OBJECTIVES/GOALS: This study aims to develop a method to examine whether blood-borne CNS-EV metal cargoes can serve as reliable biomarkers of CNS metal load and reveal a link between metal load and ALS development (i.e., neurodegenerative disease development). METHODS/STUDY POPULATION: CNS-EVs were isolated from human blood and plasma samples via direct immunoprecipitation using biotinylated antibodies for proteins known to be expressed in neurons (Contactin-2, i.e.) and astrocytes (glial-glutamate-aspartate-transporter, i.e., GLAST). Once isolated, protein concentrations in the EV samples were analyzed via ELISA assay, EV abundance was measured using ViewSizer Nanoparticle Tracking analysis, and EVs were visualized via Transmission Electron Microscopy. EVs were then analyzed for metal contents using a Perkin-Elmer NexION 350S via an ICP-MS/MS dynamic reaction cell method. RESULTS/ANTICIPATED RESULTS: Preliminary results demonstrate that it is feasible to quantify the metal contents of these CNS-derived EVs, particularly in terms of toxic metals known to be associated with neurodegenerative disorders, including copper, zinc, lead, aluminum, manganese, and iron. DISCUSSION/SIGNIFICANCE: CNS-derived EVs isolated from peripheral blood draws show promise as a potential biomarker of real-time metal load in the brain and spinal cord, with promising applications in predicting future development of neurodegenerative disorders (i.e., ALS) among patients with relevant elevated CNS metal loads.