WO2021213302A1 - 检测非小细胞肺癌患者外周血循环肿瘤细胞cea基因突变的免疫荧光试剂盒及检测方法 - Google Patents

检测非小细胞肺癌患者外周血循环肿瘤细胞cea基因突变的免疫荧光试剂盒及检测方法 Download PDF

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WO2021213302A1
WO2021213302A1 PCT/CN2021/088029 CN2021088029W WO2021213302A1 WO 2021213302 A1 WO2021213302 A1 WO 2021213302A1 CN 2021088029 W CN2021088029 W CN 2021088029W WO 2021213302 A1 WO2021213302 A1 WO 2021213302A1
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peripheral blood
solution
filter
lung cancer
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李胜
王振丹
李�浩
黄宁
高德海
马莹
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山东第一医科大学(山东省医学科学院)
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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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

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  • the invention provides an immunofluorescence kit and a detection method for detecting CEA gene mutations in peripheral blood circulating tumor cells of patients with non-small cell lung cancer, and belongs to the technical field of molecular biology.
  • NSCLC Non-small cell lung cancer
  • Circulating tumor cells are tumor cells that fall off from solid tumors and enter the peripheral blood circulation. Since they were discovered in 1989, there have been a variety of methods for detecting circulating tumor cells in the peripheral blood. Recent studies have shown that its detection has important clinical significance for evaluating the prognosis of cancer patients, especially advanced cancer patients, and selecting appropriate individualized treatments. Because CTC detection has the characteristics of minimally invasive and real-time detection, it is called "liquid biopsy" of tumors.
  • CEA carcinoembryonic antigen refers to carcinoembryonic antigen, which is an acid glycoprotein, 45% of which is protein, containing fucose, mannose, galactose and sialic acid, with a molecular weight of 150,000 to 300,000 daltons, and its code The gene is located on chromosome 19.
  • gastrointestinal tumors such as colon cancer, gastric cancer, and pancreatic cancer
  • CEA is widely used as a diagnostic and detection index for various digestive system tumors. Subsequent studies found that there are varying degrees of positive rates in liver cancer, breast cancer, ovarian cancer, uterine cancer, urinary system tumors and other malignant tumors.
  • the specimens tested by CEA in NSCLC patients are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection.
  • Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have Research on the industrialization of key technologies for tumor cell detection and identification.
  • This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Provincial Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system.
  • the identification and diagnosis kits are industrialized and promoted through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd., which are registered in Jinan.
  • the present invention provides a non-diagnostic purpose of non-diagnostic purpose of CEA gene mutations in circulating tumor cells in peripheral blood of patients with non-small cell lung cancer.
  • Detection method Separate CTCs in the peripheral blood of patients with advanced or recurrent non-small cell lung cancer from which tissue samples cannot be obtained by using a membrane filtration device, and further use immunofluorescence technology to detect the CEA expression of CTCs.
  • the present invention provides an immunofluorescence kit for detecting CEA gene mutations in circulating tumor cells in the peripheral blood of patients with non-small cell lung cancer.
  • the primary antibody working solution is composed of mouse anti-CK, rat anti-CD45, and rabbit anti-CEA, which are diluted and mixed with BD wash buffer at 1:100&1:500&1:400;
  • the second antibody working solution is It is composed of fluorescently labeled mouse anti-goat, fluorescently labeled rat anti-goat, and fluorescently labeled rabbit anti-goat.
  • the BD wash buffer is diluted 1:500 and mixed.
  • the diluent is composed of 1 mmol/L EDTA + 0.1% BSA + 0.1% trehalose + 0.2% polyoxyethylene polyoxypropylene ether block copolymer, and the base liquid is a Tris-HCl buffer.
  • the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
  • the staining solution A is a DAB staining solution
  • the staining solution B is a hematoxylin staining solution.
  • the present invention also provides a method for detecting CEA gene mutations in circulating tumor cells in the peripheral blood of patients with non-small cell lung cancer for non-diagnostic purposes using the immunofluorescence kit, which includes the following steps:
  • the specific method for detecting the CEA expression of CTC by the immunofluorescence method in the present invention is as follows:
  • Secondary antibody incubation add 100 ⁇ l of the secondary antibody diluent dropwise, and incubate at room temperature for 30 minutes;
  • the membrane filtration device for separating tumor cells used in the present invention includes a filter, a blood sample container, a waste liquid cylinder, and an iron stand.
