Saliva and Saliva Extracellular Vesicles for Biomarker Candidate Identification—Assay Development and Pilot Study in Amyotrophic Lateral Sclerosis

Abstract

Saliva is gaining increasing attention as a source of biomarkers due to non-invasive and undemanding collection access. Extracellular vesicles (EVs) are nano-sized, cell-released particles that contain molecular information about their parent cells. In this study, we developed methods for saliva biomarker candidate identification using EV-isolation and proteomic evaluation. We used pooled saliva samples for assay development. EVs were isolated using membrane affinity-based methods followed by their characterization using nanoparticle tracking analysis and transmission electron microscopy. Subsequently, both saliva and saliva-EVs were successfully analyzed using proximity extension assay and label-free quantitative proteomics. Saliva-EVs had a higher purity than plasma-EVs, based on the expression of EV-proteins and albumin. The developed methods could be used for the analysis of individual saliva samples from amyotrophic lateral sclerosis (ALS) patients and controls (n = 10 each). The starting volume ranged from 2.1 to 4.9 mL and the amount of total isolated EV-proteins ranged from 5.1 to 42.6 µg. Although no proteins were significantly differentially expressed between the two groups, there was a trend for a downregulation of ZNF428 in ALS-saliva-EVs and an upregulation of IGLL1 in ALS saliva. In conclusion, we have developed a robust workflow for saliva and saliva-EV analysis and demonstrated its technical feasibility for biomarker discovery.

Quantitative Detection of Biological Nanovesicles in Drops of Saliva Using Microcantilevers

“…While the natural concentration of native EVs in saliva is not known, various studies estimate a range 1 or 2 orders of magnitude greater than the present sensitivity achieved. 49,50 Interestingly, a rapid uptake is visible over the first 5−10 min, followed by a slower uptake also present in the control experiment (pure saliva). This suggests that a quantitative readout is possible in less than 30 min despite the small sample volume and the absence of any sample preparation or conditioning of the sample.…”

Salivary Extracellular Vesicles: Biomarkers and Beyond in Human Diseases

“…Sjoqvist et al established a workflow for the analysis of saliva and sEVs to compare protein profiles between ALS patients and healthy individuals. The study revealed a downregulation trend in ZNF428 (zinc finger protein 428) in sEVs from ALS patients compared with the control group [144].…”

Characterization of Extracellular Vesicles from Human Saliva: Effects of Age and Isolation Techniques

“…Salivary EVs have immense potential as biomarkers of non-communicable chronic diseases [ 26 , 27 , 28 ], due to their non-invasive and relatively simple collection methods and to the ability of EVs to cross epithelial barriers [ 29 , 30 , 31 ] and reach the salivary glands via peripheral blood [ 32 ]. However, the lack of studies assessing the efficiency and purity of different EV isolation techniques hinders the use of salivary EVs in clinical settings.…”