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WO2023040908A1 - 一种脂肪肝检测试剂及其在脂肪肝检测中的应用 - Google Patents

一种脂肪肝检测试剂及其在脂肪肝检测中的应用 Download PDF

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WO2023040908A1
WO2023040908A1 PCT/CN2022/118792 CN2022118792W WO2023040908A1 WO 2023040908 A1 WO2023040908 A1 WO 2023040908A1 CN 2022118792 W CN2022118792 W CN 2022118792W WO 2023040908 A1 WO2023040908 A1 WO 2023040908A1
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reagent
fatty liver
preparation
prepared
liver
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PCT/CN2022/118792
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French (fr)
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陈翠英
王蕾
谈宗男
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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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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/98—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

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  • the invention belongs to the technical field of biomedicine and relates to a method for detecting fatty liver, in particular to a method for detecting fatty liver based on the specific fingerprint of serum glycoprotein oligosaccharide chain detection (G-Test).
  • G-Test serum glycoprotein oligosaccharide chain detection
  • Fatty liver refers to the lesion of excessive fat accumulation in liver cells due to various reasons, resulting in lipid accumulation in liver cells exceeding 5% of the wet weight of the liver, which is called fatty liver (Fatty liver).
  • Fatty liver disease has become the second largest liver disease after viral hepatitis, and its incidence is increasing, and the age of onset is getting younger and younger, which is seriously threatening people's health.
  • Fatty liver is generally divided into alcoholic fatty liver and non-alcoholic fatty liver two categories. According to the extent of fatty degeneration in the liver, it can be divided into three types: mild, moderate, and severe.
  • the etiology of fatty liver includes two aspects: the condition of fatty liver (incentive) and the cause of fatty liver (pathogenic factor).
  • the body's immune status, nutritional factors, genetic factors, lifestyle, age and gender all play a very important role, which are the conditional factors for the onset of fatty liver.
  • the pathogenic factors of fatty liver include chemical factors, nutritional factors, endocrine and metabolic factors, biological pathogenic factors, genetic factors and so on.
  • Fatty liver clinical manifestations are asymptomatic in mild cases, and severe in severe cases. Laboratory tests lack specificity, and the diagnosis is made by liver biopsy. Generally speaking, fatty liver is a reversible disease, early diagnosis and timely treatment can often return to normal. Fatty liver was diagnosed by medical history and auxiliary examination. The severity of fatty liver can be judged comprehensively through liver function, blood lipids, B-ultrasound, clinical symptoms, etc. If it is suspected that the disease has developed to the stage of liver cirrhosis, "four items of liver fibrosis" and other items should be tested.
  • fatty liver The clinical manifestations of fatty liver are diverse, mild fatty liver is mostly asymptomatic, and more patients are found incidentally during physical examination. Fatigue is the most common subjective symptom in patients with fatty liver, but it is not correlated with the severity of histological damage. Moderate and severe fatty liver has symptoms similar to chronic hepatitis, such as loss of appetite, fatigue, nausea, vomiting, dull pain in the liver area or right upper quadrant, etc. Fatty liver is a common clinical phenomenon rather than an independent disease, including pathological changes such as steatosis, steatohepatitis, and cirrhosis.
  • fatty liver patients are obese, mildly enlarged liver may be tender, slightly tough, blunt edge, smooth surface, and a few patients may have splenomegaly and liver palms.
  • patients may develop jaundice, edema, asterixis, and signs of portal hypertension.
  • Laboratory examination showed mild fatty liver, and liver function was basically normal.
  • Moderate to severe fatty liver manifests as mild to moderate elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), reaching 2 to 5 times the normal upper line.
  • ALT is higher than AST in obese fatty liver
  • AST is higher than ALT in alcoholic fatty liver.
  • Alkaline phosphatase (ALP) and ⁇ -glutamyl transpeptidase (GGT) can be increased by 2 to 3 times in half of the patients. More than 80% of patients had elevated serum cholinease and abnormal serum bilirubin. B-ultrasound is more sensitive to the detection of fatty liver. It is mainly based on the clarity of liver vessels and the degree of ultrasound attenuation to diagnose fatty liver. Epidemiological Investigation. However, B-ultrasound cannot determine the degree of liver function damage, and it is difficult to detect early liver cirrhosis. Liver/spleen CT value ratio can be used to measure the degree of fatty liver.
