An exosome species containing CD63 as a marker of melanoma was isolated from bulk exosome population and used as a sample for detecting malignant melanoma. A calcium binding protein (CBP) was produced and then used to raise monoclonal antibody. The antibody was sensitive to a conformational change of CBP caused by Ca binding. Immuno-magnetic beads were prepared by immobilizing the conformation-sensitive binder and subsequent binding of CBP conjugated with the capture antibody specific to CD63. These immuno-beads were used to isolate CD63-positive exosome from a bulk exosome sample (normal or melanoma) based on the 'calcium switch-on/off' mechanism through magnetic separation. After recovery, the subpopulation sample was analyzed by immunoassays for cavelion1 (Cav1), CD81, and CD9 as sub-subpopulation markers. Normalized signals of Cav1 and/or CD81 over CD9 were higher in melanoma samples than in normal samples, depending on clinical stages (I, II, and IV) of patients. This was in contrast to assay results for the bulk exosome population that showed a completely mixed state of melanoma and normal samples. These results showed that an exosome subpopulation sample prepared using a 'Ca-dependent switch' technology might be useful for diagnosing malignant melanoma at an early stage to increase 5-year survival rates.
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