WO2021213298A1 - 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的免疫荧光试剂盒及检测方法 - Google Patents
一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的免疫荧光试剂盒及检测方法 Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Definitions
- the invention relates to an immunofluorescence kit and a detection method for detecting the expression of CA199 in peripheral blood circulating tumor cells of patients with pancreatic cancer, and belongs to the technical field of molecular biology.
- Pancreatic cancer is a common malignant tumor of the digestive system and one of the worst-prognostic malignancies. Among deaths caused by malignant tumors, pancreatic cancer ranks fourth. 90% of patients die within one year after diagnosis, with a 5-year survival rate. Less than 5%. Because the early symptoms of pancreatic cancer are insidious, not specific, aggressive, early metastasis, most of them are in the middle and late stages at the time of diagnosis, the surgical resection rate is low, only 15% at the time of diagnosis, so the selection of treatment methods for patients with pancreatic cancer is particularly important , Can make some patients avoid unnecessary surgical trauma and risks.
- CAl99 (carbohydrate antigenl99), also known as tumor-associated 199 antigen, gastrointestinal cancer-associated antigen or carbohydrate antigen, is a mucin-like tumor marker, which is a glycolipid on the cell membrane and exists in the form of mucin in the blood.
- CAl99 is an antigenic substance highly related to adenocarcinoma. It is produced by adenocarcinoma cells and drained into the blood through the thoracic duct.
- the normal reference value of serum CAl99 is 39kU/L.
- the concentration of CAl99 in serum of malignant tumors increases significantly, especially in patients with pancreatic cancer and colon cancer. It is used for the early diagnosis of pancreatic cancer.
- the specimens detected by CA199 in pancreatic cancer patients are mainly tumor tissues, which are derived from surgery or needle biopsy, which 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 an immunofluorescence kit and a detection method for detecting the expression of CA199 in circulating tumor cells in the peripheral blood of patients with pancreatic cancer.
- the membrane filtration device separates and obtains CTC in the peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue samples, and further uses immunofluorescence technology to detect the expression of CTC CA199.
- An immunofluorescence kit for detecting the expression of CA199 circulating tumor cells in the peripheral blood of patients with pancreatic cancer including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, 200 ⁇ l methanol, 200 ⁇ l 2% PFA, 100 ⁇ l 10% goat serum , 100 ⁇ l of primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-CA199, and a mixture of secondary antibodies composed of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit Suspension 100 ⁇ L, DAPI mounting tablets;
- mice anti-CK, rat anti-CD45 and rabbit anti-CA199 in the primary antibody suspension were diluted 1:100, 1:400 and 1:500 respectively, and the total volume was 100 ⁇ L;
- Fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit in the secondary antibody suspension were diluted 1:500.
- the diluent is potassium chloride 19g/L, dipotassium edetate 4.8g/L, sodium pyridinium 2.6g/L, potassium dihydrogen phosphate 17g/L, sodium hydroxide 9g/L, hydroxyethyl Piperazine ethanesulfonic acid 0.2g/L, dihydroxymethyl urea 2g/L, the balance is purified water.
- the decolorizing liquid is composed of 0.5% hydrochloric acid-70% ethanol solution.
- the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.
- the method for detecting the expression of CA199 in circulating tumor cells in the peripheral blood of patients with pancreatic cancer for non-diagnostic purposes of the above immunofluorescence kit includes the following steps:
- Peripheral blood sample pretreatment Dilute the collected peripheral blood sample with 45ml of diluent, add paraformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, and fix the final concentration to 0.25%;
- step (7) The specific method for detecting the CA199 expression of CTC in step (7) is as follows:
- the goat serum is diluted with PBS, 100 ⁇ l 10% goat serum is added dropwise to the filter membrane, placed at room temperature for 30 minutes, and the excess serum is aspirated after completion;
- S3 primary antibody incubation drop 100 ⁇ l of mouse anti-CK, rat anti-CD45 and rabbit anti-CA199 primary antibody suspension on the filter membrane, incubate at 37°C for 1h or 4°C overnight, and wash with PBS for 3min ⁇ 3 times after completion ;
- S4 secondary antibody incubation add 100 ⁇ l of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit secondary antibody suspension to the filter membrane, incubate at room temperature for 30 minutes, and wash with PBS after completion 2min ⁇ 3 times;
- S5 uses DAPI-containing mounting tablets to mount, read and take pictures
- the Wright Giemsa staining is performed after stripping, and the results are compared with the IF results.
- 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 the expression of CA199 in patients with advanced or recurrent pancreatic 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 the immunofluorescence staining image of circulating tumor cells in the peripheral blood of patients with advanced pancreatic cancer.
