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WO2018157832A1 - 一种胃癌监测试剂盒及其使用方法 - Google Patents

一种胃癌监测试剂盒及其使用方法 Download PDF

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WO2018157832A1
WO2018157832A1 PCT/CN2018/077714 CN2018077714W WO2018157832A1 WO 2018157832 A1 WO2018157832 A1 WO 2018157832A1 CN 2018077714 W CN2018077714 W CN 2018077714W WO 2018157832 A1 WO2018157832 A1 WO 2018157832A1
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gastric cancer
reagent
monitoring kit
cancer monitoring
reaction
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PCT/CN2018/077714
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French (fr)
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陈翠英
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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
常州吉泰生物科技有限公司
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Publication of WO2018157832A1 publication Critical patent/WO2018157832A1/zh

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the invention belongs to the technical field of biomedicine, and particularly relates to a gastric cancer monitoring kit and a using method thereof.
  • Gastric cancer is a malignant tumor disease that threatens the health of human beings around the world. Although the incidence of gastric cancer has generally declined in recent decades, the incidence rate in all cancers still ranks second, with mortality accounting for the first place. The incidence of gastric cancer in men worldwide is second only to lung, prostate and colorectal cancer, accounting for the fourth place; women have the fifth highest rate of gastric cancer, after breast cancer, cervical cancer, colorectal cancer and lung cancer. . According to statistics, there are about 380,000 new cases of gastric cancer in China each year, and 160,000 deaths. From the age of onset, there is a trend of younger age, and the incidence of gastric cancer in young people is gradually increasing. Different from gender, male patients are more than female, and young patients are mostly female patients.
  • the status quo of gastric cancer patients in China is characterized by “three highs and three lows”, namely, high incidence rate, metastasis rate and mortality rate; early diagnosis rate, radical resection rate, and 5-year survival rate are low.
  • the incidence and mortality of gastric cancer showed a decreasing trend from 2000 to 2011, the incidence of gastric cancer in men still accounted for the second place in all cancers, and the mortality rate was the third.
  • the incidence of gastric cancer in women is only the fifth in all cancers, the mortality rate is the second.
  • the pathological type of gastric cancer is based on histomorphological structure and cell biological characteristics. Different types of gastric cancer have different morphological structures and biological behaviors, and the epidemiology and molecular mechanism are different, so that the existing gastric cancer pathological scores There are many types of systems. With the continuous development of science and technology, the understanding of gastric cancer has gradually deepened. From the initial histomorphology study of gastric cancer to the current study of gastric cancer at the molecular level, the classification of gastric cancer has also changed with the change from general form to tissue. Types are based on environmental, genetic and epidemiological typing, to genotyping. At present, the most important and most common are Lauren classification and WHO classification.
  • the current diagnostic kit for gastric cancer detects some common tumor markers with low sensitivity and accuracy.
  • the main reason is that the single detection of selected tumor markers often has great limitations and it is difficult to meet the requirements of rapid diagnosis.
  • a glycoprotein molecule consists of a polypeptide chain and a sugar moiety. Any change in serum glycoproteins can reflect physiological changes in the body. Recent studies in glycomics have also shown that changes in oligosaccharide chains are closely related to the occurrence and development of various tumors (References: Liu, XE, Desmyter, L, Gao, CF, Laroy, W, Dewaele, S, Vanhooren, V, Wang, L, Zhuang, H, Callewaert, N, Libert, C, Contreras, R, Chen, CN-Glycomic changes in hepatocell ⁇ Lar carcinoma patients with liver cirrhosis induced by hepatitis B virus. Hepatology, 46, 1426-1435, 2007.).
  • the object of the present invention is to solve the uncertainty of monitoring of gastric cancer by the existing detection method, and to provide a gastric cancer monitoring kit and a method for using the same, which are capable of detecting carbohydrates (oligosaccharides) linked by glycosidic bonds of blood glycoproteins
  • the change in content is used to assess the progression of gastric cancer and the recurrence of tumors.
