Ultrasensitive ELISA detection of proteins in separated lumen and membrane fractions of cancer cell exosomes

Abstract

Exosomes transfer molecules horizontally to surrounding cells and therefore have a key role in cancer progression. To clarify the role of exosomes in cancer progression, trace amounts of proteins in their lumen and membrane fractions should be analyzed separately. For this purpose, an adequate and easy-to-use method of separating the lumen and membrane fractions of exosomes must be developed. Further, because exosomes contain only trace amounts of proteins, an ultrasensitive protein detection method is necessary. To develop an adequate and easy-to-use lumen and membrane fraction separation method, we applied a commercially available kit originally developed for cells to exosomes and examined the validity of the results compared with those obtained using a conventional, complicated Na₂CO₃ method. To develop an ultrasensitive protein detection method, we designated GRP78, which is upregulated in cancer cells and contributes to cancer progression, as the target protein and detected it at the subattomolar level using an ultrasensitive ELISA combined with thio-NAD cycling. By applying these methods together, GRP78 was successfully quantified in both the lumen and membrane fractions of exosomes obtained from cultured cancer cells. The present results will facilitate studies to broaden our understanding of the tumor microenvironment.

Keywords: Exosome; GRP78; Lumen-membrane separation; Thio-NAD cycling; Ultrasensitive ELISA.