WO2023040909A1 - 一种食管癌检测试剂及其在食管癌检测中的应用 - Google Patents

一种食管癌检测试剂及其在食管癌检测中的应用 Download PDF

Info

Publication number
WO2023040909A1
WO2023040909A1 PCT/CN2022/118794 CN2022118794W WO2023040909A1 WO 2023040909 A1 WO2023040909 A1 WO 2023040909A1 CN 2022118794 W CN2022118794 W CN 2022118794W WO 2023040909 A1 WO2023040909 A1 WO 2023040909A1
Authority
WO
WIPO (PCT)
Prior art keywords
reagent
esophageal cancer
prepared
preparation
add
Prior art date
Application number
PCT/CN2022/118794
Other languages
English (en)
French (fr)
Inventor
陈翠英
王蕾
谈宗男
Original Assignee
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
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 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 filed Critical 江苏先思达生物科技有限公司
Publication of WO2023040909A1 publication Critical patent/WO2023040909A1/zh

Links

Images

Classifications

    • 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
    • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • 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
    • G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a detection method for esophageal cancer, in particular to a detection method for esophageal cancer based on the specific fingerprint of serum glycoprotein oligosaccharide chain detection (G-Test).
  • G-Test serum glycoprotein oligosaccharide chain detection
  • Esophageal cancer is a common malignant tumor in humans, accounting for more than 90% of esophageal tumors, ranking second only to gastric cancer in the retrospective survey of all malignant tumor deaths. It is estimated that about 200,000 people die of esophageal cancer every year in the world, and it is one of the most common malignant tumors. Early esophageal cancer has no obvious clinical symptoms. It is mostly caused by local lesions stimulating abnormal esophageal motility or spasm, or local inflammation, erosion, superficial ulcers, and tumor infiltration. It often occurs repeatedly, and the intermittent period can be asymptomatic for several years. . The typical symptom of mid-stage esophageal cancer is progressive dysphagia.
  • the esophageal wall has good elasticity and expansion ability, when the cancer does not involve more than half of the esophagus, the symptom of dysphagia is not obvious.
  • the degree of dysphagia is related to the pathological type, and the narrow type and medullary type are more serious than other types. 20% to 40% of the initial symptoms are not dysphagia, which causes delay in the diagnosis of esophageal cancer.
  • Symptoms of advanced esophageal cancer It is mostly caused by compression and complications, and lymphatic and hematogenous metastasis can occur. If there is ulcer, inflammation or tumor invasion in the esophageal lesion, it will produce persistent dull pain behind the sternum or in the back.
  • Cancer compresses the trachea, causing cough and dyspnea. Sometimes due to the high degree of esophageal obstruction, reverse peristalsis occurs so that the contents of the esophagus are inhaled into the airway and cause infection. Cancer tissue invades the mediastinum, trachea, bronchi, and aorta, forming mediastinitis, tracheoesophageal fistula, pneumonia, lung abscess, and even fatal hemorrhage. The patient suffered from malnutrition, dehydration and other cachexia due to dysphagia. If bone, liver, brain and other important organs are transferred, bone pain, jaundice, ascites, coma and other symptoms may occur.
