Application of composition in preparation of oligosaccharide chain hepatitis B liver cancer detection reagent

Abstract

The invention provides a detection reagent for detecting hepatitis B and liver cancer based on an oligosaccharide chain and a preparation method and application thereof, the detection reagent is prepared by mixing the following reagents: a reagent A, which 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. According to the present invention, the natural oligosaccharide N-glycome map in serum is determined by using the reagent, the peak value is quantified, and statistical analysis is performed, such that the establishment method of the hepatitis B liver cancer serum natural oligosaccharide N-glycome map model is provided, and the hepatitis B liver cancer is detected by determining the change of the natural glycosylation modification of the protein under the physiological and pathological state.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a detection method for hepatitis B liver cancer, specifically to a detection method for hepatitis B liver cancer based on the specific fingerprint of serum oligosaccharide chain G-Test. Background Art

[0002] Primary liver cancer (PLC) refers to cancer that occurs in hepatocytes or intrahepatic bile duct cells. Every year, 450,000 to 1 million people die from liver cancer globally. The etiology and pathogenesis of primary liver cancer have not been fully understood and may be related to the combined action of multiple factors. The etiological factors of liver cancer in China mainly include hepatitis B virus infection, food contamination with aflatoxin, long-term alcoholism, and contamination of rural drinking water with blue-green algal toxins. Other factors include liver metabolic diseases, autoimmune diseases, and cryptogenic liver diseases or cryptogenic cirrhosis.

[0003] The clinical diagnosis of liver cancer mainly relies on histopathological and cytological examinations, imaging examinations, enzymological examinations, and tumor marker examinations. At present, pathological tissue and cytological examinations are the gold standard for diagnosing liver cancer, mainly by taking liver puncture samples for pathological diagnosis. However, this method still has deficiencies: (1) Since pathological biopsy is an invasive examination, patients are at risk of complications such as bleeding and infection; (2) The biopsy specimen only accounts for a very small part of the liver, and sampling has limitations and cannot reflect the overall liver lesion, so there is a possibility of missed diagnosis; (3) The result of a single biopsy has limited reference value. If repeated examinations are required, most patients have poor compliance. Imaging examinations including B-ultrasound, CT, and MRI are used as auxiliary examinations for the diagnosis of liver cancer, and their specificity needs to be further improved. Tumor marker examinations such as alpha-fetoprotein (AFP) are widely used in the general survey, diagnosis, judgment of treatment effects, and prediction of recurrence of liver cancer. Compared with histopathology, the advantage of AFP is non-invasiveness, only one tube of blood needs to be drawn; the disadvantage is that the specificity and sensitivity for diagnosing liver cancer are relatively low, and about 30-40% of liver cancer patients have a negative AFP test. Enzymological examinations include gamma-glutamyl transferase isoenzyme II (γ-GT2), abnormal prothrombin (AP), alpha-L-fucosidase (AFU), acidic isoferritin (AIF), aldolase A (ALD-A), 5'-nucleotide phosphodiesterase isoenzyme V (5'-NPDV), etc. They cannot be used alone as independent factors for diagnosing liver cancer and need to be used in combination with 2-3 of them to improve the diagnostic rate of liver cancer, thereby assisting in the diagnosis of liver cancer. All of the above diagnostic methods have their own limitations, and there is an urgent need to explore a new non-invasive method for assisting in the diagnosis of liver cancer and having high sensitivity and specificity at the same time.

[0004] Glycoproteins are a class of conjugated proteins formed by post-translational modification of proteins, namely glycosylation. Glycosylation of proteins is one of the most common post-translational modifications of proteins. It is a process in which sugars are transferred to proteins and specific amino acid residues on proteins under the action of glycosyltransferases to form glycosidic bonds. Most glycoproteins are secreted proteins and are widely present in cell membranes, extracellular matrices, plasma, and mucus. The N-glycans on proteins regulate the structure, stability, and activity of proteins through processing and modification. Therefore, the sugar chains in glycoproteins play an important role in maintaining the biological functions of the body, thereby endowing glycoproteins with various biological functions. Therefore, understanding the changes in sugar chains helps to clarify the molecular mechanisms of abnormal biological behaviors such as inflammation, invasion, and metastasis of tumor cells to surrounding tissues.

