Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain.
Laboratori d'Immunologia Cel·lular, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain.
Talanta. 2020 May 1;211:120657. doi: 10.1016/j.talanta.2019.120657. Epub 2019 Dec 24.
The exosomes are emerging as biomarkers for the detection of cancer in early stages, as well as for the follow-up of the patients under treatment. This paper describes the characterization of exosomes derived from three different breast cancer cell lines (MCF7, MDA-MB-231 and SKBR3), and the quantification based on a magneto-actuated immunoassay. The exosomes are separated and preconcentrated on magnetic particles by immunomagnetic separation and labelled with a second antibody conjugated with an enzyme for the optical readout performed with a standard microplate reader. Several molecular biomarkers, including the general tetraspanin CD9, CD63 and CD81, and the receptors related with cancer (CD24, CD44, CD54, CD326 and CD340) were studied either for the immunomagnetic separation or the labelling, in different formats. After a rational selection of the biomarkers, this immunoassay is able to detect 10 exosomes μL directly in human serum without any treatment, such as ultracentrifugation. The interference from free receptors in the samples could easily be prevented by performing the immunomagnetic separation with antiCD81 modified magnetic particles and the labeling based on either CD24 or CD340. Furthermore, the differentiation of healthy donors and breast cancer individuals was also demonstrated. This approach is a highly suitable alternative method for flow cytometry, providing a sensitive method for the multiplex detection but using instrumentation widely available in resource-constrained laboratories and requiring low-maintenance, as is the case of a microplate reader operated by filters.
外泌体正成为早期癌症检测以及治疗后患者随访的生物标志物。本文描述了从三种不同乳腺癌细胞系（MCF7、MDA-MB-231 和 SKBR3）中提取的外泌体的特征，并基于磁激活免疫测定进行了定量。通过免疫磁分离将外泌体分离并预浓缩在磁性颗粒上，并使用与酶结合的第二抗体进行标记，用于使用标准微孔板读取器进行光学读取。几种分子标志物，包括一般四跨膜蛋白 CD9、CD63 和 CD81，以及与癌症相关的受体（CD24、CD44、CD54、CD326 和 CD340），在不同的格式下，无论是用于免疫磁分离还是标记，都进行了研究。在对生物标志物进行合理选择后，该免疫测定法能够直接在未经任何处理（如超速离心）的人血清中检测到 10 个外泌体 μL。通过使用抗 CD81 修饰的磁性颗粒进行免疫磁分离，并基于 CD24 或 CD340 进行标记，可以轻松防止样品中游离受体的干扰。此外，还证明了健康供体和乳腺癌个体的区分。这种方法是流式细胞术的一种高度合适的替代方法，提供了一种用于多重检测的敏感方法，但使用的仪器在资源有限的实验室中广泛可用，并且需要低维护，如使用过滤器操作的微孔板读取器。