  • etiology of esophageal cancer is not yet fully understood, it is related to strong carcinogens, carcinogens, lack of some anticancer factors, and genetic susceptibility. It involves nitrosamines, nutrition, trace elements, fungi and viruses, genetics and many other aspects. It is generally believed that the occurrence of esophageal cancer may be the result of a combination of various factors, and is related to living and eating habits such as smoking and drinking.
  • Diagnosis methods for esophageal cancer 1. Cytological examination of esophageal exfoliation, which is valuable for diagnosis, simple and convenient, with less pain for the examinee and low false positive rate. 2. There are no highly sensitive and specific markers for tumor markers. Squamous cell carcinoma-associated antigen (SCC-RA) is the most sensitive immune marker, which is often negative in benign esophageal tumors, while the seropositive rate in esophageal cancer patients is 40% to 52%, but it is sensitive in early cancer esophageal cancer. low, the display is negative. Other tumor markers such as carcinoembryonic antigen (CEA), CA-50, CA19-9, etc. cannot provide reliable prognostic indicators. 3.
  • CEA carcinoembryonic antigen
  • DNA ploidy is closely related to tumor histology, but has nothing to do with clinical stage. Therefore, there is a lack of simple, rapid, highly sensitive and specific biomarkers for the diagnosis of esophageal cancer to assist in early diagnosis.
  • the serum G-Test glycomics detection technology just meets these conditions, so it has a good application prospect.
  • 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 reagent for monitoring esophageal cancer 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 of an esophageal cancer 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 esophageal cancer through the ratio of NG1A2F/NA3.
  • the invention provides a method for establishing a serum glycoprotein N-glycan group map model of esophageal cancer, and performs statistical analysis by measuring the specific fingerprint of the serum glycoprotein oligosaccharide chain G-Test.
  • Test samples Sera from patients with esophageal cancer and normal controls were collected.
  • 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 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 esophageal cancer and normal controls.
  • -Model of the glycome map In the follow-up application, the N-glycan profile of the serum to be tested is calculated using the profile model established by this method, which can detect the serum of the person to be tested who is highly suspected of esophageal cancer. Compared with the existing technology, it has a higher The specificity and accuracy, the sensitivity and specificity for the detection of esophageal cancer reached 80.4% and 82.6% respectively.
  • the N-glycan profile 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 progression of esophageal cancer in a timely manner, and is expected to be popularized and used in clinic.
  • Figure 1 is the serum glycoprotein N-glycan map of the normal control group and esophageal cancer group; the abbreviations of the oligosaccharides in the map are respectively expressed as: NG1A2F, single agalacto core- ⁇ -1, 6-fucosylated biantennary); NA3, triantennary.
  • Test samples Sera from patients with esophageal cancer and normal controls were collected.
  • 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 131 esophageal cancer patients and normal controls were processed using G-Test detection technology, including 61 esophageal cancer patients and 70 normal controls. Statistical analysis was carried out on the N-glycan profile obtained from samples measured by G-Test detection technology.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种食管癌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，提供一种食管癌血清糖组图谱的模型的建立方法来检测食管癌。

