基于磁分选技术和间接免疫荧光技术的肺癌A549细胞的分离检测

摘要

采用磁分选技术及间接免疫荧光技术分离检测肺癌循环肿瘤细胞(CTCs).将表皮生长因子受体(EGFR)抗体连接到四氧化三铁(Fe3O4)磁珠表面构建免疫磁珠(MNPs-Anti-EG-FR),通过EGFR抗体和肺癌A549细胞的表面抗原的特异性结合,将免疫磁珠与肺癌细胞结合.通过磁场作用,使肺癌细胞在磁场的作用下从混合细胞中分离富集.分离出的细胞利用间接荧光技术在其细胞表面连接结合PE荧光团的特异性抗体,并用4',6-二脒基-2-苯基吲哚(DAPI)对细胞核染色以定位细胞位置.通过倒置荧光显微镜的观察,从而实现对肿瘤细胞的定量计数检测.实验结果显示该方法对肺癌A549细胞具有较好的检测灵敏度和检测下限,检测下限可达3 cells/mL.%This article reports the development of a combining immunomagnetic separation technology with indirect immunofluorescence technology for separating and determining of the carcinoma A549 cells.Magnetic beads weresynthesized by chemical precipitation.Immunomagneticbeads were constructed by grafted EGFR to the surface of Fe3O4 nanoparticles coated by lipid.The carcinoma A549 cells could specifically bind with the EGFR antibody on the immunomagnetic's surface.The carcinoma A549 cells could be isolatedand enriched from the mixed cellsunder the action of in the magnetic field.The separated cells could connect with the specific antibody contained PE fluorophore and the location of cellnucleus can be confirmed by DAPI to stain the cell nucleus.The carcinoma A549 cells could be quantitatively detectedbyobserving inversion fluorescence microscope.All those results indicate that the method of combining Immunomagnetic separation technology and indirect immunofluorescence technology have a good specificity and high sensitivity for isolationand detection of the carcinoma A549 cells,thedetection limit is three cells per milliliter.