver fibrosis in the middle area cannot be clearly staged, and it is affected by patient transaminase levels, bilirubin, ascites, obesity and Due to the influence of different operator experience, deviations are caused, and 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 evaluating the degree of liver fibrosis, but due to invasiveness and high risk of complications And the consistency is poor, it is not suitable for routine application, its patient acceptance is poor, and the cost is high. The invention provides a reagent for detecting liver fibrosis in patients with chronic hepatitis B, which is used to measure the natural oligosaccharide N-glycan group spectrum in serum, and then quantify the peak value and conduct statistical analysis, thereby providing a chronic hepatitis B A method for establishing a serum natural oligosaccharide N-glycan map model of liver fibrosis in patients with hepatitis B, which detects liver fibrosis in patients with chronic hepatitis B by measuring the changes in natural glycosylation modifications of proteins under physiological and pathological conditions.

The technical scheme that the present invention adopts is as follows:

A detection reagent for detecting liver fibrosis in patients with chronic hepatitis B based on oligosaccharide chains, consisting of the following reagents:

Reagent A: prepared by adding SDS with a mass concentration of 1 to 5% in an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3;

Reagent B: It is prepared by mixing 0.05-10 units/10 μl of glucosamidase, NP-40 with a mass concentration of 10%, and an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the mixed solution is 5-9;

Reagent C: an organic reducing agent prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO with a concentration of 0.02mM～1M;

Reagent D: stop solution.

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

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

Preferably, the reagent D is ultrapure water.

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

Step 1 Preparation of oligosaccharide chains

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, react at 37°C for 3 hours, and then dry;

Step 2 Labeling of oligosaccharide chains

Add 5 μl of reagent C to the sample obtained after drying in step 1, react for 1 hour at 60°C for fluorescent labeling, then add 100 μl of reagent D to terminate the labeling reaction;

Step 3: Separation and analysis of oligosaccharide chains

Take 10 μl of the liquid labeled with oligosaccharide chains, and use an analyzer to separate and detect N-oligosaccharide chains to obtain the N-glycan map of natural oligosaccharides;

Step 4 Data processing and analysis

Peak quantification of the N-glycan profile: the peak height of each peak is divided by the sum of the heights of all peaks to calculate the relative content of each peak.

Application of a composition in the preparation of reagents for detecting liver fibrosis in patients with chronic hepatitis B with oligosaccharide chains, said composition is composed of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum, said The composition detects liver fibrosis in patients with chronic hepatitis B through the value of G2S2+G2S1+G2S2F.

The G1F is an isomer.

The invention provides a new reagent and an application method for detecting liver fibrosis of patients with chronic hepatitis B by natural oligosaccharide chains, and performs statistical analysis by measuring the specific fingerprint of serum natural oligosaccharide chains G-Test.

Materials and methods:

1. Test samples: serum from patients with chronic hepatitis B without fibrosis and patients with chronic hepatitis B with fibrosis.

2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

3. Reagent preparation:

1. Reagent A: prepared by adding SDS with a mass concentration of 1 to 5% in an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3;

2. Reagent B: It is prepared by mixing 0.05-10 units/10 μl of glucosamidase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3. The pH value of the mixed solution is 5-5 9;

3. Reagent C: Dissolve 8-aminopyrene-1,3,6-trisulfonic acid in DMSO to make a reducing agent with a concentration of 0.02mM～1M;

4. Reagent D: stop solution.

4. Detection of N-glycan profile

1. Preparation of oligosaccharide chains

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, react at 37°C for 3 hours, and then dry.

2. Labeling of oligosaccharide chains

1) Add 5 μl of reagent C to the dried sample, react at 60°C for 1 hour, and perform fluorescent labeling;

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

3. Separation analysis of oligosaccharide chains

Take 10 μl of the oligosaccharide chain-labeled liquid, and perform N-oligosaccharide chain separation and detection under the ABI3500dx instrument, so as to obtain the natural oligosaccharide N-glycan map.

4. Data processing and analysis

(1) Peak quantification of the N-glycan profile: divide the peak height value of each peak by the sum of the heights of all peaks to calculate the relative content of each peak.

(2) Analysis of N-glycan group data of patients with chronic hepatitis B without fibrosis and patients with chronic hepatitis B with fibrosis: compare the N-glycan data of patients with chronic hepatitis B without fibrosis and patients with chronic hepatitis B with fibrosis Conduct comparative analysis. Divide the peak height value of each peak by the sum of the heights of all peaks to calculate the relative content of each peak, that is, the peak quantification of the N-glycan profile, and then quantify the chronic hepatitis B with fibrosis group Statistical analysis was performed on the 9 N-oligosaccharide chain peaks in the N-glycogroup profile of the chronic hepatitis B non-fibrosis group. The composition of the N-glycan profile consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2, where G1F is an isomer; chronic B is detected by the composition G2S2+G2S1+G2S2F value Liver fibrosis in patients with hepatitis.