Description

一种食管癌检测试剂及其在食管癌检测中的应用 技术领域
本发明属于生物医药技术领域，涉及一种食管癌的检测方法，具体涉及一种基于血清糖蛋白寡糖链检测(G-Test)特异指纹图谱的食管癌检测方法。
背景技术
食管癌是(Esophageal Carcinoma)人类常见的恶性肿瘤，占食管肿瘤的90％以上，在全部恶性肿瘤死亡回顾调查中仅次于胃癌而居第2位。据估计全世界每年大约有20万人死于食管癌，是最常见的恶性肿瘤之一。早期食管癌无明显临床症状，多是因局部病灶刺激食管蠕动异常或痉挛，或因局部炎症、糜烂、表浅溃疡、肿瘤浸润所致，常反复出现，间歇期可无症状可持续几年时间。中期食管癌的典型症状是进行性吞咽困难，由于食管壁具有良好的弹性及扩张能力，在癌未累及食管全周一半以上时，吞咽困难症状尚不显著。咽下困难的程度与病理类型有关，缩窄型和髓质型较其他型为严重。初发症状不是咽下困难者占20％～40％，而造成食管癌的诊断延误。晚期食管癌的症状：多因压迫及并发症引起，并且可以发生淋巴及血行转移。食管病变段有溃疡、炎症或是肿瘤外侵，则产生胸骨后或背部持续性隐痛。癌肿压迫气管，可出现咳嗽及呼吸困难，有时由于食管高度梗阻，产生逆蠕动使食管内容物误吸入气道造成感染。癌组织侵透纵隔、气管、支气管、主动脉，形成纵隔炎、气管食管瘘，发生肺炎、肺脓肿，甚至致命性大出血等。病人因咽下困难出现营养不良，脱水等恶病质。若有骨、肝、脑等重要脏器转移，可出现骨痛、黄疸、腹水、昏迷等症状。
目前食管癌的病因虽尚未完全明了，但与强致癌物、促癌物，缺乏一些抗癌因素以及有遗传易感性等相关。涉及亚硝胺、营养、微量元素、真菌及病毒、遗传等多方面。一般认为食管癌的发生可能是多种因素综合作用的结果，并且与生活饮食习惯有关如吸烟与饮酒。
食管癌疾病诊断方法：1.食管脱落细胞学检查，有确诊价值，方法简便，受检者痛苦小，假阳性率低。2.对肿瘤标志物现在还没有高灵敏度和特异性的的标记物。鳞状细胞癌相关抗原(SCC-RA)是最敏感的免疫标记物，在良性食管瘤中常为阴性，而在食管癌病人血清阳性率为40％～52％，但是在早期癌食管癌中灵 敏度低，显示是阴性。其他肿瘤标记物如癌胚抗原(CEA)、CA-50、CA19-9等都不能提供可靠的预后指标。3.DNA倍体与肿瘤之组织学关系密切，但与临床病期无关。因此缺乏简单，快速，高灵敏度和特异度的食管癌疾病诊断的生物标志物进行无创快速的诊断方法来辅助早期诊断。而血清G-Test糖组学检测技术正好满足这些条件，因此具有良好的应用前景。
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：
一种食管癌监测试剂，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。
一种食管癌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℃。
一种组合物在制备食管癌监测试剂中的应用，所述组合物通过NG1A2F/NA3的比值来检测食管癌。
本发明提供一种食管癌的血清糖蛋白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：收集食管癌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，然后对量化后的食管癌组和正常对照组N-糖组图谱中9个寡糖峰进行比对统计分析。N-糖组图谱的组合物通过NG1A2F/NA3的比值来检测食管癌。
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，建立了食管癌患者和正常对照人具有显著 差异的N-糖组图谱模型。在后续应用中，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，能够对高度怀疑食管癌的待检测人员的血清进行检测，与现有技术相比，具有更高的特异性以及准确度，对食管癌症检测的灵敏度以及特异性分别达到80.4％和82.6％。
(2)基于本发明方法构建的N-糖组图谱模型，能够让众多患者接受常规、无创检测，帮助医生及患者及时监测食管癌的发生和病情进展，有望在临床中推广使用。
附图说明
图1是正常对照组和食管癌组的血清糖蛋白N-糖组图谱；图谱中的寡糖缩写分别表示为：NG1A2F，半乳糖单一缺失的核心岩藻糖两天线(single agalacto core-α-1，6-fucosylated biantennary)；NA3，三天线(Triantennary)。
图2是模型建立后的ROC曲线图；检测样本通过函数NG1A2F/NA3用于鉴别食管癌的ROC曲线；检测样本总数为131例，其中食管癌患者血清61例，正常人对照组血清70例，得到曲线下面积AUC＝0.804。
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 检测食管癌
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：收集食管癌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131例食管癌患者和正常人对照组的血清样本进行处理，其中食管癌患者血清61例，正常人对照组血清70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，然后对量化后的食管癌组和正常对照组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为正常对照组，图1B为食管癌组。N-糖组图谱的通过NG1A2F/NA3的比值来检测食管癌。
对G-Test指纹图谱的各个峰值进行量化，然后对食管癌组(61例)和正常对照组(70例)进行统计学分析，通过九个峰建立模型来预测食管癌，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，模型在检测食管癌患者时具有显著的临床意义，即AUC可达0.804(图2)。由此说明血清中九个寡糖峰可以作为食管癌的标志物。
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. 一种食管癌监测试剂，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所述的食管癌检测试剂，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所述的食管癌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所述的食管癌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所述的食管癌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. 一种组合物在制备食管癌监测试剂中的应用，其特征在于，所述组合物通过NG1A2F/NA3的比值来检测食管癌。
PCT/CN2022/118794 2021-09-15 2022-09-14 一种食管癌检测试剂及其在食管癌检测中的应用 WO2023040909A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111079842.7 2021-09-15
CN202111079842.7A CN114032284A (zh) 2021-09-15 2021-09-15 一种食管癌检测试剂及其在食管癌检测中的应用