[0005] Currently, abnormal changes in protein N-glycans have been found in various tumors, and changes in the terminal sialic acid modification of N-glycans are one of them. Sialic acid is a class of negatively charged nine-carbon sugar compounds that are widely present in living organisms and are often located at the end of glycan chains. Under the action of sialidase, it forms polysialic acid chains by connecting to galactose or N-acetylgalactosamine on the sugar chain through α-2,3 or α-2,6 glycosidic bonds. Studies have found that sialic acid at the end of glycan chains plays an important role in regulating cell-cell recognition, molecular interactions, viral infections, immune responses, and signal transduction, and the sialic acid modification of cell surface glycoproteins is tissue- and cell-specific. Numerous studies have shown that abnormal sialic acid modification is involved in the occurrence and development of diseases and is closely related to various diseases such as infectious diseases and the invasion and metastasis of tumors. Therefore, detecting changes in sialic acid-related oligosaccharides has potential clinical value for the diagnosis of diseases and the development of therapeutic drugs as targets. Summary of the Invention

[0006] Aiming at the problems existing in the current clinical detection of hepatitis B-related liver cancer, such as low specificity and accuracy of blood tests and imaging tests, invasiveness of pathological examinations, sampling errors, and poor patient compliance, the present invention provides a detection reagent for hepatitis B-related liver cancer. By using this reagent to determine the natural oligosaccharide N-glycome profile in serum, and then quantifying the peaks and performing statistical analysis, a method for establishing a serum natural oligosaccharide N-glycome profile model for hepatitis B-related liver cancer is provided, and hepatitis B-related liver cancer is detected by measuring the changes in the natural glycosylation modification of proteins under physiological and pathological conditions.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A detection reagent for detecting hepatitis B-related liver cancer based on oligosaccharide chains, which is composed of the following reagents:

[0009] Reagent A: Prepared by adding SDS with a mass concentration of 1-5% to an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3;

[0010] Reagent B: It is prepared by mixing 0.05 - 10 units / 10 μl of glycosaminidase, 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. The pH value of the mixed solution is 5 - 9;

[0011] Reagent C: 8 - aminopyrene - 1,3,6 - trisulfonic acid is dissolved in DMSO to prepare an organic reducing agent with a concentration of 0.02 mM - 1 M;

[0012] Reagent D: Termination solution.

[0013] Preferably, the volume ratio of Reagent A, Reagent B and Reagent C is 1:1:1.

[0014] Preferably, the volumes of Reagent A, Reagent B and Reagent C are all 5 μl.

[0015] Preferably, Reagent D is ultrapure water.

[0016] A preparation method of a detection reagent for detecting hepatitis B and liver cancer based on oligosaccharide chains, comprising the following steps:

[0017] Step 1: Preparation of oligosaccharide chains

[0018] 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.

[0019] Step 2: Labeling of oligosaccharide chains

[0020] Add 5 μl of Reagent C to the sample obtained after drying in Step 1, react at 60 °C for 1 h for fluorescence labeling, and then add 100 μl of Reagent D to terminate the labeling reaction;

[0021] Step 3: Separation and analysis of oligosaccharide chains

[0022] Take 10 μl of the liquid after oligosaccharide chain labeling, and use an analyzer to perform N - oligosaccharide chain separation detection to obtain a natural oligosaccharide N - glycome map;

[0023] Step 4: Data processing and analysis

[0024] Peak quantification of N - glycome map: 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.

[0025] Use of a composition in the preparation of an oligosaccharide chain hepatitis B liver cancer detection reagent, wherein the composition consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum, and the composition detects hepatitis B liver cancer by the value of (G3S3 - G2S2) / G4S4.

