WO2018233617A1 - Procédé d'établissement d'un modèle de profil de glycome de glycoprotéines sériques d'une hépatite chronique et d'une atteinte hépatique - Google Patents

Procédé d'établissement d'un modèle de profil de glycome de glycoprotéines sériques d'une hépatite chronique et d'une atteinte hépatique Download PDF

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WO2018233617A1
WO2018233617A1 PCT/CN2018/091920 CN2018091920W WO2018233617A1 WO 2018233617 A1 WO2018233617 A1 WO 2018233617A1 CN 2018091920 W CN2018091920 W CN 2018091920W WO 2018233617 A1 WO2018233617 A1 WO 2018233617A1
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reagent
serum
liver damage
peak
chronic hepatitis
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PCT/CN2018/091920
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陈翠英
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江苏先思达生物科技有限公司
陈翠英
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a method for establishing a serum glycoprotein glycoprotein pattern model for chronic hepatitis liver injury.
  • Chronic Hepatitis is a general term for chronic liver inflammation. It is caused by various pathogenic factors such as viruses, chemical poisons, drugs, alcohol, autoimmune factors, etc., causing damage to liver cells and causing damage to the liver. A series of discomfort symptoms, as well as abnormalities in liver function indicators.
  • Chronic hepatitis B (CHB) is caused by prolonged infection with hepatitis B virus (HBV). More than 400 million people worldwide are infected with hepatitis B virus (HBV), and nearly 200 million people in China are chronically infected with hepatitis B virus. Most infected people have no symptoms and are not infected, but three out of every 10 chronically infected people may have serious life-threatening complications such as cirrhosis and liver cancer. Chronic hepatitis B causes more than 330,000 cancer-related deaths each year in China. Testing and treatment before chronically infected people have not developed a more serious disease is currently a vital task.
  • Clinical testing for chronic hepatitis relies mainly on blood testing and imaging testing.
  • the gold standard for diagnosis is liver biopsy, but the following defects: pathological examination is traumatic, easy to cause bleeding, infection and other complications; there is sampling error, because the liver tissue biopsy center only accounts for 1/200,000-1/5 of the liver. 10,000, can not reflect the entire liver lesions; poor patient dependence, a single pathological examination results can not reflect the degree of liver damage.
  • the specificity test for viral markers should be the initial identification test for chronic liver injury.
  • the above diagnostic methods are not specific and can only play a supporting role in the diagnosis of liver damage.
  • the above diagnostic methods have their own limitations, and it is urgent to explore a new non-invasive index for the method of assisting diagnosis of liver damage caused by hepatitis B virus and having high sensitivity and specificity.
  • Glycoproteins are a class of binding proteins formed by post-translational modification of proteins, ie, glycosylation. Glycosylation of proteins is one of the most common post-translational modifications of proteins. It is the process of transferring sugars to specific amino acid residues on proteins and proteins to form glycosidic bonds under the action of glycosyltransferases. Most glycoproteins are secreted proteins that are widely found in cell membranes, interstitial cells, plasma, and mucus. Glycoproteins have a variety of biological functions. Because of the importance of sugar chains in glycoproteins to maintain the biological functions of the body, changes in sugar chains help to elucidate the molecular mechanisms of abnormal biological behavior such as inflammation, invasion and metastasis of surrounding cells by surrounding cells. At present, changes in N-glycans have been found in a variety of tumors.
  • the structure of the sugar chain is very complicated and has microscopic heterogeneity.
  • the analysis methods of the sugar chain structure mainly include: (1) High performance liquid chromatography (HPLC): the method has high resolution, high detection speed and high repeatability, high-performance liquid phase. Columns can be used repeatedly, but the column efficiency will decrease with the increase in the number of uses, and the mobile phase is toxic. Equipment operation requires highly trained professionals, and the equipment is relatively expensive, and the solvent needs to be strictly purified.