Compared with prior art, the beneficial effect of the present invention:

(1) Quantify each peak of the G-Test fingerprint, and then compare the relative content of each peak in patients with chronic hepatitis B with hepatic fibrosis (149 cases) and patients with chronic hepatitis B without hepatic fibrosis (136 cases) The results were compared and analyzed, and it was found that the N-glycan compositions G2S2, G2S1 and G2S2F had significant statistical differences (p<0.05) between the two groups; When patients with hepatitis B have liver fibrosis, the AUC value under the ROC curve reaches 0.859 (Fig. 2). When the cutoff value of the G2S2+G2S1+G2S2F model detection is 0.405, the sensitivity for the detection of liver fibrosis in chronic hepatitis B is 81.2%. The specificity is 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.

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

(3) The G-Test natural N-glycan map model constructed based on the method of the present invention can allow many patients to receive routine and non-invasive testing, and help doctors and patients monitor the occurrence and disease progression of liver fibrosis in patients with chronic hepatitis B in a timely manner , the detection technology of the present invention has the advantages of high sensitivity, simple operation, trace amount (5 μl serum), high repeatability, good stability, high throughput (96-well plate), etc., and can be popularized and used in clinic.

Description of drawings

Fig. 1 is the serum natural oligosaccharide N-glycan group map of chronic hepatitis B patient without hepatic fibrosis and chronic hepatitis B patient with hepatic fibrosis;

Fig. 2 is the ROC curve of the model based on the N-sugar composition G2S2+G2S1+G2S2F established to identify chronic hepatitis B patients with hepatic fibrosis; the total number of test samples is 285 cases, of which the serum of chronic hepatitis B patients with hepatic fibrosis There were 149 samples, including 136 serum samples from patients with chronic hepatitis B without hepatic fibrosis, and the area under the ROC curve AUC = 0.859.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings. It should be noted that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not specify specific conditions in the following examples, they are usually tested according to conventional conditions, or according to the conditions suggested by the manufacturer.

To detect the state of liver fibrosis in patients with chronic hepatitis B, measure the N-glycan profile of serum natural oligosaccharides and perform statistical analysis. The materials and methods used are as in the following examples.

Embodiment one

1. Test samples: Serum from chronic hepatitis B patients with liver fibrosis and chronic hepatitis B patients without liver fibrosis.

2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

3. Reagent preparation:

1) Reagent A: prepared by adding SDS with a mass concentration of 3% in ammonium bicarbonate solution with a concentration of 10 mM;

2) Reagent B: prepared by mixing 5 units/10 μl of glucosamidase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the mixed solution is 7;

3) Reagent C: an organic reducing agent prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO at a concentration of 10 mM;

4) Reagent D: ultrapure water.

4. N-glycan map detection:

(1) Preparation of oligosaccharide chains

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 hours, and then dried.

(2) Labeling of oligosaccharide chains

1) Add 5 μl of reagent C to the dried sample, react at 60°C for 1 hour, and perform fluorescent labeling;

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

(3) Separation analysis of oligosaccharide chains

Take 10 μl of the oligosaccharide chain-labeled liquid, and perform N-oligosaccharide chain separation and detection under the ABI3500dx instrument, so as to obtain the natural oligosaccharide N-glycan map.

(4) Data processing and analysis

1) Peak quantification of the N-glycan profile: divide the peak height value of each peak by the sum of the heights of all peaks to calculate the relative content of each peak.

2) Analysis of N-glycan group data of patients with chronic hepatitis B with liver fibrosis and patients with chronic hepatitis B without liver fibrosis: N-glycan group of patients with chronic hepatitis B with liver fibrosis and patients with chronic hepatitis B Glycogroup data were compared and analyzed. As shown in Figure 1, the N-glycan profile of human serum shows nearly nine peaks of N-oligosaccharide chains, and different oligosaccharide chains show different mobility due to the different charges and molecular sizes, that is, the Different peaks on the N-glycan map represent different oligosaccharide chains, and the peak heights represent the relative content of oligosaccharide chains. In Figure 1, A is the serum N-glycan profile of chronic hepatitis B patients without liver fibrosis, and B is the serum N-glycan profile of chronic hepatitis B patients with liver fibrosis. The composition of the N-glycan profile is composed of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2, of which G1F is an isomer; by calculating the composition G2S2+G2S1+G2S2F value to assist in judging chronic Liver fibrosis status in patients with hepatitis B.