[0026] The G1F is an isomer.

[0027] The present invention provides a method for establishing a serum natural oligosaccharide N-glycome map model for hepatitis B liver cancer, by measuring the G-Test specific fingerprint map of serum natural oligosaccharide chains and performing statistical analysis.

[0028] Materials and methods:

[0029] I. Detection samples: sera of hepatitis B liver cancer patients and healthy people.

[0030] II. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0031] III. Reagent preparation:

[0032] 1. Reagent A: prepared by adding SDS with a mass concentration of 1-5% to an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3;

[0033] 2. Reagent B: prepared by mixing 0.05-10 units / 10 μl of glycosaminidase, 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;

[0034] 3. Reagent C: prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO to form an organic reducing agent with a concentration of 0.02 mM-1 M;

[0035] 4. Reagent D: termination solution.

[0036] IV. N-glycome map detection

[0037] 1. Preparation of oligosaccharide chains

[0038] 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 h, and then dry.

[0039] 2. Labeling of oligosaccharide chains

[0040] (1) Add 5 μl of reagent C to the dried sample, react at 60 °C for 1 h for fluorescence labeling;

[0041] (2) After the fluorescence labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

[0042] 3. Oligosaccharide chain separation and analysis

[0043] Take 10 μl of the liquid after oligosaccharide chain labeling and perform N - oligosaccharide chain separation and detection on an ABI3500dx instrument to obtain a natural oligosaccharide N - glycome map.

[0044] 4. Data processing and analysis

[0045] (1) Peak quantification of the N - glycome map: 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.

[0046] (2) Data analysis of N - glycome between hepatitis B liver cancer patients and healthy people: Compare and analyze the N - glycome data of hepatitis B liver cancer patients and healthy people. Divide the peak height value of each peak by the sum of the heights of all peaks, and quantitatively calculate the relative content of each peak, that is, the peak quantification of the N - glycome map. Then, perform comparative statistical analysis on 9 N - oligosaccharide chain peaks in the quantified N - glycome maps of the hepatitis B liver cancer group and the normal control group. The composition of the N - glycome map consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, where G1F is an isomer; detect hepatitis B liver cancer through the value of the composition (G3S3 - G2S2) / G4S4.

[0047] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0048] (1) Quantify each peak of the G - Test fingerprint map, and then compare and analyze the relative content results of each peak of hepatitis B liver cancer patients (178 cases) and healthy people (202 cases). It is found that there are significant statistical differences in N - glycan compositions G3S3, G2S2, and G4S4 between the two groups (p < 0.05); therefore, the AUC value under the ROC curve of the model established based on the composition (G3S3 - G2S2) / G4S4 reaches 0.902 when distinguishing hepatitis B liver cancer patients Figure 2 ). When the cutoff value detected by the (G3S3 - G2S2) / G4S4 model is 4.05, the detection of hepatitis B liver cancer has a sensitivity of 85.70% and a specificity of 86.90%, indicating that the N - glycan composition (G3S3 - G2S2) / G4S4 in serum can be used as a marker for the auxiliary diagnosis of hepatitis B liver cancer.

[0049] (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 glycoprotein N-glycans in a serum sample, they are separated by capillary microelectrophoresis, and the content of the glycoprotein obtained by measuring the fluorescence signal is the natural oligosaccharide N-glycome map. By detecting the correlation between the changes in sialic acid oligosaccharide chains in physiological and pathological states and the disease state, according to these changes, a prediction model of N-glycan composition can be established to assist in the diagnosis of the state of hepatitis B liver cancer.