  • Mass spectrometry has the advantages of high sensitivity, various structural information and suitable for analysis of mixtures, but the mass spectrometer is precise, the equipment operation is complicated, and the mass spectrometer is expensive, which is not suitable for popularization and promotion in clinical practice;
  • Capillary electrophoresis Capillary electrophoresis has low cost, high column efficiency, high sensitivity, high speed, low injection volume, and simple operation, but the repeatability is not high and the stability is not as good as HPLC.
  • G-Test is a capillary micro-electrophoresis technique (DSA-FACE) based on DNA sequencer.
  • DSA-FACE capillary micro-electrophoresis technique
  • the N-glycan of glycoprotein in serum samples is fluorescently labeled and separated by capillary microelectrophoresis.
  • the content of the N-oligosaccharide chain obtained by measuring the fluorescence signal is a fingerprint (referred to as G-Test map).
  • the detection technology has the advantages of high sensitivity, simple operation, trace (2 ⁇ l serum), high reproducibility, good stability, high-throughput (96-well plate) and other sugar chain analysis techniques, which is suitable for general laboratory. It is expected to be used for clinical promotion.
  • the present invention provides chronic hepatitis liver damage.
  • a method for establishing a serum glycoprotein glycoprotein map model was established by screening the model to screen for NG1A2F (single agalacto ( ⁇ 1,6Fuc) two antennas (single agalacto, core- ⁇ -1,6- Fucosylated biantennary)) can be used as a specific marker for the diagnosis of chronic hepatitis liver damage.
  • the method for establishing a serum glycoprotein glycoprotein map model for chronic hepatitis liver injury is as follows:
  • Step 1 Collect serum from patients with liver injury and normal controls
  • Step 2 preparing a solution containing 5% SDS (sodium dodecyl sulfate) at a concentration of 10 mM, a pH of 8.3, NH 4 HCO 3 as reagent A, and reagent B from 2.2 U/ ⁇ L of PNGaseF and 3.33% of NP-40.
  • SDS sodium dodecyl sulfate
  • reagent B 2.2 U/ ⁇ L of PNGaseF and 3.33% of NP-40.
  • reagent C was prepared by mixing 20 mM APTS (8-aminoindole-1,3,6-trisulphonic acid) and 1 M NaCNBH 3 in equal volumes.
  • Reagent D consisted of 100 mM NH 4 AC, 2 mU/ ⁇ L of sialidase and hydrogen peroxide are mixed in a volume ratio of 5:1:14;
  • Step 3 preparation of oligosaccharide chain: adding half volume of reagent A to the diluted serum, denaturation at 95 ° C for 5 min, then adding reagent B with the same volume of serum, reacting at 37 ° C for 3 h and then drying;
  • Step 4 labeling of the oligosaccharide chain: adding the reagent C of the same volume as the reagent A in the liquid of the step 3, reacting at 65 ° C for 3 h for fluorescent labeling, and then adding water to terminate the labeling reaction;
  • Step 5 desialic acid treatment: take an equal volume of step 4 fluorescently labeled liquid and reagent D at 45 ° C for 3 h, then add water to terminate the reaction;
  • Step 6 Oligosaccharide chain separation analysis: the liquid after the sialic acid treatment in step 5 is taken, and the fragment analysis is performed by a DNA sequencer to obtain a sugar group map;
  • step 7 the obtained sugar group map is subjected to peak quantification, and the peak height value of each peak is divided by the sum of the heights of all the peaks, and the relative content of each peak is quantitatively calculated, and then the quantified liver damage group and The peak of NG1A2F in the sugar group map of the normal control group was subjected to statistical analysis.
  • step 1 the serum is inactivated.
  • step 4 the fluorescent labeling time of the serum sample is 3h to ensure the success rate of the label. Under the general experimental conditions, the test requirement can be met within 2.5 hours.
  • step 5 the reaction time for removing the terminal sialic acid is 3 hours, and in order to ensure sufficient contact reaction of the enzyme, the reaction can be made more complete by extending to 4 hours.
