NOX2-Targeted Immunocapture Enables High-Specificity Lysosome Isolation from Low Cell Numbers

Abstract

Research on lysosomal composition and function is critical for understanding cellular homeostasis and pathogenesis of diverse diseases, including cancer. However, current isolation methods often require millions of input cells, limiting their application to rare cell populations or clinical samples. Here, we present Lysolation, a high-specificity antibody-based isolation method optimized for low cell numbers. By combining digitonin-mediated selective plasma membrane permeabilization with immunocapture targeting the lysosomal membrane protein NOX2, we developed both magnetic bead- and plate-based isolation formats. We first identified clone 7D5 as the superior antibody for capturing native lysosomal NOX2 across multiple cell types. In the bead-based workflow, we demonstrated near-exhaustive recovery of lysosomes (>99.5%) even from targets with low epitope density. To further downscale input requirements, we implemented a plate-based system combined with papain-mediated proteolytic release, achieving an isolation efficiency of 60.1% from as few as 15,000 cells. Overall, Lysolation provides a robust, tag-free platform for the isolation of intact, functional lysosomes from limited clinical samples and rare material, bridging the gap between standard biochemical assays and low-input sample availability.