Efficient Isolation of Biofluid-Derived Exosomes Using Ammonium Sulfate: A Promising Alternative to Commercial Kits for Clinical Diagnostics

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Abstract Background and Objective: Exosomes are nanoscale, lipid bilayer vesicles secreted by nearly all cell types, possessing diverse origins and abundant molecular cargo. This combination makes them exceptionally promising carriers for non-invasive diagnostic applications—particularly for neurodegenerative diseases like Alzheimer's disease (AD) and Parkinson's disease (PD). Given that the pathological lesions of these diseases are primarily concentrated in the central nervous system (CNS), achieving early and accurate diagnosis poses a significant challenge. However, existing exosome isolation methods—including ultracentrifugation, size exclusion chromatography, density gradient centrifugation, and commercial kits—face inherent limitations. These approaches are often cumbersome, time-consuming, involve high costs, and frequently yield low purity due to the difficulty in removing residual reagent components, which severely hampers downstream experimental processes. Such limitations hinder the application of exosomes in the clinical diagnosis of these diseases. Therefore, the present study aims to develop a novel, cost-effective, and efficient Ammonium sulfate (AS)-based exosome precipitation method and investigate its potential utility in the diagnosis of neurodegenerative diseases. Methods and Results: We systematically optimized the ammonium sulfate concentration for exosome precipitation, ultimately identifying 2.66 M as the optimal working concentration. This method demonstrated remarkable time efficiency, completing the entire isolation process in approximately 1.5 hours. Furthermore, it only requires centrifugation at 12000 g, 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 total exosomes from saliva. Comparative analyses against the commercially available ExoQuick kit demonstrated that our ammonium sulfate-based precipitation method achieved comparable efficacy in isolating total exosomes from the plasma of AD patients and in purifying astrocyte-derived exosomes. Furthermore, comprehensive validation using nanoparticle tracking analysis (NTA), electron microscopy, and detection of canonical exosomal markers (CD63, CD9, CD171) confirmed the functional equivalence of the two isolation methods. Conclusion: The cumulative evidence from this study firmly establishes the ammonium sulfate-based exosome isolation protocol as a viable and robust alternative 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 exosomes from plasma, a conventional biofluid widely utilized in clinical diagnostics, as well as from saliva, a non-invasive and easily accessible sample matrix. 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 exosome-based diagnostic research and clinical practice.

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