  • the iron stand is provided with a base, a stand and a bracket.
  • the blood sample container is set on the upper part of the iron stand through the bracket.
  • a filter which is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.
  • the filter includes a filter upper port, a filter membrane, a filter membrane platform and a filter lower port.
  • the filter membrane is placed on the filter membrane platform; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion device.
  • the filter membrane is made of a hydrophobic material, and the filter holes with a diameter of 8 micrometers are evenly spread on it.
  • the detection method provided by the present invention can detect CEA expression in patients with advanced or recurrent non-small cell lung cancer without puncture biopsy to obtain tissue samples. This technology is minimally invasive and can be detected in real time.
  • the method provided by the present invention can avoid false positive results caused by edge effects that may occur during the staining process, has good stability, reduces cell loss, and improves detection accuracy.
  • Figure 1 is a schematic diagram of the structure of the membrane filtration device of the present invention.
  • FIG. 2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention
  • FIG. 3 is a schematic diagram of the structure of the filter membrane of the membrane filtration device of the present invention.
  • Figure 4 shows CEA immunofluorescence images of circulating tumor cells in the peripheral blood of patients with advanced non-small cell lung cancer.
  • Component content Diluent 45mL Decolorizing liquid 1mL Staining solution A 0.5mL Staining Solution B 1mL Primary antibody working fluid 100 ⁇ L Secondary antibody working fluid 100 ⁇ L Methanol 200 ⁇ L 2% PFA 200 ⁇ L 10% goat serum 100 ⁇ L DAPI Mounting Tablets H-1200
  • the membrane filtration device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;
  • the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, after the peripheral blood containing CTC is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is larger than the diameter. The filter hole 10 is trapped on the filter membrane 7.
  • the primary antibody working solution is composed of mouse anti-CK, rat anti-CD45 and rabbit anti-EGFR, using BD respectively. Dilute and mix the wash buffer at 1:100&1:500&1:400, and incubate at 37°C for 60 minutes in a humid box (or overnight at 4°C, and reheat at 37°C for 30 minutes the next day);
  • Secondary antibody incubation Drop 100 ⁇ l of the secondary antibody dilution.
  • the working solution of the secondary antibody is composed of fluorescently labeled mouse anti-goat, fluorescently labeled rat anti-goat, and fluorescently labeled rabbit anti-goat, each using BD wash buffer Dilute and mix at 1:500, incubate at room temperature for 30 minutes;
  • Figure 4 is an immunofluorescence staining image of circulating tumor cells in the peripheral blood of patients with advanced non-small cell lung cancer. According to immunological and morphological findings, it is found that the tumor cells are large in size and have abnormal nucleo-cytoplasmic ratios. The immunological manifestations are typical CTCs, where A is merge; B is CD45; C is CEA; D is DAPI; E is CK.
  • the detected circulating tumor cells were confirmed by immunohistochemistry to confirm the expression of CEA and compared with the CEA results of the gross specimens of non-small cell lung cancer, to observe the differences, mainly for patients with negative CEA expression in gross specimens and positive expression of circulating tumor cells, to guide non-small cells
  • Targeted therapy of lung cancer provides new ideas for targeted therapy of non-small cell lung cancer.

