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Raman Spectroscopy Characterization of Antibody-Ligand Association at Supported Phospholipid Bilayers

Year: 2023

Presenter Name: Clista Galecki

There are fundamental questions about the chemistry of antibody-ligand interactions, Antibodies are proteins generated by the immune response to target pathogens through their specific binding to proteins or other ligands on cell membranes. One of the fundamental questions that is being addressed in this research is to determine how the accessibility of ligands at supported-lipid bilayers (models of cell membranes) varies with ligand coverage, thereby impacting their association with antibodies in solution. We detect the binding of proteins by measuring inelastic light scattering from their characteristic molecular vibrations using Raman microscopy. The use of Raman spectroscopy allows label-free and quantitative analysis of antibody-to-ligand binding. We are able to determine the concentration of antibody that has accumulated on the surfaces of supported-lipid bilayers. Unlike current methodologies, Raman spectroscopy can detect changes in the binding state of the targeted ligand. Understanding the binding state is crucial in assessing how ligand accessibility may influence its interaction with solution-phase proteins. We prepare lipid bilayers on the interior surfaces of porous silica particles, whose high surface area provides a sufficiently high concentration of both ligand and its antibody to allow detection of quite modest mol-fractions (2-mol%) of ligand-modified (2,4-dinitrophenylated) lipid. Capture of antibodies requires the presence of ligand in the bilayer, where lipid bilayers prepared without dinitrophenylated-lipid produce no detectable signal from the antibody. By varying the dinitrophenylated-lipid density, the surface concentration of captured antibody increases proportionally to a level that is limited by antibody size and packing density of antibody at the lipid-bilayer surface. After establishing specific protein accumulation and detectable changes in ligand state following binding, we plan to determine how the coverage of antibody impacts the binding state of the recognition ligands at supported lipid-bilayer interfaces and determine the stoichiometry of ligand-to-antibody binding.
University / Institution: University of Utah
Type: Poster
Format: In Person
Presentation #D66
SESSION D (3:30-5:00PM)
Area of Research: Science & Technology
Faculty Mentor: Joel Harris