Hetero SandwichImmunoassay as Tool to Probe the Compositionof the Extracellular Vesicles Membranes: The Case Study of L1CAM Localization

Lateral flow immunoassays (LFIAs) are widely used for point-of-care diagnostic devices due to their simplicity, low cost, and rapid results. In this work, we demonstrate that a heterosandwich design LFIA can be an effective tool for verifying the presence of different proteins on the same particles. As a case study, we address a recent controversy regarding the presence of the protein L1CAM on the extracellular vesicles (EVs). EVs are crucial for cell communication and may serve as valuable disease biomarkers, including for neurodegenerative disorders. EVs from neuronal cells can cross the blood-brain barrier and be selectively isolated from plasma. Although L1CAM has been suggested as a marker for neuron-derived EVs, recent studies report that L1CAM exists as a cleaved soluble protein in plasma, not associated with EVs. We propose a heterosandwich LFIA to detect and quantify L1CAM and a confirmed EV marker, tetraspanin CD63 or CD9, on the same EV. This assay, together with several control experiments on EVs isolated from plasma by size exclusion chromatography (SEC), demonstrates that although most L1CAM in plasma is present as soluble cleaved proteins, 13% of the EVs are strongly associated with this protein. This evidence is confirmed by dynamic light scattering measurements, showing a significant size increase of gold nanoparticles conjugated with L1CAM antibodies when exposed to EVs but not to cleaved soluble L1CAM. Our results validate the selective immune-isolation of L1CAM-EVs, resolving the controversy by confirming that L1CAM is indeed associated with a significant fraction of EVs despite the presence of its soluble form in plasma.