Ammonium sulfate provides an efficient method for isolating small extracellular vesicles from human biofluids

Small extracellular vesicles (EVs) are nanosized vesicles (< 200 nm) secreted from various tissues, including the central nervous system (CNS), into diverse biofluids. Due to their ability to carry molecular cargo that reflects the physiological state of their parental cells, small EVs represent promising diagnostic carriers for neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). While biofluid-derived small EVs offer a "liquid biopsy" solution, their clinical translation is severely hindered by the limitations of conventional isolation methods, which are often time-consuming, costly, or yield low purity. Building upon the "ExoPRISM" (Exosome Precipitation by Ionic Strength Modulation) framework, we developed and validated an optimized, cost-effective ammonium sulfate (AS)-based pipeline for small EVs isolation. We have refined and established the optimal AS ratio specifically for plasma and further demonstrated its feasibility for isolating small EVs from saliva. Subsequently, we introduced a targeted purification step using glutamate aspartate transporter (GLAST) antibodies to specifically isolate central nervous system (CNS)-derived EVs. This step robustly demonstrated that our AS-based approach preserves small EVs integrity and enables the effective isolation of Astrocyte-Derived Extracellular Vesicles (ADEVs) for downstream applications. Finally, the practical utility of this optimized protocol was validated in a clinical cohort. Our findings highlight the robustness, high efficiency, and significant translational potential of this AS-based method for the early and accurate diagnosis of neurodegenerative diseases. We optimized the AS concentration for small EVs precipitation, ultimately identifying 2.66 M as the optimal working concentration. This method demonstrated remarkable time efficiency, completing the entire isolation process in approximately 90 min. Furthermore, it only requires centrifugation at 12,000 xg, significantly enhancing its practicality in clinical diagnostic scenarios. To expand its potential application scope, we also tested the method's efficacy on other bodily fluids and found that the concentration of 2.66 M was equally effective in isolating small EVs from saliva. Comparative analyzes against the commercially available ExoQuick kit demonstrated that our AS-based precipitation method achieved comparable efficacy in isolating small EVs from the plasma of AD patients and in purifying ADEVs. Furthermore, comprehensive validation using nanoparticle tracking analysis (NTA), electron microscopy, and detection of canonical small EVs markers (CD63, CD9) confirmed the functional equivalence of the two isolation methods. The cumulative evidence from this study firmly establishes the AS-based small EVs isolation protocol as a viable and robust efficacy comparable to commercially available kits. This method confers three distinct advantages: enhanced temporal efficiency, reduced cost burden, and minimal interference with downstream assays, rendering it highly amenable to clinical implementation. Notably, it enables the effective isolation of small EVs from plasma, a conventional biofluid widely utilized in clinical diagnostics, as well as from saliva. These attributes underscore the method's potential in advancing the early detection of neurodegenerative disorders. Looking ahead, the versatility of this approach suggests its applicability across a diverse array of biological specimens, thereby facilitating the expansion of small EVs-based diagnostic research and clinical practice.