  • a gastric cancer monitoring kit consisting of the following reagents:
  • Reagent A a solution of 10% by mass of ammonium bicarbonate in a solution of 5% by mass of SDS;
  • Reagent B a mass concentration of 10% NP40 is prepared by adding not less than 2.4 units/ ⁇ L of a glycoside exonuclease
  • Reagent C a mixture of 1.2 M citric acid in an equal volume of 20 mM APTS and 2 M organic reducing agent in DMSO;
  • Reagent D 0.5 ⁇ L of NH 4 AC at a concentration of 100 mM, pH 5.6, not less than 0.2 ⁇ L of sialidase, 2.50 ⁇ L of hydrogen peroxide.
  • organic reducing agent is NaCNBH 3 .
  • the volume of the reagent A may be 3 ⁇ L
  • the volume of the reagent B may be 3 ⁇ L
  • the volume of the reagent C may be 4 ⁇ L
  • the volume of the reagent D may be 3 ⁇ L.
  • the method for using the above gastric cancer monitoring kit includes the following steps:
  • Step 1 adding 3 ⁇ L of reagent A to the diluted 3 ⁇ L serum, performing denaturation reaction, adding 3 ⁇ L of reagent B, reacting at 37 ° C for 4 hours, and then drying to obtain a sample;
  • Step 2 adding 4 ⁇ L of reagent C to the obtained sample, performing fluorescent labeling, and then adding 150 ⁇ L of water to terminate the labeling reaction to obtain a fluorescently labeled sample;
  • Step 3 adding 3 ⁇ L of reagent D to 5 ⁇ L of the fluorescently labeled sample, performing a terminal sialic acid reaction, and adding 100 ⁇ L of water to terminate the labeling reaction to obtain a sample of the terminal sialic acid reaction;
  • step 4 8 ⁇ L of the end-sialic acid-reacted sample was taken, and the oligosaccharide chain was separated by an ABI sequencer to obtain a map.
  • condition of the step 1 denaturation reaction is heating not lower than 95 °C.
  • condition of fluorescent labeling in step 2 is 60-70 ° C heating.
  • condition of the terminal sialic acid reaction in the step 3 is heating not higher than 45 °C.
  • the above gastric cancer monitoring kit can be used for detecting blood or human body fluid containing oligosaccharide chain components.
  • the gastric cancer monitoring kit of the present invention is based on the detection of a fingerprint of oligosaccharide chains in blood glycoproteins as a diagnostic index for evaluating gastric cancer patients to diagnose tumor staging of gastric cancer, and the detection method can allow many gastric cancer patients to receive routine, Non-invasive testing helps doctors detect stomach cancer and monitor disease progression in a timely manner.
  • Example 1 is a schematic flow chart of the analysis of the N-oligosaccharide chain fingerprinting technique using serum in Example 1;
  • Example 2 is a G-Test map of human serum used in Example 1;
  • Figure 3 is a serum G-Test spectrum of the healthy control group in Example 1;
  • Example 4 is a serum G-Test chart of a lung cancer patient in Example 1.
  • the study found that there is a significant correlation between the change in glycoside-linked carbohydrate content of blood glycoproteins and the histology of gastric cancer patients. Further, the clinical monitoring method for assessing gastric cancer can effectively screen and regularly evaluate the progress of gastric cancer and the recurrence of tumor after treatment.
  • serum oligosaccharide chain fingerprinting technology (G-Test method for short) is used as a diagnostic index for gastric cancer patients.
  • the main steps of the method are: releasing and fluorescently labeling the oligosaccharide chain of the glycoprotein in the serum or plasma sample; separating the content or fingerprint of the fluorescently labeled oligosaccharide chain in the measurement sample (referred to as G-Test map); analyzing and comparing the oligosaccharide Fingerprint map, get the test index parameters, the specific process is shown in Figure 1.
  • This method can enable many patients with gastric cancer to receive routine, non-invasive testing, help doctors to detect gastric cancer, and timely monitor the occurrence of disease and the progress of the disease.
  • the composition for monitoring the risk of developing gastric cancer or gastric cancer is selected from the group consisting of oligosaccharide chains: NA3F, NA2F, NA2FB, NGA2F, NGA2FB, and NA3.
  • the ratio of (NA3+NA2FB)/NA3F was used to diagnose gastric cancer detection.