The model established based on the composition G2S2+G2S1+G2S2F can distinguish chronic hepatitis B patients with liver fibrosis with an AUC value under the ROC curve of 0.859 (Figure 2). When the cutoff value of the G2S2+G2S1+G2S2F model is 0.405, The sensitivity for detecting liver fibrosis in chronic hepatitis B is 81.2%, and the specificity is 78.5%.

Embodiment two

1) Reagent A: prepared by adding SDS with a mass concentration of 1% to ammonium bicarbonate solution with a concentration of 10 mM;

2) Reagent B: prepared by mixing 0.05 units/10 μl of glucosaminylase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the solution is 5;

3) Reagent C: an organic reducing agent prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO with a concentration of 0.02mM;

4) Reagent D: ultrapure water.

4. The detection of the N-glycan profile is the same as in Example 1.

Embodiment Three

1) Reagent A: prepared by adding SDS with a mass concentration of 5% into ammonium bicarbonate solution with a concentration of 10 mM;

2) Reagent B: prepared by mixing 10 units/10 μl of glucosaminylase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the solution is 9;

3) Reagent C: a reducing agent prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO with a concentration of 1M;

4) Reagent D: ultrapure water.

4. The detection of the N-glycan profile is the same as in Example 1.

Compared with the existing technology, this detection technology detects the correlation between changes in sialic acid oligosaccharide chains and disease states under physiological and pathological conditions, and can establish a prediction model of N-glycan composition based on these changes to assist in the judgment of chronic hepatitis B. Liver fibrosis status in patients with hepatitis. The G-Test natural N-glycan map model constructed based on the method of the present invention can allow many patients to receive routine and non-invasive testing, and help doctors and patients to monitor the occurrence and disease progression of liver fibrosis in patients with chronic hepatitis B in a timely manner. Promoted and used in clinical practice.

The purpose, technical solutions and beneficial effects of the present invention have been further described in detail in conjunction with the specific embodiments described in the accompanying drawings. For the limitation of the scope of protection, those skilled in the art should understand that any modification, equivalent replacement, improvement, etc. that can be made within the spirit and principles of the present invention without creative work shall be included in the protection of the present invention. within range.

Claims (7)

1. A detection reagent based on oligosaccharide chains to detect liver fibrosis in patients with chronic hepatitis B, characterized in that it consists of the following reagents: Reagent A: prepared by adding SDS with a mass concentration of 1 to 5% in an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3; Reagent B: It is prepared by mixing 0.05-10 units/10 μl of glucosamidase, NP-40 with a mass concentration of 10%, and an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the mixed solution is 5-9; Reagent C: Dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO to make a reducing agent with a concentration of 0.02mM～1M; Reagent D: stop solution. 2. The detection reagent for detecting liver fibrosis in patients with chronic hepatitis B based on oligosaccharide chains according to claim 1, wherein the volume ratio of the reagent A, reagent B and reagent C is 1:1:1. 3 . The detection reagent for detecting liver fibrosis in patients with hepatitis B based on oligosaccharide chains according to claim 2 , wherein the volumes of the reagent A, reagent B and reagent C are all 5 μl. 4. The detection reagent for detecting liver fibrosis in patients with hepatitis B based on oligosaccharide chains according to claim 1, wherein the reagent D is ultrapure water. 5. the preparation method of the detection reagent based on oligosaccharide chain detection hepatitis B hepatitis patient liver fibrosis according to claim 1, is characterized in that, comprises the following steps: Step 1 Preparation of oligosaccharide chains 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, react at 37°C for 3 hours, and then dry; Step 2 Labeling of oligosaccharide chains Add 5 μl of reagent C to the sample obtained after drying in step 1, react for 1 hour at 60°C for fluorescent labeling, then add 100 μl of reagent D to terminate the labeling reaction; Step 3: Separation and analysis of oligosaccharide chains Take 10 μl of the liquid labeled with oligosaccharide chains, and use an analyzer to separate and detect N-oligosaccharide chains to obtain the N-glycan map of natural oligosaccharides; Step 4 Data processing and analysis Peak quantification of N-glycan profiles: divide the peak height value of each peak by the sum of the heights of all peaks to calculate the relative content of each peak. 6. The application of a composition in the preparation of reagents for detection of liver fibrosis in patients with chronic hepatitis B with oligosaccharide chains, characterized in that the composition consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F in serum and G2F2, the composition detects liver fibrosis in patients with chronic hepatitis B through the value of G2S2+G2S1+G2S2F. 7. The application of a composition according to claim 6 in the preparation of reagents for detecting liver fibrosis in patients with chronic hepatitis B with oligosaccharide chains, characterized in that the G1F is an isomer.

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