WO2023040868A1 - Réactif de détection de l'insuffisance hépatique et son utilisation dans la détection de l'insuffisance hépatique - Google Patents

Réactif de détection de l'insuffisance hépatique et son utilisation dans la détection de l'insuffisance hépatique Download PDF

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WO2023040868A1
WO2023040868A1 PCT/CN2022/118624 CN2022118624W WO2023040868A1 WO 2023040868 A1 WO2023040868 A1 WO 2023040868A1 CN 2022118624 W CN2022118624 W CN 2022118624W WO 2023040868 A1 WO2023040868 A1 WO 2023040868A1
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reagent
liver failure
preparation
failure detection
prepared
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PCT/CN2022/118624
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Chinese (zh)
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陈翠英
王蕾
谈宗男
徐蕾
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江苏先思达生物科技有限公司
先思达（南京）生物科技有限公司
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    • 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
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/80—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for detecting, monitoring or modelling epidemics or pandemics, e.g. flu
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a detection method for liver failure, in particular to a detection method for liver failure based on the specific fingerprint of serum glycoprotein oligosaccharide chain detection (G-Test).
  • G-Test serum glycoprotein oligosaccharide chain detection
  • Liver failure refers to severe liver damage caused by a variety of factors, resulting in serious impairment or decompensation of its synthesis, detoxification, excretion and biotransformation functions, resulting in coagulation disorder, jaundice, and hepatic encephalopathy , ascites, etc. as the main manifestations of a group of clinical syndrome.
  • ALF acute Liver Failure
  • CLF chronic liver failure
  • the clinical manifestations are extreme fatigue, and severe gastrointestinal symptoms such as obvious anorexia, abdominal distension, nausea, and vomiting.
  • the liver is also called a "processing plant” because of its functions of synthesis, detoxification, metabolism, secretion, biotransformation, and immune defense.
  • processing plant When severe damage is caused by various factors (such as viruses, alcohol, drugs, etc.), a large amount of necrosis of liver cells is caused, resulting in serious impairment or decompensation of the above-mentioned functions, and then the coagulation mechanism disorder and jaundice, hepatic encephalopathy, ascites A group of clinical syndromes mainly manifested as liver failure.
  • liver failure The main cause of liver failure is hepatitis virus (mainly hepatitis B virus, accounting for about 80-85%), non-hepatitis virus, drugs and toxic substances (isoniazid, carbon tetrachloride, alcohol, etc.), acute pregnancy fat Liver/HELLP syndrome, autoimmune hepatitis, and extensive tumor cell infiltration.
  • Clinical diagnosis The clinical diagnosis of liver failure needs to be determined based on a comprehensive analysis of medical history, clinical manifestations, and auxiliary examinations.
  • the following indicators are commonly used in the clinical diagnosis of liver failure: total bilirubin, albumin or prealbumin significantly decreased, aspartate aminotransferase (AST) / alanine aminotransferase (ALT) ratio > 1, serum cholinesterase activity significantly decreased, blood coagulation Enzyme activity (PTA) ⁇ 40%, branched-chain amino acid/aromatic amino acid ratio (BCAA/AAA) decreased significantly, blood ammonia level increased significantly, blood endotoxin level increased, imaging examination showed that the liver volume was progressively reduced, Blood cholesterol levels were significantly lowered.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • PTA blood coagulation Enzyme activity
  • BCAA/AAA branched-chain amino acid/aromatic amino acid ratio
  • Protein glycosylation is the most common post-translational modification of proteins. It is a process in which sugars are transferred to proteins and special amino acid residues on proteins to form glycosidic bonds under the action of glycosyltransferases. Most glycoproteins are secreted proteins, widely present in cell membranes, interstitial cells, plasma, and mucus. Some enzymes and hormones are glycoproteins. Glycoproteins have a variety of biological functions. Some glycoproteins such as trocollagen are structural proteins.
  • glycoproteins are glycoproteins
  • fiber Proproteins are glycoproteins
  • Lectins have the ability to aggregate cells, and sugar chains can also stabilize peptide chains. Another important function of glycoprotein is to directly or indirectly participate in various recognition phenomena on the cell surface.
  • sugar chains Due to the importance of sugar chains in glycoproteins for maintaining biological functions of the body, changes in sugar chains help to elucidate the molecular mechanisms of abnormal biobehaviors such as inflammation, tumor cell invasion and metastasis of surrounding tissues. At present, changes in N-glycan chains have been found in various tumors.
  • Sugar chains are important bioinformatics molecules that play unique roles in many physiological and pathological processes.
  • the sugar chain structure is very complex and has microscopic heterogeneity. Its analysis and structural elucidation have always been the bottleneck of glycobiology research.
  • the analysis methods of sugar chain structure are developing rapidly, mainly including (1) high performance liquid chromatography (HPLC): high resolution, fast detection speed, high repeatability, high performance liquid chromatography column can be used repeatedly, but column efficiency will vary with time
  • HPLC high performance liquid chromatography
  • MS mass spectrometry
  • mass spectrometry mass spectrometry has high sensitivity, can be It is an ideal method for the qualitative and quantitative analysis of sugar chains due to the advantages of obtaining a variety of structural information and being suitable for analyzing mixtures.
  • capillary electrophoresis capillary electrophoresis is low in cost, high in column efficiency, high in sensitivity, fast in speed, and easy to inject. The amount is small and the operation is simple, but the repeatability is not high and the stability is not as good as HPLC.
  • the G-Test detection method is based on the capillary microelectrophoresis technology (DSA-FACE) of the DNA analyzer. After the N-sugar chain of the glycoprotein in the prostatic fluid sample is fluorescently labeled, it is separated by capillary microelectrophoresis. The content of the N-oligosaccharide chain obtained by measuring the fluorescent signal is the fingerprint spectrum (G-Test spectrum for short).
  • This detection technology has the advantages of high sensitivity, simple operation, trace volume (2 ⁇ L serum), high repeatability, good stability, high throughput (96-well plate) and other sugar chain analysis technologies, and is suitable for general laboratory departments. It is expected to be used in clinical promotion.
  • Serum oligosaccharide chain G-Test detection is a non-invasive, fast, simple, high-sensitivity, high-specificity reliable method, providing services for early detection of liver failure patients.
  • a reagent for detecting liver failure consisting of the following reagents:
  • Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
  • Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
  • Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
  • Reagent D stop solution.
  • the volume ratio of the reagent A, reagent B and reagent C is 2:2:1.
  • a preparation method for liver failure detection reagent comprising the following steps:
  • Step 3 Separation and analysis of oligosaccharide chains
  • the denaturation temperature in the preparation of the step 1 oligosaccharide is not lower than 75°C, and the incubation temperature is not lower than 25°C.
  • the temperature of fluorescent labeling in the second step is 50-90°C.
  • composition detects liver failure through the ratio of (NGA2F+NG1A2F+NA2F)/NA2.
  • the invention provides a method for establishing a serum glycoprotein N-glycan group pattern model of liver failure, and performs statistical analysis by measuring the serum glycoprotein oligosaccharide chain G-Test specific fingerprint.
  • Test samples collect serum from patients with liver failure and normal controls.
  • Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
  • Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
  • Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
  • Reagent D stop solution.
  • Step 3 Separation and analysis of oligosaccharide chains
  • the composition detects liver failure by the ratio of (NGA2F+NG1A2F+NA2F)/NA2.
  • the method of the present invention adopts the G-Test detection method with high sensitivity, simple operation, only needs a small amount of sample, high repeatability, good stability and high throughput, and has established a significant difference between patients with liver failure and normal controls.
  • -Model of the glycome map In subsequent applications, the N-glycan profile of the patient's serum to be tested is calculated using the profile model established by this method, which can detect highly suspected liver failure samples. Compared with the existing technology, it has higher specificity and Accuracy, the AUC area of the ROC curve for the detection model of liver failure detection reached 0.914.
  • the 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 detect the occurrence and progression of liver failure in a timely manner, and is expected to be popularized and used in clinical practice.
  • Figure 1 is the serum glycoprotein N-glycogroup map of the normal control group and the liver failure group; the abbreviations of the oligosaccharides in the map are respectively expressed as: NGA2F, galactose lacking two antennae containing core fucose (Agalacto core- ⁇ -1 , 6-fucosylated biantennary); NG1A2F, single agalacto core- ⁇ -1, 6-fucosylated biantennary); NA2, two antennas (Biantennary); NA2F, core fucose two Antenna (Bigalacto core- ⁇ -1, 6-fucosylated biantennary).
  • Embodiment 1 detects liver failure
  • Test samples collect serum from patients with liver failure and normal controls.
  • Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
  • Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
  • Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
  • Reagent D stop solution.
  • Step 3 Separation and analysis of oligosaccharide chains
  • Serum samples collected from 106 patients with liver failure and normal controls were processed by using G-Test detection technology, including 56 serum samples from patients with liver failure and 50 serum samples from normal controls. Statistical analysis was carried out on the N-glycan profile obtained from samples measured by G-Test detection technology.