  • the sensitivity of CT to diagnose fatty liver is lower than that of B-ultrasound, but the specificity is better than that of B-ultrasound.
  • MRI is mainly used for those who are difficult to diagnose by ultrasound and CT examination, especially when it is difficult to distinguish focal fatty liver from liver tumors.
  • Liver histopathological examination can be used for clinicopathological classification of non-alcoholic fatty liver disease.
  • Liver puncture is generally performed under the guidance of B-ultrasound to improve the accuracy of puncture and minimize liver damage.
  • invasive liver biopsy is required for liver tissue pathological examination, so a non-invasive, rapid, high-sensitivity and specific method is needed to assist in the early diagnosis of fatty liver.
  • the G-Test oligosaccharide chain fingerprint based on glycomics satisfies these conditions when diagnosing fatty liver, so it is an excellent new method for assisting early diagnosis of fatty liver.
  • 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.
  • a fatty liver monitoring reagent consists 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 of a fatty liver 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 fatty liver through the ratio of (NGA2F+NA2F)/NA2.
  • the invention provides a method for establishing a serum glycoprotein N-glycan group map model of fatty liver, and performs statistical analysis by measuring the specific fingerprint of serum glycoprotein oligosaccharide chain G-Test.
  • Test samples collect serum from patients with fatty liver 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 fatty liver by the ratio of (NGA2F+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 establishes the N-Test with significant difference between fatty liver and normal control people.
  • the N-glycan group map of the serum to be tested is calculated by the map model established by this method to detect whether there is fatty liver. Compared with the existing technology, it has higher specificity and accuracy.
  • the AUC area of the ROC curve made by the detection model reached 0.829.
  • the N-glycan atlas model constructed based on the method of the present invention can allow many fatty liver patients to receive routine and non-invasive testing, and help doctors and patients monitor the occurrence and disease progression of fatty liver in a timely manner, and is expected to be popularized and used in clinical practice.
