Quantification of CD81 in mouse serum-derived extracellular vesicles via inductively coupled plasma - mass...

Extracellular vesicles (EVs) released by cells are one of the main mechanisms of intercellular communication. Nowadays it is known that they can play important roles in diseases such as cancer or neurodegenerative processes. Therefore, it is of high interest to develop proper procedures for analyzing EVs in order to exploit their potential to understand biological dysregulations and identifying biomarkers, such as proteins. However, the determination of specific proteins in EVs typically require highly sensitive methods, particularly when working with limited sample volumes, such as mouse serum. The combined use of metal labelled immunoprobes and detection by inductively coupled plasma - mass spectrometry (ICP-MS) has proved to be an advantageous strategy for the analysis of proteins in biological samples. In this work, a methodology based on an immunoassay using antibodies labelled with gold nanoclusters, to achieve a high signal amplification, and ICP-MS detection was developed to determine target proteins in EVs from mouse blood serum. As a proof of concept, CD81, a generic marker of EVs, was investigated, and a limit of detection as low as 56 fM was obtained. CD81 levels in EVs isolated from aliquots of 0.5 mL of commercial mouse blood serum were determined after applying an optimized differential centrifugation protocol. Results obtained with the developed analytical procedure were in good agreement with those achieved with a commercial ELISA kit.