  • step 7 the cut-off value of the relative content of NG1A2F is 6.85.
  • the present invention has the following advantages:
  • the method of the invention adopts the G-Test detection method with high sensitivity, simple operation, only a small amount of sample, high repeatability, good stability and high throughput, and establishes a sugar group map with significant difference between liver injury patients and normal control personnel.
  • the model was screened for NG1A2F with significant difference in expression between the liver injury group and the normal control group.
  • the degree of liver damage of the test subject can be detected by the peak value of the single peak NG1A2F in the map model established by the method in the glycoform map of the serum of the test subject, compared with the prior art, With higher specificity and accuracy, the sensitivity and specificity of detection for liver injury reached 80.0% and 74.4%, respectively.
  • the glycoform map model constructed based on the method of the invention can enable many patients with chronic hepatitis and liver injury to receive routine and non-invasive detection, and help doctors and patients to timely monitor the occurrence and progression of chronic hepatitis liver injury, and is expected to be promoted in clinical use.
  • Figure 1 is a graph of serum glycoprotein glycans of normal control group (A) and liver injury (B).
  • Figure 2 is a plot of ROC for differential diagnosis of liver injury with NG1A2F.
  • Serum samples of 158 liver injury and control group were collected by G-Test detection technique. Among them, 80 patients with liver injury caused by hepatitis B virus and 78 patients with normal control group without hepatitis B virus. The glycan profiles obtained from the G-Test detection technique were statistically analyzed.
  • liver injury caused by hepatitis B virus There were 80 patients with liver injury caused by hepatitis B virus, and 78 patients with normal control group without hepatitis B virus.
  • Reagent A SDS was dissolved in 10 mM NH 4 HCO 3 to prepare a NH 4 HCO 3 solution containing 5% SDS and a pH of 8.3.
  • Reagent B 2.2 U/ ⁇ L of PNGaseF and 3.33% of NP-40 were mixed at a volume ratio of 1:20;
  • Reagent C mixing equal volumes of 20 mM APTS and 1 M NaCNBH 3 ;
  • Reagent D 100 mM NH4AC, sialidase (2 mU/ ⁇ L) and hydrogen peroxide were mixed at a volume ratio of 5:1:14.
  • Step 1 Preparation of oligosaccharide chain: 2 ⁇ L of reagent A was added to 4 ⁇ L of diluted serum, and denatured at 95 ° C for 5 minutes, then an equivalent volume (4 ⁇ L) of reagent B was added, and reacted at 37 ° C for 3 hours and then dried;
  • Step 2 labeling of the oligosaccharide chain: adding 2 ⁇ L of reagent C to the liquid of step 1, reacting at 65 ° C for 3 hours for fluorescent labeling, and then adding 200 ⁇ L of water to terminate the labeling reaction;
  • Step 3 post-labeling treatment: take 2 ⁇ L of fluorescently labeled step 2 liquid, add 2 ⁇ L of reagent D, react at 45 ° C for 3 hours, then add 200 ⁇ L of water to terminate the reaction;
  • Step 4 Oligosaccharide chain separation analysis: 10 ⁇ L of the liquid after the step 3 reaction was taken, and the N-oligosaccharide chain was separated by an ABI 3500dx sequencer to obtain a sugar group map.
  • step 5 the obtained sugar group map is subjected to peak quantification, and the relative content of each peak is quantitatively calculated by dividing the peak height value of each peak by the sum of the heights of all the peaks, and statistical analysis is performed.
  • the glycan profile of human serum probably shows nearly 9 N-oligosaccharide chain peaks.
  • the oligosaccharide chains exhibit different mobility due to different molecular sizes, that is, they are expressed on the glycan map.
  • the different peaks represent different oligosaccharide chains.
  • the measured peak height represents the relative concentration of oligosaccharide chains.
  • A is the normal control group and B is the hepatitis B injury group.