  • Figure 1A is the N-glycan map of the normal control group
  • Figure 1B is the serum glycoprotein N-glycan map of the fatty liver group
  • the abbreviations of the oligosaccharides in the map are respectively expressed as: NGA2F, galactose deficiency containing core fucose Two antennas (Agalacto core- ⁇ -1, 6-fucosylated biantennary); NA2, two antennas (Biantennary); NA2F, core fucose two antennas (Bigalacto core- ⁇ -1, 6-fucosylated biantennary).
  • Test samples collect serum from patients with fatty liver 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 1084 fatty liver patients and normal controls were processed using G-Test detection technology, including 584 serum samples from fatty liver patients and 500 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

一种脂肪肝检测试剂及其制备方法，检测试剂由以下试剂混合而成，试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；试剂D：终止液。通过检测试剂测定血清中糖组图谱，将峰值量化进行统计学分析，提供一种脂肪肝血清糖组图谱的模型的建立方法来检测脂肪肝。

Description

一种脂肪肝检测试剂及其在脂肪肝检测中的应用 技术领域
本发明属于生物医药技术领域，涉及一种脂肪肝的检测方法，具体涉及一个基于血清糖蛋白寡糖链检测(G-Test)特异指纹图谱的脂肪肝检测方法。
背景技术
脂肪肝(Fatty liver)是指由于各种原因引起的肝细胞内脂肪堆积过多的病变，导致肝细胞内脂质积聚超过肝湿重的5％，称之为脂肪肝(Fatty liver)。脂肪性肝病成为仅次于病毒性肝炎的第二大肝病，发病率在不断升高，且发病年龄日趋年轻化，正严重威胁人们的健康。脂肪肝一般分为酒精性脂肪肝和非酒精性脂肪肝两大类。根据脂肪变性在肝脏累及的范围，又可分为轻、中、重三型，通常脂肪含量超过肝脏重量的5％～10％时被视为轻度脂肪肝，超过10％～25％为中度脂肪肝，超过25％为重度脂肪肝。脂肪肝的病因学包括脂肪肝发生的条件(诱因)和导致脂肪肝的原因(致病因素)两个方面。在脂肪肝的发生发展过程中，机体的免疫状态、营养因素、遗传因素、生活方式以及年龄和性别等均起相当重要的作用，即为脂肪肝发病的条件因素。脂肪肝的致病因素有化学因素、营养因素、内分泌代谢因素、生物性致病因素、遗传因素等。脂肪肝临床表现轻者无症状，重者病情凶猛。实验室检查缺乏特异性，确诊靠肝穿刺活检。一般而言，脂肪肝属可逆性疾病，早期诊断并及时治疗常可恢复正常。通过病史及辅助检查诊断脂肪肝。即可通过肝功能、血脂、B超、临床症状等综合判断脂肪肝的严重程度，如果怀疑病情发展到肝硬化阶段，还应检测“肝纤四项”等项目。
脂肪肝的临床表现多样，轻度脂肪肝多无临床症状，患者多于体检时偶然发现。疲乏感是脂肪肝患者最常见的自觉症状，但与组织学损伤的严重程度无相关性。中、重度脂肪肝有类似慢性肝炎的表现，可有食欲不振、疲倦乏力、恶心、呕吐、肝区或右上腹隐痛等。脂肪肝是一个常见的临床现象，而不是一个独立的疾病，包括脂肪变性、脂肪肝炎和肝硬化等病理改变。多数脂肪肝患者存在肥胖，肝脏轻度肿大可有触痛，质地稍韧、边缘钝、表面光滑，少数患者可有脾肿大和肝掌。进展至肝硬化时，患者可出现黄疸、水肿、扑翼样震颤以及门脉高压体征。实验室检查，轻度脂肪肝，肝功能基本正常。中、重度脂肪肝，表现为谷丙转氨 酶(ALT)、谷草转氨酶(AST)轻中度升高，达正常上线的2～5倍。一般肥胖性脂肪肝ALT高于AST，反之，酒精性脂肪肝AST高于ALT。半数患者碱性磷酸酶(ALP)和γ-谷氨酰转肽酶(GGT)可升高2～3倍。80％以上患者血清胆碱酶升高，血清胆红素异常。B超对脂肪肝的检出比较灵敏，主要依据肝血管的清晰度、超声衰减程度等对脂肪肝进行分级诊断，现已作为脂肪肝的首选诊断方法，并广泛用于人群脂肪肝发病率的流行病学调查。但B超不能确定肝功能受损的程度，也很难发现早期肝硬化。肝/脾CT值比值可用于衡量脂肪肝程度。CT诊断脂肪肝的敏感性低于B超，但特异性优于B超。磁共振主要用于超声及CT检查诊断困难者，特别是局灶性脂肪肝难以与肝脏肿瘤鉴别时。肝组织病理检查，能对非酒精性脂肪肝病进行临床病理分型。一般在B超的引导下进行肝穿刺，以提高穿刺准确性，最大限度地减少肝脏损伤。
现有脂肪肝诊断方法由于特异度低，需要有创肝穿刺进行肝组织病理检查，所以需要一种无创、快速、高灵敏度和特异度的方法进行辅助早期诊断脂肪肝。基于糖组学的G-Test寡糖链指纹图谱由于在诊断脂肪肝时满足这些条件，因此是一种极好的进行辅助早期诊断脂肪肝的新方法。