  • the G-Test spectrum of human serum probably shows nearly 10 N-oligosaccharide chain peaks. Different oligosaccharide chains exhibit different mobility due to different molecular sizes, that is, they are expressed in G-Test.
  • the different peaks on the map represent different oligosaccharide chains; the relative concentration of oligosaccharide chains is expressed in the measured peak height.
  • NGA2F galactose-deficient two antennas containing core fucose ( ⁇ 1,6Fuc);
  • NGA2FB galactose-deficient core fucose modified with halved acetylglucosamine (GlcNAc) ( ⁇ 1, 6Fuc) two antennas;
  • NG1A2F galactose single deletion core fucose ( ⁇ 1,6Fuc) two antennas (single agalacto, core- ⁇ -1,6-fucosylated biantennary); NA2, two antennas (bigalacto, biantennary NA2F, core fucose ( ⁇ 1,6Fuc) two antennas;
  • NA2FB core fucose ( ⁇ 1,6Fuc) antenna with halved acetylglucosamine (GlcNAc) modification;
  • the invention provides a gastric cancer monitoring kit consisting of the following reagents:
  • Reagent A denaturation buffer: 10 mM ammonium bicarbonate SDS;
  • Reagent B (glycosidase reaction buffer): final concentration of exoglycosidase 2.4 units / ⁇ L at 10% NP40;
  • Reagent C (APTS Labeling Buffer): Mix an equal volume of 20 mM APTS (dissolved in 1.2 M citric acid) and 2 M organic reducing agent (dissolved in DMSO);
  • Reagent D sialidase reaction solution: 0.5 ⁇ L of 100 mM NH 4 AC, pH 5.6; 0.2 ⁇ L of sialidase, 2.50 ⁇ L of hydrogen peroxide.
  • the organic reducing agent is preferably NaCNBH 3 .
  • a total of 49 patients with gastric cancer were collected from the serum of this patient.
  • the serum was collected from Zhenjiang First People's Hospital.
  • the serum of 57 healthy controls was from Zhenjiang First People's Hospital.
  • HAV human immunodeficiency virus
  • the equipment is mainly an ABI sequencer (Applied Biosystems) and a capillary electrophoresis system with the same principle of action.
  • the reagent mainly contains reagent A (denaturation buffer): 10 mM ammonium bicarbonate 5% SDS; reagent B (PNGaseF reaction buffer): final concentration of exoglycosidase 2.4 units / ⁇ L at 10% NP40; reagent C (APTS label buffer) Liquid): Mix equal volumes of 20 mM APTS (dissolved in 1.2 M citric acid) and 2 M NaCNBH 3 (dissolved in DMSO); Reagent D (sialidase reaction solution): 0.5 ⁇ L 100 mM NH 4 AC; 0.2 ⁇ L sialidase, 2.50 ⁇ L of hydrogen peroxide.
  • reagent A denaturation buffer
  • reagent B PNGaseF reaction buffer
  • APTS label buffer APTS label buffer
  • the G-Test Atlas analysis procedure consists of four steps:
  • Step 1 Prepare a free oligosaccharide chain with a specific N-glycosidic bond hydrolase: add 3 ⁇ L of reagent A to 3 ⁇ L of diluted serum, and denature at 95 ° C for 5 minutes; then equal volume (3 ⁇ L) of reagent B , reacted at 37 ° C for 4 hours and then dried;
  • Step 2 fluorescently labeling the free oligosaccharide chain: adding 4 ⁇ L of reagent C to the liquid of step 1, heating at 65 ° C for 2 hours for fluorescent labeling, and then adding 150 ⁇ L of water to terminate the labeling reaction;
  • Step 3 remove the terminal sialic acid: take 5 ⁇ L of the fluorescently labeled liquid of step 2, then add 3 ⁇ L of reagent D, heat at 45 ° C for 3 hours to carry out the terminal sialic acid reaction, and then add 100 ⁇ L of water to terminate the labeling reaction;
  • Step 4 Separation and analysis of the fluorescently labeled N-oligosaccharide chain: 8 ⁇ L of the end-to-sialic acid reaction liquid obtained in the step 3 was taken, and the N-oligosaccharide chain fragment was separated by an ABI 3500dx sequencer to obtain a G-Test spectrum.