  • the relative content of NG1A2F in the normal control group was 3%
  • the relative content of NG1A2F in the hepatitis B injury group was 6%. It can be seen that the oligosaccharide content of the single peak NG1A2F in the hepatitis B group and the normal control group has Significant gap.
  • ROC curve analysis showed that unimodal NG1A2F has significant clinical significance in detecting patients with liver injury, ie AUC up to 0.813 ( Figure 2).
  • the cut-off value is 6.85
  • the sensitivity and specificity of detection for liver injury are 80.0% and 74.4%, respectively, while the sensitivity and specificity of the existing conventional two-dimensional ultrasound imaging method are detected.

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Abstract

L'invention concerne un procédé d'établissement d'un modèle de profil de glycome de glycoprotéines sériques d'une hépatite chronique et d'une atteinte hépatique. Ce procédé utilise un procédé de détection de test G pour détecter un profil de glycome de glycoprotéines sériques, établit un modèle de profil de glycome, sur des patients présentant une atteinte hépatique et des témoins normaux ayant une différence significative entre eux, réalise un criblage pour détecter NG1A2F, dont l'expression est significativement différente entre le groupe de patients présentant une atteinte hépatique et le groupe de témoins normaux, et peut détecter le degré d'atteinte hépatique de personnes à tester par comparaison de la valeur de pic du NG1A2F singulet dans le profil N-glycome du sérum des personnes à tester avec la valeur de pic du NG1A2F singulet dans le modèle de profil établi. Ce procédé peut permettre à de nombreux patients souffrant d'une hépatite chronique et d'une atteinte hépatique d'être soumis à une détection de routine, non invasive, et de surveiller l'occurrence et la progression de la maladie d'une hépatite chronique et d'une atteinte hépatique d'une manière opportune.
PCT/CN2018/091920 2017-06-20 2018-06-20 Procédé d'établissement d'un modèle de profil de glycome de glycoprotéines sériques d'une hépatite chronique et d'une atteinte hépatique WO2018233617A1 (fr)

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CN201710468389.6A CN109100507A (zh) 2017-06-20 2017-06-20 慢性肝炎肝损伤的血清糖蛋白n-糖组图谱模型的建立方法
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CN109682975B (zh) * 2018-12-29 2022-02-22 江苏先思达生物科技有限公司 一种乙型肝炎检测试剂及其在乙型肝炎检测中的应用
CN109596842B (zh) * 2018-12-29 2022-02-22 江苏先思达生物科技有限公司 一种阿尔兹海默症检测试剂及其在阿尔兹海默症检测中的应用
CN109490548A (zh) * 2018-12-29 2019-03-19 江苏先思达生物科技有限公司 一种肝硬化检测试剂及其在肝硬化检测中的应用
CN109682974A (zh) * 2018-12-29 2019-04-26 江苏先思达生物科技有限公司 一种胰腺癌检测试剂及其在胰腺癌检测中的应用
CN114032284A (zh) * 2021-09-15 2022-02-11 陈翠英 一种食管癌检测试剂及其在食管癌检测中的应用
CN114032282A (zh) * 2021-09-15 2022-02-11 陈翠英 一种前列腺癌检测试剂及其在前列腺癌检测中的应用
CN114032283A (zh) * 2021-09-15 2022-02-11 陈翠英 一种肠癌检测试剂及其在肠癌检测中的应用
CN114058673A (zh) * 2021-09-15 2022-02-18 江苏先思达生物科技有限公司 一种脂肪肝检测试剂及其在脂肪肝检测中的应用
CN114034752A (zh) * 2021-09-15 2022-02-11 先思达（南京）生物科技有限公司 一种肝衰竭检测试剂及其在肝衰竭检测中的应用
CN114032281A (zh) * 2021-09-15 2022-02-11 陈翠英 一种丙肝肝癌检测试剂及其在丙肝肝癌检测中的应用

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