[0050] (3) The G-Test natural N-glycome map model constructed based on the method of the present invention enables many patients to receive routine and non-invasive detection, helping doctors and patients to monitor the occurrence and disease progression of hepatitis B liver cancer in a timely manner. The detection technology of the present invention has the advantages of high sensitivity, simple operation, microscale (5 μl of serum), high repeatability, good stability, high throughput (96-well plate), etc., and can be popularized and used in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is the natural oligosaccharide N-glycome map of the sera of hepatitis B liver cancer patients and healthy people;

[0052] Figure 2 is the ROC curve graph of the discrimination model for the state of hepatitis B liver cancer established based on the N-glycan composition (G3S3 - G2S2) / G4S4; the total number of detected samples is 380, including 178 serum samples of hepatitis B liver cancer patients and 202 serum samples of healthy people, and the area under the ROC curve AUC = 0.902 is obtained. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] The present invention will be further described in detail below in conjunction with the embodiments and the drawings. It should be noted that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0054] To detect the state of hepatitis B liver cancer, by measuring the natural oligosaccharide N-glycome map of the serum and performing statistical analysis, the materials and methods used are as follows in the following embodiments.

[0055] Example 1

[0056] 1. Detection samples: Sera of 178 hepatitis B liver cancer patients and 202 healthy people.

[0057] 2. Experimental equipment: Capillary electrophoresis analyzer, PCR, centrifuge.

[0058] 3. Reagent preparation:

[0059] 1) Reagent A: Prepared by adding SDS with a mass concentration of 2.5% to an ammonium bicarbonate solution with a concentration of 10 mM;

[0060] 2) Reagent B: Prepared by mixing 2.5 units / 10 μl of glycosaminidase, 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 solution is 7;

[0061] 3) Reagent C: An organic reducing agent with a concentration of 10 mM prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO;

[0062] 4) Reagent D: Ultra-pure water.

[0063] 4. N-glycome map detection:

[0064] (1) Preparation of oligosaccharide chains

[0065] 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 h, and then dry.

[0066] (2) Labeling of oligosaccharide chains

[0067] 1) Add 5 μl of Reagent C to the dried sample and react at 60 °C for 1 h for fluorescence labeling;

[0068] 2) After the fluorescence labeling is completed, add 100 μl of Reagent D to terminate the labeling reaction.

[0069] (3) Separation and analysis of oligosaccharide chains

[0070] Take 10 μl of the liquid after oligosaccharide chain labeling and perform N-oligosaccharide chain separation detection under an ABI3500dx instrument to obtain the natural oligosaccharide N-glycome map.

[0071] (4) Data processing and analysis

[0072] 1) Peak quantification of N-glycome map: 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.

[0073] 2) N-glycome data analysis of hepatitis B liver cancer patients and healthy people: Compare and analyze the N-glycome data of hepatitis B liver cancer patients and healthy people. As Figure 1 shown, the N-glycome map of human serum shows nearly 9 N-oligosaccharide chain peaks. Different oligosaccharide chains show different mobilities due to different charges and molecular sizes, that is, different peaks on the N-glycome map represent different oligosaccharide chains, and their peak heights represent the relative contents of the oligosaccharide chains. Figure 1In this, A is the N-glycan map of healthy human serum, and B is the N-glycan map of serum from patients with hepatitis B liver cancer. The composition of the N-glycome map consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, where G1F is an isomer; the value of the composition (G3S3 - G2S2) / G4S4 is calculated to assist in judging the status of hepatitis B liver cancer.

[0074] The AUC value under the ROC curve of the model established based on the composition (G3S3 - G2S2) / G4S4 reached 0.902 when distinguishing patients with hepatitis B liver cancer ( Figure 2 ), and when the cutoff value detected by the (G3S3 - G2S2) / G4S4 model was 4.05, the detection of hepatitis B liver cancer had a sensitivity of 85.70% and a specificity of 86.90%, indicating that the N-glycan composition (G3S3 - G2S2) / G4S4 in serum can be used as a marker for the auxiliary diagnosis of hepatitis B liver cancer.

[0075] Example 2

[0076] 1. Test samples: Serum from 168 patients with hepatitis B liver cancer and 196 healthy individuals.

[0077] 2. Experimental equipment: Capillary electrophoresis analyzer, PCR, centrifuge.