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Abstract

一种检测非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变的免疫荧光试剂盒及检测方法。该试剂盒包括稀释液45mL、、脱色液1mL、染色液A 0.5mL、染色液B 1mL、甲醇200μl、2％PFA 200μl、10％山羊血清100μl、一抗工作液100μl、二抗工作液100μl、DAPI封片剂。该检测方法不用穿刺活检获取组织标本即可检测到晚期或复发非小细胞肺癌患者CEA表达情况。该检测方法属于微创并能够实时检测，且能够避免染色过程中可能产生的边缘效应导致的假阳性结果，稳定性好，降低细胞的损失，提高检测的准确性。

Description

检测非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变的免疫荧光试剂盒及检测方法 技术领域
本发明提供了一种检测非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变的免疫荧光试剂盒及检测方法，属于分子生物学技术领域。
背景技术
肺癌是导致癌症患者死亡的主要恶性肿瘤之一，在我国，肺癌的发病率和死亡率均居第一位。非小细胞肺癌(NSCLC)约占全部肺癌的85％，而这其中超过70％的NSCLC患者在确诊时已为晚期。尽管手术和放化疗等治疗技术不断提高，NSCLC患者5年生存率仍低于20％，主要死因包括局部复发与远处转移。
循环肿瘤细胞(Circulating tumor cell，CTC)是从实体肿瘤脱落进入外周血液循环的肿瘤细胞，自1989年被发现以来，目前已有多种方法用于外周血循环肿瘤细胞的检测。近期研究表明，其检测对于评估肿瘤患者尤其是晚期肿瘤患者的预后以及选择合适的个体化治疗具有重要的临床意义。因CTC检测具有微创、实时检测等特点，被称为肿瘤的“液态活检”。
CEA(carcinoembryonic antigen)是指癌胚抗原，是一种酸性糖蛋白，45％为蛋白质，含有岩藻糖、甘露糖、半乳糖及唾液酸等成分，分子量为150000-300000道尔顿，其编码基因位19号染色体。既往研究表明，结肠癌、胃癌、胰腺癌等消化道肿瘤患者血清中CEA均呈现不同程度的升高，CEA被广泛用作各种消化系统肿瘤的诊断及检测指标。随后研究发现，在肝癌、乳腺癌、卵巢癌、子宫癌、泌尿系肿瘤等其他恶性肿瘤中也有不同程度的阳性率，在恶性肿瘤的鉴别诊断、病情监测、疗效评价等方面仍有重要作用。而目前临床实践中，NSCLC患者CEA检测的标本主要为肿瘤组织，来源于手术或穿刺活检，很难做到多次或实时检测。
目前，山东省第一医科大学、山东省药物研究院联合山东祺欣生物科技有限公司、山东喻晓生物科技有限公司、济南杏恩生物科技有限公司、山东发现生物技术有限公司等单位，对于循环肿瘤细胞检测鉴定关键技术进行产业化推广的研究，本项目为山东省重大科技创新工程项目，本项目将以山东第一医科大学济南校区的山东省药物研究院为核心，落实注册人制度，依托循环肿瘤细胞检测鉴定核心诊断技术，进一步注册鉴定诊断试剂盒，以包括PD1、PD-L1、ER、PR、Her-2、GPC-3、VEGF、P53、Vimentin、TKI-EGFR、RAS、CK、 ALK-D5F3、CD20、ALK/EML4、Beta-catenin、E-Cadherin、EP-CAM、HPV、IDH-1、PSA、PSMA、VEGF、GFAP、细胞角蛋白、AE1/AE3、雌激素受体、孕激素受体、BCA-225、CA 125、CEA、EMA、ERCC1、HPV、Ki-67、P53、TOP2A等作为CTCs表达的示踪剂，注册超灵敏、超快速、高覆盖、低成本、准确特异的鉴定诊断试剂盒，通过与在济南注册的山东祺欣生物科技有限公司、山东喻晓生物科技有限公司、济南杏恩生物科技有限公司、山东发现生物技术有限公司合作进行产业化推广。
发明内容
为了克服晚期或复发非小细胞肺癌患者无法实时或反复穿刺获取组织标本、进而不能评估患者CEA状态的不足，本发明提供了一种非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变非诊断目的的检测方法：利用膜过滤装置分离获得无法获取组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC，进一步运用免疫荧光技术检测CTC的CEA表达情况。