  • the G-Test map of human serum represents different N-oligosaccharide chain peaks, showing different mobility due to different molecular sizes, ie different peaks on the G-Test map represent different oligosaccharide chains.
  • the relative concentration of the oligosaccharide chain is expressed in the measured peak height. As shown in Fig. 3 and Fig.
  • NA2FB of gastric cancer group and normal control group with two oligoacetylglucosamine (GlcNAc) modified core fucose ( ⁇ 1,6Fuc) antennas; NA3, three antennas; NA3F
  • the three antennas modified by branched fucose ( ⁇ 1,3/1,2Fuc) have a significant gap, the change of N-glycosidically linked carbohydrate (N-oligosaccharide) content of blood glycoprotein and the histology of gastric cancer patients There is a significant correlation between them.
  • the setting of (NA3+NA2FB)/NA3F was greater than 4 for the normal group, and 51 of the normal group were satisfactory. It is 89.5%.
  • the threshold of (NA3+NA2FB)/NA3F was less than 2, it was the gastric cancer group, and in the gastric cancer group, 43 cases met the requirement, and the accuracy was 87.8%.
  • the results indicate that there is a significant correlation between the change in the N-glycosidically linked carbohydrate (N-oligosaccharide) content of the blood glycoprotein and the histology of the gastric cancer patient.

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Abstract

本发明提供了一种胃癌监测试剂盒及其使用方法，该试剂盒包括：试剂A：浓度为10mM的碳酸氢铵溶液中加入5％的SDS配制而成；试剂B：浓度为10％NP40中加入不少于2.4单位/μL糖苷外切酶配制而成；试剂C：等体积20mM APTS的1.2M柠檬酸和2M有机物还原剂的DMSO的混合液；试剂D：0.5μL浓度为100mM、pH 5.6的NH 4AC，0.2μL的唾液酸酶，2.50μL双氧水。该试剂盒基于检测血液糖蛋白中寡糖链的指纹图谱作为诊断指标对胃癌患者进行了评估，以诊断胃癌的肿瘤分期。

Description

一种胃癌监测试剂盒及其使用方法 技术领域
本发明属于生物医药技术领域，具体涉及一种胃癌监测试剂盒及其使用方法。
背景技术
