Quantification and immunoprofiling of bladder cancer cell-derived extracellular vesicles with microfluidic chemiluminescent ELISA

Abstract

The functional membrane proteins on tumor-cell-derived EVs contain a large amount of biomolecular information, and can serve as a comprehensive marker to delineate the molecular nature of cancer. However, due to low secretion rates, it is difficult to perform accurate quantification and biomolecular analysis with conventional EV analysis technologies such as the Western blots. Here, we introduce a multifunctional EV analysis technology based on an automated microfluidic chemiluminescent ELISA (Enzyme-Linked ImmunoSorbent Assay) platform. With this system, we were able to achieve rapid EV quantification (<1 h) with relatively small sample volume (~8 μL) and high sensitivity (optimal LOD = 8.7×107 EV/mL). In addition to the EV quantification, we evaluated the expression levels for a panel of four cancer-related EV membrane proteins (EGFR, HER2, MHC-I, and EpCAM) using a newly developed immunoprofiling assay that combines immunoprecipitation and sandwich ELISA. Due to high sensitivity, this immunoprofiling assay only requires a very small amount of input protein (<40 ng/marker). Our studies show that the expression level of functional EV membrane proteins is stable under external stimulation, which suggests that the expression profile of the EV membrane proteins may serve as a robust and unique “molecular fingerprint” for the immunophenotyping of cancer cell lines.