[0078] 3. Reagent preparation:

[0079] 1) Reagent A: Prepared by adding 1% SDS by mass concentration to a 10 mM ammonium bicarbonate solution;

[0080] 2) Reagent B: Prepared by mixing 0.05 units / 10 μl of glycosaminidase, 10% NP-40 by mass concentration, and a 10 mM ammonium bicarbonate solution with a pH of 8.3, and the pH value of the solution is 5;

[0081] 3) Reagent C: Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO to form an organic reducing agent with a concentration of 0.02 mM;

[0082] 4) Reagent D: Ultrapure water.

[0083] 4. N-glycome map detection:

[0084] (1) Preparation of oligosaccharide chains

[0085] Add 5 μl of reagent A to 5 μl of the inactivated serum sample for denaturation; after cooling to room temperature, add 5 μl of reagent B, react at 37 °C for 3 h, and then dry.

[0086] (2) Labeling of oligosaccharide chains

[0087] 1) Add 5 μl of Reagent C to the dried sample, react at 60 °C for 1 h for fluorescence labeling;

[0088] 2) After the fluorescence labeling is completed, add 100 μl of Reagent D to terminate the labeling reaction.

[0089] (3) Oligosaccharide chain separation and analysis

[0090] Take 10 μl of the liquid after oligosaccharide chain labeling and perform N - oligosaccharide chain separation and detection on the ABI3500dx instrument to obtain the natural oligosaccharide N - glycome map.

[0091] (4) Data processing and analysis

[0092] 1) Peak quantification of the N - glycome map: 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.

[0093] 2) Data analysis of N - glycome between hepatitis B liver cancer patients and healthy people: Compare and analyze the N - glycome data of hepatitis B liver cancer patients and healthy people. As Figure 1 shown, the N - glycome map of human serum shows nearly 9 N - oligosaccharide chain peaks. Different oligosaccharide chains show different mobilities due to different charges and molecular sizes, that is, different peaks on the N - glycome map represent different oligosaccharide chains, and their peak heights represent the relative contents of the oligosaccharide chains. Figure 1 In it, A is the N - glycan map of healthy human serum, and B is the N - glycan map of hepatitis B liver cancer patient serum. The composition of the N - glycome map consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, where G1F is an isomer; the value of (G3S3 - G2S2) / G4S4 of the composition is calculated to assist in judging the state of hepatitis B liver cancer.

[0094] To further verify the accuracy of the model, 168 hepatitis B liver cancer patients and 196 healthy normal control samples are used to verify the model established based on the composition (G3S3 - G2S2) / G4S4. Based on Example 1, when the cutoff value is 4.05, the discrimination is better. The sensitivity and specificity of the sample model in this example are 85.12% and 86.22% respectively (Table 1).

[0095] Table 1 Verification results of 168 hepatitis B liver cancer patients and 196 healthy people

[0096] Type Sensitivity Specificity Hepatitis B and liver cancer 85.12％(143 / 168) - Health - 86.22％(169 / 196)

[0097] Compared with the prior art, the present detection technology can, by detecting the correlation between the changes in sialic acid oligosaccharide chains under physiological and pathological conditions and the disease state, establish a prediction model of N-glycan composition based on these changes to assist in judging the state of hepatitis B liver cancer. The G-Test natural N-glycome atlas model constructed based on the method of the present invention enables many patients to receive routine and non-invasive detection, helps doctors and patients to monitor the occurrence and disease progression of hepatitis B liver cancer in a timely manner, and can be popularized and used in clinical practice.

[0098] The specific embodiments described above in conjunction with the accompanying drawings have further detailed the object, technical solution and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention, but not a limitation on the protection scope of the present invention. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. that can be made without creative labor within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a composition in the preparation of an oligosaccharide chain hepatitis B liver cancer detection reagent, characterized in that, The composition consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum, and the composition detects hepatitis B liver cancer by the value of (G3S3 - G2S2) / G4S4.

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

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