Molecular and Cell Biology Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Immunogenetics Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
Biotechnol Lett. 2019 May;41(4-5):523-531. doi: 10.1007/s10529-019-02667-5. Epub 2019 Apr 8.
Exosomes are nano-scaled carriers of miRNA, mRNA and proteins which are secreted from viable cells. Exosome detection within serum, saliva and semen offers diagnostic value for detection of various diseases including cancer. In the present study, we have lunched an in vitro study to develop a more efficient method for exosome detection. In this regard, the recombinant LAMP-DARPins (capable of Her2 targeting) gene was packed within the lentiviral particles and stably transferred into the HEK293T cells. The morphology and sizes of the obtained exosomes were characterized by TEM and zeta sizer. The expression of LAMP-DARPins antigen on the exosome surface was verified by western blotting. Ultimately, the efficiency of cell surface ELISA in suspension method was examined for exosome detection.
The exosome particles were successfully harvested and purified from transfected cells. The sizes of exosome particles were determined to be 90 nm using zeta sizer instrument. The TEM scan showed that the exosomes are cap like shaped and their sizes range between 40 and 150 nm. An observed 120 kDa band on western blotting paper indicated the LAMP-DARPins antigen expression on exosome surfaces. The results of cell surface ELISA in suspension method were superior to the results of conventional cell ELISA.
It could be concluded that the cell surface ELISA in suspension method could be an amenable method to detect exosome particles within the biological samples. Moreover, the method could be modified to evaluate the ability of exosomes to interact with target cells in both diagnostic and therapeutic applications.
外泌体是 miRNA、mRNA 和蛋白质的纳米级载体，由活细胞分泌。血清、唾液和精液中外泌体的检测为包括癌症在内的各种疾病的诊断提供了有价值的信息。在本研究中，我们进行了一项体外研究，以开发一种更有效的外泌体检测方法。为此，将能够靶向 Her2 的重组 LAMP-DARPins（富含亮氨酸的重复蛋白）基因包装在慢病毒颗粒中，并稳定转染到 HEK293T 细胞中。通过 TEM 和纳米粒度仪对获得的外泌体的形态和大小进行了表征。通过 Western blot 验证了外泌体表面 LAMP-DARPins 抗原的表达。最终，通过悬浮法细胞表面 ELISA 检测评估了外泌体的检测效率。
成功地从转染细胞中收获和纯化了外泌体颗粒。通过纳米粒度仪确定外泌体颗粒的大小为 90nm。TEM 扫描显示，外泌体呈帽状，大小在 40 到 150nm 之间。Western blot 纸上观察到的 120kDa 条带表明 LAMP-DARPins 抗原在外泌体表面的表达。悬浮法细胞表面 ELISA 的结果优于常规细胞 ELISA。
可以得出结论，悬浮法细胞表面 ELISA 可能是一种可行的方法，可以检测生物样本中的外泌体颗粒。此外，该方法可以进一步修改，以评估外泌体与靶细胞相互作用的能力，用于诊断和治疗应用。