蛋白质的糖基化(Glycosylation)是一种最常见的蛋白翻译后修饰，是在糖基转移酶作用下将糖类转移至蛋白质和蛋白质上特殊的氨基酸残基形成糖苷键的过程。大多数的糖蛋白都是分泌蛋白，广泛存在于细胞膜、细胞间质、血浆以及粘液中。有些酶和激素是糖蛋白。糖蛋白具有多种生物功能。有些糖蛋白如原胶原是结构蛋白质。许多酶和激素(如黄体生成素、促甲状腺激素等)有糖蛋白结构，血液中的许多糖蛋白担负无机离子(Fe2+、Ca2+、Cu2+等)和激素等生物活性物质的运输，血液凝固(纤维蛋白原是糖蛋白)和抗体活性等生物功能。凝集素有凝集细胞的能力，糖链还可起稳定肽链的作用。糖蛋白的另一重要功能是直接或间接地参与细胞表面的种种识别现象。由于糖蛋白中糖链对于维持机体生物学功能的重要性，糖链的改变有助于阐明炎症、肿瘤细胞对周围组织侵袭及转移等异常生物行为学的分子机理。目前，己经在多种肿瘤中发现了N-糖链的改变。
糖链是重要的生物信息分子，在许多生理和病理过程中都发挥着独特作用。糖链结构非常复杂，具有微观不均一性，其分析和结构解析一直是糖生物学研究的瓶颈。目前糖链结构的分析方法发展迅速，主要包括(1)高效液相色谱法 (HPLC)：分辨率高，检测速度快，重复性高，高效液相色谱柱可以反复使用，但是柱效会随着使用次数的增加而变低，且流动相有毒，设备操作需要受过严格培训的专业人才进行，且设备相对昂贵，溶剂需要严格纯化；(2)质谱法(MS)：质谱具有灵敏度高、可获得多种结构信息和适于分析混合物等优点，是糖链定性定量分析的一种理想手段。但是质谱仪器精密，设备操作复杂，且质谱仪价格昂贵，不适合临床上普及推广使用；(3)毛细管电泳法(CE)：毛细管电泳成本低、柱效高、灵敏度高、速度快、进样量少、操作简单，但是重复性不高，稳定性不如HPLC。
G-Test检测法(Glycan-Test)是基于DNA分析仪的毛细管微电泳技术(DSA-FACE)，将前列腺液样本中糖蛋白的N-糖链进行荧光标记后，用毛细管微电泳进行分离，通过测量荧光信号得到的N-寡糖链的含量即指纹图谱(简称G-Test图谱)。该检测技术具有灵敏度高、操作简单、微量(2μL血清)、重复性高、稳定性好、高通量(96-孔板)等其他糖链分析技术无法比拟的优点，适用于一般检验科室，可望用于临床推广使用。
发明内容
本发明采用的技术方案如下：
一种脂肪肝监测试剂，由以下试剂组成：
试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；
试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；
试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；
试剂D：终止液。
优选地，所述试剂A、试剂B与试剂C的体积比为2：2：1。
一种脂肪肝检测试剂的制备方法，包括以下步骤：
步骤一 寡糖链的制备
在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；
步骤二 寡糖链的标记
在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；
步骤三 寡糖链分离分析
取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。
优选地，所述步骤一寡糖的制备中变性温度为不低于75℃加热，孵育温度为不低于25℃。
优选地，所述步骤二中荧光标记的温度为50～90℃。
一种组合物在制备脂肪肝监测试剂中的应用，所述组合物通过(NGA2F+NA2F)/NA2的比值来检测脂肪肝。
本发明提供一种脂肪肝的血清糖蛋白N-糖组图谱模型的建立方法，通过测定血清糖蛋白寡糖链G-Test特异指纹图谱，进行统计学分析。
材料和方法：
一、检测样本：收集脂肪肝患者和正常对照人的血清。
二、实验设备：糖组分析仪，PCR，离心机。
三、试剂制备：
试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；
试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；
试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；
试剂D：终止液。
四、糖测序检测步骤：
步骤一 寡糖链的制备
在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；
步骤二 寡糖链的标记
在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；
步骤三 寡糖链分离分析
取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。
五、监测对比分析
将每个峰峰高值除以所有峰高度的总和，定量计算得到每个峰的相对含量，即N-糖组图谱峰值量化，然后对量化后的脂肪肝组和正常对照组N-糖组图谱中9个寡糖峰进行比对统计分析。所述组合物通过(NGA2F+NA2F)/NA2的比值来检测脂肪肝。
与现有技术相比，本发明的有益效果：
(1)本发明方法采用灵敏度高、操作简单、仅需微量样品、重复性高、稳定性好和高通量的G-Test检测方法，建立了脂肪肝和正常对照人具有显著差异的N-糖组图谱模型。待检测血清的N-糖组图谱用本方法建立的图谱模型来计算，对是否患有脂肪肝进行检测，与现有技术相比，具有更高的特异性以及准确度，对脂肪肝的检测模型所做的ROC曲线的AUC面积达到了0.829。