本发明采用的技术方案如下：
本发明提供了一种检测非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变的免疫荧光试剂盒，包括稀释液45mL、、脱色液1mL、染色液A 0.5mL、染色液B 1mL、甲醇200μl、2％PFA 200μl 10％山羊血清100μl、一抗工作液100μl、二抗工作液100μl、DAPI封片剂。
进一步的，所述一抗工作液是由小鼠抗CK、大鼠抗CD45和兔抗CEA组成，分别采用BD wash buffer按1:100&1:500&1:400稀释混匀；所述二抗工作液是由荧光标记的小鼠抗羊、荧光标记的大鼠抗羊和荧光标记的兔抗羊组成，分别采用BD wash buffer按1:500稀释混匀。
进一步的，所述稀释液是由1mmol/L EDTA+0.1％BSA+0.1％海藻糖+0.2％聚氧乙烯聚氧丙烯醚嵌段共聚物组成，所述基础液为Tris-HCl缓冲剂。
进一步的，所述脱色液是由95％酒精与100％二甲苯按容积比1:1组成。
进一步的，所述染色液A为DAB染色液；所述染色液B为苏木素染色液。
本发明还提供了一种利用上述免疫荧光试剂盒非诊断目的检测非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变的方法，包括以下步骤：
(1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC：采集无法获取组织标本的晚期或复发非小细胞肺癌患者外周血：肘正中静脉外周血5ml；
(2)外周血样预处理：将采集的外周血样采用稀释液进行10倍稀释，稀释后加多聚甲醛固定外周血样10分钟，固定终浓度为0.25％；
(3)利用膜过滤分离肿瘤细胞装置过滤外周血样，分离获得外周血CTC：将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中，使其依靠重力自然过滤；
(4)过滤结束后，从膜过滤分离肿瘤细胞装置中取下滤器，将循环肿瘤细胞染色液A液0.5ml加入到滤器中，染色3min，PBS缓冲液冲洗干净；滤液过滤完全后加入染色液B液1ml，染色2min，纯水1ml冲洗2次，向滤器中加入200μl 2％PFA，室温固定5min，完成后0.5ml PBS漂洗3次，每次2min；再向滤器中加入200μl预冷的甲醇，4℃固定15min；取下滤膜，将滤膜下表面平整的贴附到载玻片一侧，将载玻片置于室温干燥4-5min后，使用镊子快速将滤膜从载玻片表面一次性揭起，转移至载玻片中央，滴加2ul贴片剂，干燥后在显微镜下观察，确定是否存在CTC；
(5)运用免疫荧光法检测CTC的CEA表达情况。
本发明运用免疫荧光法检测CTC的CEA表达的具体方法如下：
(1)脱色：将带有CTC的滤膜从载玻片上取下，置于脱色液中浸泡4-6小时，脱去CTC染色液，PBS洗2min×3次；
(2)封闭：向滤膜上滴加100μl 10％山羊血清，室温放置30min，完成后吸去多余的血清；
(3)一抗孵育：弃掉载玻片上的封闭液，并立即滴加100μl一抗稀释液置于湿盒37℃孵育60min(或4℃过夜孵育，次日置于37℃复温30min)；
(4)完成后PBS漂洗3次，每次3min；
(5)二抗孵育：滴加100μl二抗稀释液，室温孵育30min；
(6)完成后PBS漂洗3次，每次2min；
(7)使用含DAPI的封片剂封片，阅片，采图；
(8)采照完成后，脱片后进行瑞氏吉姆萨染色，与IF结果进行对比。
采图标准(对阳性细胞进行采图)
本发明所使用的膜过滤分离肿瘤细胞装置，包括滤器、血样容器、废液缸和铁架台，所述铁架台设有底座、立架和支架，所述血样容器通过支架设置于铁架台上部，血样容器的下方为滤器，滤器通过输液器联通至废液缸，废液缸设置于底座上。
所述滤器包括滤器上口、滤膜、载滤膜平台和滤器下口，滤膜置于载滤膜平台上；滤器上口接血样容器，滤器下口通过输液器接废液缸。
所述滤膜为疏水材料制成，其上均匀布满口径为8微米的滤孔。
本发明的有益效果是：
(1)本发明提供的检测方法，不用穿刺活检获取组织标本即可检测到晚期或复发非小细胞肺癌患者CEA表达情况。该技术属于微创，并能够实时检测。