Publications (1)

Publication Number Publication Date
WO2023040909A1 true WO2023040909A1 (zh) 2023-03-23

Family

ID=80134357

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/118794 WO2023040909A1 (zh) 2021-09-15 2022-09-14 一种食管癌检测试剂及其在食管癌检测中的应用

Country Status (2)

Country Link
CN (1) CN114032284A (zh)
WO (1) WO2023040909A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102565318A (zh) * 2012-01-11 2012-07-11 陈翠英 一种肝癌监测、分期以及预后风险评估的试剂及其方法
CN104807998A (zh) * 2015-05-12 2015-07-29 先思达（南京）生物科技有限公司 一种早期肝癌诊断试剂盒及其使用方法
CN106950379A (zh) * 2017-03-02 2017-07-14 先思达（南京）生物科技有限公司 一种肺癌监测试剂盒及其使用方法
CN106950380A (zh) * 2017-03-02 2017-07-14 先思达（南京）生物科技有限公司 一种胃癌监测试剂盒及其使用方法
CN107505295A (zh) * 2017-07-21 2017-12-22 江苏先思达生物科技有限公司 一种肝癌监测试剂盒及其使用方法
CN107894483A (zh) * 2017-06-20 2018-04-10 江苏先思达生物科技有限公司 肝衰竭的血清糖蛋白n‑糖组图谱模型的建立方法
CN109100507A (zh) * 2017-06-20 2018-12-28 江苏先思达生物科技有限公司 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
CN109100410A (zh) * 2017-06-20 2018-12-28 江苏先思达生物科技有限公司 肝硬化的血清糖蛋白n-糖组图谱模型的建立方法
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

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1310033C (zh) * 2004-03-23 2007-04-11 中国医学科学院肿瘤医院肿瘤研究所 一种检测血清蛋白指纹的新方法
AU2008263899A1 (en) * 2007-06-14 2008-12-18 Universiteit Gent Diagnostic test for the detection of early stage liver cancer
KR101484969B1 (ko) * 2014-03-26 2015-01-22 충남대학교산학협력단 Ｎ-당쇄화된 당펩타이드를 이용한 암 진단방법
ES2765673T3 (es) * 2015-07-02 2020-06-10 Univ Gent Biomarcador para predecir la falta de función primaria de un injerto de hígado
CN106814121B (zh) * 2015-11-30 2019-07-23 中国科学院大连化学物理研究所 一种基于微流控芯片的血清糖谱分型方法
EP3299818B1 (en) * 2016-09-26 2019-12-04 GA Generic Assays GmbH Method for the diagnosis of acute pancreatitis (ap) by detection of glycoprotein 2 isoform alpha (gp2a)
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

