A method to quantify ligand-neuron binding in vivo.

BACKGROUND: Neurotrophic factor signaling depends on effective engagement with neuronal cell-surface receptors, yet conventional ligand-neuron binding assays require enzymatic or mechanical neuron isolation that can disrupt native receptor landscapes. This limitation reduces the physiological relevance of binding measurements. NEW METHOD: We developed a vitreous-based in vivo ligand binding assay that quantifies interactions between vascular endothelial growth factor (VEGF) and retinal ganglion cells (RGCs) by leveraging the intact RGC layer directly exposed to the vitreous. Aflibercept, a VEGF-neutralizing agent, was used to define receptor-mediated binding. RESULTS: Following intravitreal administration, a clonal T7 phage displaying VEGF (VEGF-Phage) exhibited 11.6-, 1,493-, 4,790-, and 51-fold higher binding than background at 1, 4, 18, and 48hours, respectively. Background signals were minimal, indicating limited VEGF-Phage penetration into deeper retinal layers, consistent with immunohistochemical analyses. COMPARISON WITH EXISTING METHODS: This in vivo approach detected up to 4,790-fold VEGF-specific binding, far exceeding the ~1.4-fold enhancement achievable using ex vivo assays that require neuronal isolation. CONCLUSIONS: The unique organization of the RGC layer enables a robust, non-disruptive in vivo ligand-neuron binding assay that preserves native cell-surface receptors. This platform is compatible with ligandomics and can be applied to map disease-selective neuronal ligands, providing a foundation for developing targeted neuroprotective therapies for retinal disorders.