胃癌是一种恶性的肿瘤疾病威胁着全球人类的健康，虽然近几十年以来胃癌的发生率总体呈现降低趋势，但在所有癌症中发生率仍占第二位，死亡率占第一位。全球范围内男性的胃癌的发生率仅次于肺、前列腺癌和结直肠癌，占第四位；女性的胃癌发生率居第五位，排在乳腺癌、宫颈癌、结直肠癌和肺癌之后。据统计，我国每年新发胃癌患者病例约38万，死亡病例16万。从发病年龄分析，具有年轻化趋势，年轻人胃癌发病率逐渐上升。从性别进行区别，男性患者多于女性，年轻患者中女性患者居多。我国胃癌患者现状呈现“三高三低”的特点，即发病率、转移率、死亡率高；早诊率、根治性切除率、5年生存率低。根据WanqingChen等人的研究统计发现，从2000年到2011年期间，虽然胃癌的发生率和死亡率呈现降低的趋势，但男性的胃癌发生率仍然占所有癌症的第二位，死亡率占第三位，女性的胃癌发生率虽然只占所有癌症的第五位，但死亡率却占第二位。
胃癌的病理分型是以组织形态结构和细胞生物学特性为基础，不同类型的胃癌，其形态结构和生物学行为各异，流行病学和分子机制亦不同，以致于现有的胃癌病理分型系统众多。随着科学技术的不断发展，对胃癌的认识也逐步加深，从最初组织形态研究胃癌到目前从分子水平研究胃癌，同时胃癌的分型也随着发生不断变化，从大体形态分型到组织分型再到基于环境、遗传和流行病学分型，到基因分型。目前最主要最常见的是Lauren分型和WHO分型。
目前出现的胃癌诊断试剂盒都是检测一些常见的肿瘤标记物，灵敏性及准确性都偏低。原因主要是选定的肿瘤标记物单项检测往往有很大的局限性，难以满足快速诊断的要求。
糖蛋白分子由多肽链和糖两部分组成。血清糖蛋白中的任何变化可以反映人体的生理改变。近年来的糖组学研究也显示了寡糖链的改变与各种肿瘤的发生和发展有密切的相关性(参考文献：Liu，X-E，Desmyter，L，Gao，C-F，Laroy，W，Dewaele，S，Vanhooren，V，Wang，L，Zhuang，H，Callewaert，N，Libert，C，Contreras，R，Chen，C.N-Glycomic changes in hepatocell μ Lar carcinoma patients with liver cirrhosisinduced by  hepatitis B virus.Hepatology，46，1426-1435，2007.)。
鉴于胃癌监测的不确定性，目前迫切需要一种能有效监测胃癌的试剂和监测评估方法。
发明内容
解决的技术问题：本发明的目的是解决现有检测方法对胃癌监测的不确定性，提供一种胃癌监测试剂盒及其使用方法，通过检测血液糖蛋白的糖苷键连接的碳水化合物(寡糖)含量的改变来评估胃癌的进展以及愈后肿瘤的复发。
技术方案：一种胃癌监测试剂盒，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5％的SDS配制而成；
试剂B：质量浓度为10％NP40中加入不少于2.4单位/μL糖苷外切酶配制而成；
试剂C：等体积20mM APTS的1.2M柠檬酸和2M有机物还原剂的DMSO的混合液；
试剂D：0.5μL浓度为100mM、pH 5.6的NH 4AC，不少于0.2μL的唾液酸酶，2.50μL双氧水。
其中，所述有机物还原剂为NaCNBH 3。
其中，试剂A的体积可以为3μL，试剂B的体积可以为3μL，试剂C的体积可以为4μL，试剂D的体积可以为3μL。
上述胃癌监测试剂盒的使用方法，包括以下步骤：
步骤1，往稀释一倍的3μL血清加入3μL的试剂A，进行变性反应，加入3μL试剂B，37℃反应4小时后干燥，得到样品；
步骤2，在所得样品中加入4μL试剂C，进行荧光标记，然后加入150μL水终止标记反应，得到荧光标记后的样品；
步骤3，将3μL试剂D加至5μL荧光标记后的样品，进行去末端唾液酸反应，加入100μL的水终止标记反应，得到去末端唾液酸反应的样品；
步骤4，取8μL去末端唾液酸反应的样品，用ABI测序仪进行寡糖链片分离，得到图谱。
进一步地，步骤1变性反应的条件为不低于95℃加热。
进一步地，步骤2中荧光标记的条件为60-70℃加热。
进一步地，步骤3中去末端唾液酸反应的条件为不高于45℃加热。
上述胃癌监测试剂盒可用于检测包含有寡糖链成分的血液或人体体液。
有益效果：本发明的胃癌监测试剂盒基于检测血液糖蛋白中寡糖链的指纹图谱作为 诊断指标对胃癌患者进行了评估，以诊断胃癌的肿瘤分期，该检测方法可以让众多胃癌患者接受常规、无创检测，帮助医生检测胃癌，并能够及时监测疾病进展。