- Component content Diluent 45mL Decolorizing liquid 1mL Staining solution A 0.5mL Staining Solution B 1mL Methanol 200 ⁇ l 2% PFA 200 ⁇ l 10% goat serum (diluted in PBS) 100 ⁇ L Primary antibody suspension 100 ⁇ L Secondary antibody suspension 100 ⁇ L DAPI Mounting Tablets H-1200
- the primary antibody suspension is composed of mouse anti-CK, rat anti-CD45 and rabbit anti-CA199.
- Mouse anti-CK, rat anti-CD45 and rabbit anti-CA199 are respectively used with BD wash buffer at 1:100, 1:500 and 1 :Dilute with 400. After dilution, take 10 ⁇ L mouse anti-CK, 50 ⁇ L rat anti-CD45 and 40 ⁇ L rabbit anti-E-Cadherin to form the primary antibody suspension;
- the secondary antibody suspension is composed of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit. They are respectively commercially available Alexa Fluor 546 goat Anti-mouse, Alexa Fluor 488 goat Anti -rat and Alexa Fluor 647 goat Anti-rabbit, take the same amount of the above three fluorescently labeled secondary antibodies, dilute them with BD wash buffer at 1:500 and mix to obtain the secondary antibody suspension.
- the configuration of the diluent potassium chloride 19g, dipotassium edetate 4.8g, sodium pyridinium 2.6g, potassium dihydrogen phosphate 17g, sodium hydroxide 9g, hydroxyethylpiperazine ethanesulfonic acid 0.2g, Dihydroxymethyl urea 2g, purified water 945.4g.
- 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 prepared blood sample has poor stability, and some blood samples will also form stratification, and blood cells are prone to aggregation and adhesion, which affects the final The detection effect.
- the decolorizing solution prepared from a 0.5% hydrochloric acid-70% ethanol solution has a better decolorizing effect than a decolorizing solution composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
- Figure 4 is an immunofluorescence staining image of circulating tumor cells in the peripheral blood of patients with advanced pancreatic cancer. According to immunological and morphological findings, it is found that the tumor cells are large in size and have abnormal nucleus-to-cytoplasmic ratios. The immunological findings are typical CTCs.
- the detected circulating tumor cells were confirmed by immunofluorescence to confirm the expression of CA199 and compared with the results of CA199 expression in pancreatic cancer surgery or puncture tissue specimens to observe their differences, verifying that CTCs have great potential as a non-invasive biopsy for real-time evaluation of pancreatic cancer CA199 expression. Provide an important basis for evaluating the prognosis of pancreatic cancer chemotherapy combined with immunotherapy.
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Abstract
Description
组分 | 含量 |
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(PBS稀释) | 100μL |
一抗混悬液 | 100μL |
二抗混悬液 | 100μL |
DAPI封片剂 | H-1200 |
Claims (6)
- 一种检测胰腺癌患者外周血循环肿瘤细胞CA199表达的免疫荧光试剂盒,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,200μl甲醇、200μl 2%PFA,100μl10%山羊血清,小鼠抗CK、大鼠抗CD45和兔抗CA199组成的一抗混悬液100μl,荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液100μL,DAPI封片剂;一抗混悬液中小鼠抗CK、大鼠抗CD45和兔抗CA199分别按1:100、1:400和1:500稀释,总体积为100μL;二抗混悬液中荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔按1:500稀释。
- 根据权利要求1所述的免疫荧光试剂盒,其特征在于,所述稀释液为氯化钾19g/L,乙二胺四乙酸二钾4.8g/L,吡啶鎓钠2.6g/L,磷酸二氢钾17g/L,氢氧化钠9g/L,羟乙基哌嗪乙硫磺酸0.2g/L,二羟基甲基脲2g/L,余量为纯化水。
- 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0.5%盐酸-70%乙醇溶液组成。
- 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。
- 一种利用权利要求1-4任一项所述的免疫荧光试剂盒非诊断目的检测胰腺癌患者外周血循环肿瘤细胞CA199表达的方法,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胰腺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发胰腺癌患者外周血: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45ml进行稀释,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;(5)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;(6)再向滤器中加入200μl预冷的甲醇,4℃固定15min,取下滤膜,放置在载玻片上,干 燥后在显微镜下观察,确定是否存在CTC;(7)运用免疫荧光方法检测外周血CTC的CA199表达情况。
- 根据权利要求5所述的检测方法,其特征在于,步骤(7)所述检测CTC的CA199表达的具体方法如下:S1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;S2封闭:羊血清用PBS稀释,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;S3一抗孵育:向滤膜上滴加100μl小鼠抗CK、大鼠抗CD45和兔抗CA199组成的一抗混悬液,37℃孵育1h或4℃过夜,完成后PBS洗3min×3次;S4二抗孵育:向滤膜上滴加100μl荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液,室温孵育30min,完成后PBS洗2min×3次;S5使用含DAPI的封片剂封片,阅片,采图;S6采图完成后,脱片后进行瑞氏吉姆萨染色,与IF结果进行对比。
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