(2)本发明提供的方法，能够避免染色过程中可能产生的边缘效应导致的假阳性结果，稳定性好，降低细胞的损失，提高检测的准确性。
附图说明
图1为本发明的膜过滤装置结构示意图；
图2为本发明膜过滤装置的滤器的结构示意剖视图；
图3为本发明膜过滤装置的滤器滤膜的结构示意图；
图4为晚期非小细胞肺癌患者外周血循环肿瘤细胞CEA免疫荧光图像。
图中：1铁架台、2血样容器、3滤器、4输液器、5废液缸、6滤器上口、7滤膜、8载滤膜平台、9滤器下口、10滤孔、11底座、12立架、13支架。
具体实施方式
下面结合附图和实施例对本发明阐述如下。
本发明所使用的试剂盒具体规格如表1所示：
表1
组分 含量
稀释液 45mL
脱色液 1mL
染色液A 0.5mL
染色液B 1mL
一抗工作液 100μL
二抗工作液 100μL
甲醇 200μL
2％PFA 200μL
10％山羊血清 100μL
DAPI封片剂 H-1200
运用此技术方法分离获取并鉴定10例非小细胞肺癌患者(同时检测10例正常人样本做阴性 对照)外周血循环肿瘤细胞的实施例。
实施例1
一、利用膜过滤装置分离获取无法获得组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC，确定CTC是否存在：
自肘正中静脉采集空腹8-12小时的空腹血5ml，用45ml稀释液(成分：1mmol/L EDTA+0.1％BSA+0.1％海藻糖+0.2％聚氧乙烯聚氧丙烯醚嵌段共聚物)稀释外周血，然后加入3ml的4％多聚甲醛固定稀释后的血样10分钟；
在固定的间期，组装膜过滤装置：如附图1、图2、图3所示，该过滤装置由滤器3、滤膜7、血样容器2、废液缸5、铁架台1构成；
用10mlPBS润湿滤器3，然后将固定好的外周血样加入到膜过滤装置的血样容器2中，使其依靠重力自然过滤，CTC被截留在滤膜7上；
肿瘤细胞直径一般大于15微米，而血细胞(包括红细胞、白细胞)直径一般小于8微米，因此当含有CTC的外周血经过滤后，血细胞因直径小于滤孔10能够被滤过，而CTC因直径大于滤孔10被截留在滤膜7上。
过滤结束后，从过滤装置中取下滤器3，打开并移走滤器上口6，将将循环肿瘤细胞染色液A液0.5ml加入到滤器中，染色3min，PBS缓冲液冲洗干净；滤液过滤完全后加入B液，1ml，染色2min，纯水1ml,PBS缓冲液将滤器3冲洗干净，用眼科镊子取下滤膜7，细胞面朝上，放置在载玻片上；
将滤膜干燥后在显微镜下观察,确定是否存在CTC。
通过观察，10例健康志愿者均未查到CTC；除2例复发非小细胞肺癌患者未检测到CTC外，其余8例均检测到CTC(表2)，本次检测阳性率为80％，值得注意的是，当稀释液不添加0.1％海藻糖或者不添加0.2％聚氧乙烯聚氧丙烯醚嵌段共聚物时，单一的采用0.3％海藻糖或者0.3％聚氧乙烯聚氧丙烯醚嵌段共聚物，制备的血样稳定性差，部分血样还会形成分层，血液细胞容易发生聚集和粘连，影响最终的检测效果。
表2实施例CTC检测结果
Figure PCTCN2021088029-appb-000001
二、运用免疫荧光技术检测CTC的CEA表达情况：
(1)脱色：将带有CTC的滤膜从载玻片上取下，置于脱色液中浸泡4-6小时，脱去CTC染色液，PBS洗2min×3次；
(2)封闭：向滤膜上滴加100μl 10％山羊血清，室温放置30min，完成后吸去多余的血清(注：山羊血清采用用pH值为7.4的PBS缓冲液稀释)；
(3)一抗孵育：弃掉载玻片上的封闭液，并立即滴加100μl一抗稀释液，一抗工作液是由 小鼠抗CK、大鼠抗CD45和兔抗EGFR组成，分别采用BD wash buffer按1:100&1:500&1:400稀释混匀，置于湿盒37℃孵育60min(或4℃过夜孵育，次日置于37℃复温30min)；
(4)完成后PBS漂洗3次，每次3min；
(5)二抗孵育：滴加100μl二抗稀释液，二抗工作液是由荧光标记的小鼠抗羊、荧光标记的大鼠抗羊和荧光标记的兔抗羊组成，分别采用BD wash buffer按1:500稀释混匀，室温孵育30min；
(6)完成后PBS漂洗3次，每次2min；
(7)使用含DAPI的封片剂封片，阅片，采图；
(8)采照完成后，脱片后进行瑞氏吉姆萨染色，与IF结果进行对比。
图4为晚期非小细胞肺癌患者外周血循环肿瘤细胞免疫荧光染色图像，根据免疫学及形态学表现，发现肿瘤细胞细胞体积大，核质比异常，免疫学表现为典型的CTCs，其中，A为merge；B为CD45；C为CEA；D为DAPI；E为CK。
