Abstract
BackgroundSerum exosomal proteins have great potential as indicators of disease status in cancer, inflammatory or metabolic diseases. The association of a fraction of various serum proteins such as carcinoembryonic antigen (CEA) with circulating exosomes has been debated. The establishment of a method to measure the exosomal fraction of such proteins might help resolve this controversy. The use of enzyme-linked immunosorbent assays (ELISAs) to measure serum exosomal molecules, for example CEA, is rare in research laboratories and totally absent in clinical biology. In this study, we optimized a method for assessment of serum exosomal molecules combining a treatment by volume-excluding polymers to isolate the exosomes, their subsequent solubilization in an assay buffer and ELISA.MethodsOne hundred sixteen consecutive patients with colorectal cancer were enrolled for this study between June 2015 and June 2016 at Wakayama Medical University Hospital (WMUH). Whole blood samples were collected from patients during surgery. Exosomes were isolated using the ExoQuick reagent, solubilized in an assay buffer and subjected to CEA detection by ELISA. The procedure of serum exosome isolation and the formulation of the assay buffer used for the ELISA were optimized in order to improve the sensitivity and specificity of the assay.ResultsA five-fold increase in the concentration of the exosomes in the assay buffer (using initial serum volume as a reference) and the addition of bovine serum albumin (BSA) resulted in more accurate measurements of the serum exosomal CEA. The thawing temperature of frozen serum samples before exosome extraction was also optimized. A validation study that included one hundred sixteen patients with colorectal cancer demonstrated that serum exosomal CEA from samples thawed at 25°C exhibited a better AUC value, sensitivity, and specificity as well as a more correct classification than serum CEA.ConclusionsWe optimized an easy and rapid detection method for assessment of serum exosomal CEA. The thawing temperature of frozen serum prior to exosome extraction, the formulation of the assay buffer used for exosome solubilization and the concentration of the exosomes in this buffer were fine-tuned to enable the appropriate and accurate measurement of serum exosomal CEA.
Highlights
An exosome is a small vesicle, measuring 40–150 nm in size [1], and is characterized based on its origin [2]
The thawing temperature of frozen serum prior to exosome extraction, the formulation of the assay buffer used for exosome solubilization and the concentration of the exosomes in this buffer were fine-tuned to enable the appropriate and accurate measurement of serum exosomal carcinoembryonic antigen (CEA)
We optimized a method to measure serum exosomal CEA by utilizing volume-excluding polymers and enzyme-linked immunosorbent assays (ELISAs), and we examined whether serum exosomal CEA can better predict the existence of distant metastasis than serum CEA
Summary
An exosome is a small vesicle, measuring 40–150 nm in size [1], and is characterized based on its origin [2]. The use of enzyme-linked immunosorbent assays (ELISAs) to measure serum exosomal molecules is neither common nor clinically available. To address these issues, we investigated the feasibility of measuring serum molecules by ELISA and volume-excluding polymers for exosome isolation. Serum exosomal proteins have great potential as indicators of disease status in cancer, inflammatory or metabolic diseases. The use of enzyme-linked immunosorbent assays (ELISAs) to measure serum exosomal molecules, for example CEA, is rare in research laboratories and totally absent in clinical biology. We optimized a method for assessment of serum exosomal molecules combining a treatment by volume-excluding polymers to isolate the exosomes, their subsequent solubilization in an assay buffer and ELISA
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