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102565318A (zh) * 2012-01-11 2012-07-11 陈翠英 一种肝癌监测、分期以及预后风险评估的试剂及其方法
CN104807998A (zh) * 2015-05-12 2015-07-29 先思达（南京）生物科技有限公司 一种早期肝癌诊断试剂盒及其使用方法
CN106950379A (zh) * 2017-03-02 2017-07-14 先思达（南京）生物科技有限公司 一种肺癌监测试剂盒及其使用方法
CN106950380A (zh) * 2017-03-02 2017-07-14 先思达（南京）生物科技有限公司 一种胃癌监测试剂盒及其使用方法
CN107894483A (zh) * 2017-06-20 2018-04-10 江苏先思达生物科技有限公司 肝衰竭的血清糖蛋白n‑糖组图谱模型的建立方法
CN109100507A (zh) * 2017-06-20 2018-12-28 江苏先思达生物科技有限公司 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
CN109100410A (zh) * 2017-06-20 2018-12-28 江苏先思达生物科技有限公司 肝硬化的血清糖蛋白n-糖组图谱模型的建立方法
CN107505295A (zh) * 2017-07-21 2017-12-22 江苏先思达生物科技有限公司 一种肝癌监测试剂盒及其使用方法
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BYRNE A.-M., SHARMA R., DUGGAN G., KELLEHER D., LONG A.: "Deoxycholic acid impairs glycosylation and fucosylation processes in esophageal epithelial cells", GLYCOBIOLOGY, OXFORD UNIVERSITY PRESS, US, vol. 22, no. 5, 1 May 2012 (2012-05-01), US , pages 638 - 648, XP093048524, ISSN: 0959-6658, DOI: 10.1093/glycob/cwr190 *

Also Published As

Publication number Publication date
CN114032284A (zh) 2022-02-11

Similar Documents

Publication Publication Date Title
EP3394620B1 (en) Bladder cancer biomarker proteins
WO2018233617A1 (zh) 慢性肝炎肝损伤的血清糖蛋白糖组图谱模型的建立方法
WO2018233619A1 (zh) 肝衰竭的血清糖蛋白糖组图谱模型的建立方法
CA2614946A1 (en) Biomarkers for melanoma
CN109100410B (zh) 肝硬化的血清糖蛋白n-糖组图谱模型的建立方法
WO2018157832A1 (zh) 一种胃癌监测试剂盒及其使用方法
CN102565318A (zh) 一种肝癌监测、分期以及预后风险评估的试剂及其方法
CN107505295A (zh) 一种肝癌监测试剂盒及其使用方法
Starodubtseva et al. Label‐free cervicovaginal fluid proteome profiling reflects the cervix neoplastic transformation
WO2023040909A1 (zh) 一种食管癌检测试剂及其在食管癌检测中的应用
CN105092627B (zh) 用于检测胃癌相关代谢小分子的核磁共振模型及制备方法
WO2023040912A1 (zh) 一种前列腺癌检测试剂及其在前列腺癌检测中的应用
WO2023040911A1 (zh) 一种肠癌检测试剂及其在肠癌检测中的应用
CN116879558B (zh) 卵巢癌诊断标志物、检测试剂及检测试剂盒
WO2023040868A1 (zh) 一种肝衰竭检测试剂及其在肝衰竭检测中的应用
WO2023040908A1 (zh) 一种脂肪肝检测试剂及其在脂肪肝检测中的应用
CN114660290B (zh) 预测甲状腺癌术后复发的糖链标志物及其应用
CN114032281A (zh) 一种丙肝肝癌检测试剂及其在丙肝肝癌检测中的应用
CN113125757B (zh) 一种用于母猪早期妊娠诊断的蛋白生物标志物及其用于母猪早期妊娠检测的方法
CN114136932A (zh) 一种子宫内膜样腺癌检测试剂及其在子宫内膜样腺癌检测中的应用
CN108823308A (zh) 检测circMAN1A2和LOC284454试剂的应用及试剂盒
CN118091135A (zh) 一种生物标记物用于制备表征甲状腺乳头状癌产品的用途及其筛选方法
Mischak et al. Urinary proteome analysis using capillary electrophoresis coupled to mass spectrometry: a powerful tool in clinical diagnosis, prognosis and therapy evaluation
Borre et al. Erythrocyte Sedimentation Rate—A Predictor of Malignant Potential in Early Prostate Cancer
CN116298285A (zh) 一种基于寡糖链检测乳腺癌的检测试剂、制备方法及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22869278

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE