Multiplex detection and characterization of breast cancer exosomes by magneto-actuated immunoassay

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 10⁵ exosomes μL^-1 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.