附图说明
图1为实施例1中采用血清的N-寡糖链指纹技术分析的流程示意图；
图2为实施例1中采用的人血清G-Test图谱；
图3为实施例1中健康对照组的血清G-Test图谱；
图4为实施例1中肺癌患者的血清G-Test图谱。
具体实施方式
下面将进一步的详细说明本发明。需要指出的是，以下说明仅仅是对本发明要求保护的技术方案的举例说明，并非对这些技术方案的任何限制。本发明的保护范围以所附权利要求书记载的内容为准。
下列实施例中未注明具体实验条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。
研究发现，血液糖蛋白的糖苷键连接的碳水化合物含量的改变与胃癌患者的组织学之间存在显著地相关性。进一步地，评估胃癌临床监测方法可有效筛查、定期评估胃癌的进展以及治疗后肿瘤的复发。
在本发明中，采用血清的寡糖链指纹技术(简称G-Test方法)作为诊断指标对胃癌患者进行了评估。该方法主要步骤是，释放并荧光标记血清或血浆样品中的糖蛋白的寡糖链；分离测量样本中荧光标记的寡糖链的含量或指纹图谱(简称G-Test图谱)；分析比较寡糖指纹图谱，得到检测指标参数，具体流程见图1。该方法可以让众多胃癌患者接受常规、无创检测，帮助医生检测胃癌，并能够及时监测疾病的发生和发病的进展。
具体地，用于监测患胃癌或胃癌风险的组合物选自下述的寡糖链：NA3F、NA2F、NA2FB、NGA2F、NGA2FB以及NA3。以下实施例中，利用(NA3+NA2FB)/NA3F的比值来诊断胃癌检测。如图2所示，人血清的G-Test图谱大概显示出近10个N-寡糖链峰，不同的寡糖链因分子大小的不同而表现出不同的迁移性，即表现在G-Test图谱上的不同的峰则代表了不同的寡糖链；寡糖链的相对浓度含量则表现在所测出的峰高。
上述寡糖缩写分别表示为：NGA2F，半乳糖缺失含核心岩藻糖(α1，6Fuc)的两天线；NGA2FB，半乳糖缺失带有二等分乙酰葡糖胺(GlcNAc)修饰的核心岩藻糖(α1， 6Fuc)两天线；NG1A2F，半乳糖单一缺失的核心岩藻糖(α1，6Fuc)两天线(single agalacto，core-α-1，6-fucosylated biantennary)；NA2，两天线(bigalacto，biantennary)；NA2F，核心岩藻糖(α1，6Fuc)两天线；NA2FB，带有二等分乙酰葡糖胺(GlcNAc)修饰的核心岩藻糖(α1，6Fuc)两天线；NA3，三天线；NA3F，分支岩藻糖(α1，3/1，2Fuc)修饰的三天线。
本发明提供的一种胃癌监测试剂盒由以下试剂组成：
试剂A(变性缓冲液)：10mM的碳酸氢铵SDS；
试剂B(糖苷外切酶反应缓冲液)：糖苷外切酶终浓度2.4单位/μL在10％NP40；
试剂C(APTS标记缓冲液)：混和同等体积的20mM APTS(溶于1.2M柠檬酸)和2M有机物还原剂(溶于DMSO)；
试剂D(唾液酸酶反应液)：0.5μL 100mM NH 4AC，pH 5.6；0.2μL唾液酸酶，2.50μL双氧水。
在本发明中，有机物还原剂优选NaCNBH 3。
实施例1
一、测试样本
本实验共收集了49例胃癌患者的血清，血清收集来自镇江第一人民医院。健康对照组57例的血清来自镇江第一人民医院。
以上49例患者均接受临床实验室分析诊断和组织学检查并符合以下选择标准(a)均为胃癌患者；(b)排除人类免疫缺陷病毒(human immunodeficiencyvirus，HIV)等其他病毒感染；(c)血清采集均在患者未接受任何治疗之前，临床血常规、生化、肿瘤标志物、DNA载量等数据与血清同期收集。以上数据用于评估病人的胃功能损害的程度。
二、设备和试剂
设备主要是ABI测序仪(Applied Biosystems美国生物应用公司)，以及与之作用原理相同的毛细管电泳仪。试剂主要含有试剂A(变性缓冲液)：10mM的碳酸氢铵5％ SDS；试剂B(PNGaseF反应缓冲液)：糖苷外切酶终浓度2.4单位/μL在10％ NP40；试剂C(APTS标记缓冲液)：混和同等体积的20mM APTS(溶于1.2M柠檬酸)和2M NaCNBH 3(溶于DMSO)；试剂D(唾液酸酶反应液)：0.5μL 100mM NH 4AC；0.2μL唾液酸酶，2.50μL双氧水。