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Abstract

La présente invention concerne un réactif de détection de l'insuffisance hépatique et son procédé de préparation. Le réactif de détection est formé par mélange des réactifs suivants : le réactif A, qui est préparé par ajout de SDS à une concentration massique de 0,5 % à 5 % dans un bicarbonate d'ammonium à une concentration de 10 mM; le réactif B, qui est préparé par mélange de 0,01 à 10 U/10 μL de glycopeptidase et de 0,01 à 10 U/10 μL de sialidase, la valeur de pH de la solution mixte étant comprise entre 4 et 9; le réactif C, qui est préparé par dissolution de 8-aminopyrène-1,3,6-trisulfonate dans du DMSO, la concentration étant de 0,01 mM à 1M; et le réactif D, qui est une solution d'arrêt. Selon la présente invention, un réactif de détection est utilisé pour déterminer un profil de glycome dans le sérum et un pic est quantifié pour une analyse statistique, ce qui permet d'obtenir un procédé d'établissement d'un modèle de profil de glycome sérique d'insuffisance hépatique pour détecter une insuffisance hépatique.
PCT/CN2022/118624 2021-09-15 2022-09-14 Réactif de détection de l'insuffisance hépatique et son utilisation dans la détection de l'insuffisance hépatique WO2023040868A1 (fr)

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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
CN114540485B (zh) * 2022-03-22 2024-06-25 北京大学 用于预测hbv相关肝病患者aclf发生或预后的方法、系统及其应用
CN116223597A (zh) * 2022-12-16 2023-06-06 先思达（南京）生物科技有限公司 一种基于寡糖链检测前列腺癌的检测试剂、制备方法及应用
CN116448859A (zh) * 2022-12-16 2023-07-18 先思达（南京）生物科技有限公司 一种基于寡糖链检测肠癌的检测试剂、制备方法及应用

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