(2)基于本发明方法构建的N-糖组图谱模型，能够让众多脂肪肝患者接受常规、无创检测，帮助医生及患者及时监测脂肪肝的发生和病情进展，有望在临床中推广使用。
附图说明
图1A是正常对照组N-糖组图谱；图1B是脂肪肝组的血清糖蛋白N-糖组图谱；图谱中的寡糖缩写分别表示为：NGA2F，半乳糖缺失含核心岩藻糖两天线(Agalacto core-α-1，6-fucosylated biantennary)；NA2，两天线(Biantennary)；NA2F，核心岩藻糖两天线(Bigalacto core-α-1，6-fucosylated biantennary)。
图3是模型建立后的ROC曲线图；检测样本通过函数(NGA2F+NA2F)/NA2用于鉴别脂肪肝的ROC曲线；检测样本总数为1084例，其中脂肪肝患者血清584例，正常人对照组血清500例，得到曲线下面积AUC＝0.829。
具体实施方式
下面结合实施例和附图对本发明作进一步详述。需要说明的是，下列实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件试验，或按照制造厂商建议的条件。
实施例1 检测脂肪肝
通过测定血清糖蛋白寡糖链G-Test特异指纹图谱，进行统计学分析，采用的材料和方法：
一、检测样本：收集脂肪肝患者和正常对照人的血清。
二、实验设备：糖组分析仪，PCR，离心机。
三、试剂制备：
试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；
试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；
试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；
试剂D：终止液。
四、糖测序检测步骤：
步骤一 寡糖链的制备
在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；
步骤二 寡糖链的标记
在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；
步骤三 寡糖链分离分析
取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。
五、监测对比分析
利用G-Test检测技术对收集的1084例脂肪肝患者和正常人对照组的血清样本进行处理，其中脂肪肝患者血清584例，正常人对照组血清500例。对G-Test检测技术测定样本得到的N-糖组图谱进行统计学分析。
将每个峰峰高值除以所有峰高度的总和，定量计算得到每个峰的相对含量，即N-糖组图谱峰值量化，然后对量化后的脂肪肝组和正常对照组N-糖组图谱中9个寡糖峰进行比对统计分析。如图1和图2所示，人血清的G-Test指纹图谱显示出9个N-寡糖链峰，寡糖链因分子大小的不同而表现出不同的迁移率，即表现在G-Test指纹图谱上的不同的峰则代表了不同的寡糖链，所测出的峰高 代表了寡糖链的相对浓度含量，图1A正常对照组，图1B为脂肪肝组。N-糖组图谱组合物通过(NGA2F+NA2F)/NA2的比值来检测脂肪肝。
对G-Test指纹图谱的各个峰值进行量化，然后对脂肪肝组(584例)和正常对照组(500例)进行统计学分析，通过九个峰建立模型来预测脂肪肝，发现在两组的区分上具有统计学意义(p＜0.05)。ROC曲线分析显示，模型在检测脂肪肝患者时具有显著的临床意义，即AUC可达0.829(图2)。由此说明血清中九个寡糖峰可以作为脂肪肝的标志物。
以上所述的具体实施例，结合附图对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例并不用于限制本发明，凡在本发明的精神和原则之内，本领域技术人员所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种脂肪肝监测试剂，其特征在于，由以下试剂组成：
    试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；
    试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；
    试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；
    试剂D：终止液。
  2. 根据权利要求1所述的脂肪肝检测试剂，其特征在于，所述试剂A、试剂B与试剂C的体积比是2：2：1。
  3. 根据权利要求1所述的脂肪肝检测试剂的制备方法，其特征在于，包括以下步骤：
    步骤一 寡糖链的制备
    在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；
    步骤二 寡糖链的标记
    在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；
    步骤三 寡糖链分离分析
    取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。
  4. 根据权利要求3所述的脂肪肝检测试剂的制备方法，其特征在于，所述步骤一寡糖的制备中变性温度为不低于75℃加热，孵育温度为不低于25℃。
  5. 根据权利要求3所述的脂肪肝检测试剂的制备方法，其特征在于，所述步骤二中荧光标记的温度为50～90℃。
  6. 一种组合物在制备脂肪肝监测试剂中的应用，其特征在于，所述组合物通过(NGA2F+NA2F)/NA2的比值来检测脂肪肝。
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