三、检测血清样本中的G-Test图谱的操作步骤：
G-Test图谱分析的操作程序保括四个步骤：
步骤1，用专一性的N-糖苷键水解酶制备自由寡糖链：往稀释一倍的3μL血清加入3μL的试剂A，95℃加热5分钟变性；然后同等体积的(3μL)的试剂B，37℃反应4小时后干燥；
步骤2，荧光标记自由寡糖链：在步骤1的液体中加入4μL的试剂C，65℃加热2小时进行荧光标记，然后加入150μL的水终止标记反应；
步骤3，去除末端唾液酸：取5μL荧光标记的步骤2的液体，然后加入3μL的试剂D，45℃加热3小时进行去末端唾液酸反应，然后加入100μL的水终止标记反应；
步骤4，分离分析荧光标记的N-寡糖链：取8μL步骤3所得去末端唾液酸反应的液体，用ABI3500dx测序仪进行N-寡糖链片分离，得到G-Test图谱。
检测分析：
对测量得到的G-Test图谱中的每个峰均进行量化计算，以每个峰的相对含量来表示(％)：即用每个峰的峰高值除以所有峰的高度的总和来定量计算，计算得到G-TEST值。用统计学比较胃癌患者与健康对照组的相对含量的差异，具体结果如下表所示：
Figure PCTCN2018077714-appb-000001
人血清的G-Test图谱代表不同的N-寡糖链峰，因分子大小的不同而表现出不同的迁移性，即表现在G-Test图谱上的不同的峰则代表了不同的寡糖链；寡糖链的相对浓度含量则表现在所测出的峰高。如图3和图4所示，胃癌组与正常对照组的NA2FB，带有二等分乙酰葡糖胺(GlcNAc)修饰的核心岩藻糖(α1，6Fuc)两天线；NA3，三天线；NA3F，分支岩藻糖(α1，3/1，2Fuc)修饰的三天线有着明显差距，血液糖蛋白的N-糖苷键连接的碳水化合物(N-寡糖)含量的改变与胃癌患者的组织学之间存在显著地相关性。研究发现利用(NA3+NA2FB)/NA3F的比值来诊断胃癌检测效果显著，设定(NA3+NA2FB)/NA3F的阈值大于4时为正常组，正常组57例中有51例满足要求，准确度为89.5％。判定(NA3+NA2FB)/NA3F的阈值小于2时为胃癌组，胃癌组49例有43例满足要求，准确度为87.8％。结果说明血液糖蛋白的N-糖苷键连接的碳水化合物(N-寡糖)含量的改变与胃癌患 者的组织学之间存在显著地相关性。

Claims (7)

  1. 一种胃癌监测试剂盒，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5％的SDS配制而成；
    试剂B：质量浓度为10％NP40中加入不少于2.4单位/μL糖苷外切酶配制而成；
    试剂C：等体积20mM APTS的1.2M柠檬酸和2M有机物还原剂的DMSO的混合液；
    试剂D：0.5μL浓度为100mM、pH 5.6的NH 4AC，不少于0.2μL的唾液酸酶，2.50μL双氧水。
  2. 根据权利要求1所述的肺癌监测试剂盒，其特征在于：所述有机物还原剂为NaCNBH 3。
  3. 权利要求1所述的胃癌监测试剂盒的使用方法，其特征在于：包括以下步骤：
    步骤1，往稀释一倍的3μL血清加入3μL的试剂A，进行变性反应，加入3μL试剂B，37℃反应4小时后干燥，得到样品；
    步骤2，在所得样品中加入4μL试剂C，进行荧光标记，然后加入150μL水终止标记反应，得到荧光标记后的样品；
    步骤3，将3μL试剂D加至5μL荧光标记后的样品，进行去末端唾液酸反应，加入100μL的水终止标记反应，得到去末端唾液酸反应的样品；
    步骤4，取8μL去末端唾液酸反应的样品，用ABI测序仪进行寡糖链片分离，得到图谱。
  4. 根据权利要求3所述的胃癌监测试剂盒的使用方法，其特征在于：步骤1变性反应的条件为不低于95℃加热。
  5. 根据权利要求3所述的胃癌监测试剂盒的使用方法，其特征在于：步骤2中荧光标记的条件为60-70℃加热。
  6. 根据权利要求3所述的胃癌监测试剂盒的使用方法，其特征在于：步骤3中去末端唾液酸反应的条件为不高于45℃加热。
  7. 权利要求1或2所述的胃癌监测试剂盒用于检测包含有寡糖链成分的血液或人体体液。
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