所检测的循环肿瘤细胞应用免疫组化证实CEA的表达并与非小细胞肺癌大体标本CEA结果对比，观察其差异，主要针对大体标本CEA表达阴性而循环肿瘤细胞表达阳性的患者，指导非小细胞肺癌的靶向治疗，为非小细胞肺癌靶向治疗提供新的思路。

Claims (7)

  1. 一种检测非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变的免疫荧光试剂盒，其特征在于，包括稀释液45mL、、脱色液1mL、染色液A 0.5mL、染色液B 1mL、甲醇200μl、2％PFA 200μl 10％山羊血清100μl、一抗工作液100μl、二抗工作液100μl、DAPI封片剂。
  2. 根据权利要求1所述的免疫荧光试剂盒，其特征在于，所述一抗工作液是由小鼠抗CK、大鼠抗CD45和兔抗CEA组成，分别采用BD wash buffer按1:100&1:500&1:400稀释混匀；所述二抗工作液是由荧光标记的小鼠抗羊、荧光标记的大鼠抗羊和荧光标记的兔抗羊组成，分别采用BD wash buffer按1:500稀释混匀。
  3. 根据权利要求1所述的免疫荧光试剂盒，其特征在于，所述稀释液是由1mmol/L EDTA+0.1％BSA+0.1％海藻糖+0.2％聚氧乙烯聚氧丙烯醚嵌段共聚物组成，所述基础液为Tris-HCl缓冲剂。
  4. 根据权利要求1所述的免疫荧光试剂盒，其特征在于，所述脱色液是由95％酒精与100％二甲苯按容积比1:1组成。
  5. 根据权利要求1所述的免疫荧光试剂盒，其特征在于，所述染色液A为DAB染色液；所述染色液B为苏木素染色液1。
  6. 一种利用权利要求1-5任一项所述的免疫荧光试剂盒非诊断目的检测非小细胞肺癌患者外周血循环肿瘤细胞CEA基因突变的方法，其特征在于，包括以下步骤：
    (1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC：采集无法获取组织标本的晚期或复发非小细胞肺癌患者外周血：肘正中静脉外周血5ml；
    (2)外周血样预处理：将采集的外周血样采用稀释液进行10倍稀释，稀释后加多聚甲醛固定外周血样10分钟，固定终浓度为0.25％；
    (3)利用膜过滤分离肿瘤细胞装置过滤外周血样，分离获得外周血CTC：将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中，使其依靠重力自然过滤；
    (4)过滤结束后，从膜过滤分离肿瘤细胞装置中取下滤器，将循环肿瘤细胞染色液A液0.5ml加入到滤器中，染色3min，PBS缓冲液冲洗干净；滤液过滤完全后加入染色液B液1ml，染色2min，纯水1ml冲洗2次，向滤器中加入200μl 2％PFA，室温固定5min，完成后0.5ml PBS漂洗3次，每次2min；再向滤器中加入200μl预冷的甲醇，4℃固定15min；取下滤膜，将滤膜下表面平整的贴附到载玻片一侧，将载玻片置于室温干燥4-5min后，使用镊子快速将滤膜从载玻片表面一次性揭起，转移至载玻片中央，滴加2ul贴片剂，干燥后 在显微镜下观察，确定是否存在CTC；
    (5)运用免疫荧光法检测CTC的CEA表达情况。
  7. 根据权利要求6所述的方法，其特征在于，所述检测CTC的CEA表达的具体方法如下：
    (1)脱色：将带有CTC的滤膜从载玻片上取下，置于脱色液中浸泡4-6小时，脱去CTC染色液，PBS洗2min×3次；
    (2)封闭：向滤膜上滴加100μl 10％山羊血清，室温放置30min，完成后吸去多余的血清；
    (3)一抗孵育：弃掉载玻片上的封闭液，并立即滴加100μl一抗稀释液置于湿盒37℃孵育60min(或4℃过夜孵育，次日置于37℃复温30min)；
    (4)完成后PBS漂洗3次，每次3min；
    (5)二抗孵育：滴加100μl二抗稀释液，室温孵育30min；
    (6)完成后PBS漂洗3次，每次2min；
    (7)使用含DAPI的封片剂封片，阅片，采图；
    (8)采照完成后，脱片后进行瑞氏吉姆萨染色，与IF结果进行对比。
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