The goal of this research is to develop a quantitative label-free method to assess the association of antibodies with haptens immobilized at supported-lipid bilayers. Methodologies currently used to characterize antibody-ligand complexation are unable to report changes in the conformation of the ligand following the association reaction. This information is crucial in assessing how hapten accessibility may change with the surface coverage of bound antibody at the lipid bilayer surface. Here we employ confocal Raman microscopy to determine Immunoglobulin G (IgG) association at supported-lipid bilayers prepared with a ligand-modified (dinitrophenylated) lipid and formed on porous silica surfaces. The high specific-surface area of the wide-pore silica overcomes the poor sensitivity of Raman scattering measurements. Characterizing the composition of the lipid bilayers within individual porous silica particles with confocal-Raman microscopy, we establish our ability to detect the presence of the ligand in bilayers prepared with modest mol-fractions (2 mol%) of dinitrophenylated lipid. Demonstrating the platform's specificity, we test 1µM IgG against bilayers prepared with and without dinitrophenylated lipid and detect only IgG binding at bilayers prepared with the recognition-ligand. By varying the dinitrophenylated lipid density while maintaining the same IgG concentration, it was determined that there is a linear dependence of antibody binding to the bilayer surface as a function of ligand density to the point when the surface is saturated with antibody. Equilibrating the IgG-bound samples with 100μM 2,4-dinitophenol resulted in complete competitive dissociation of surface-bound IgG. The 1527 cm-1 NO2 stretch of the hapten exhibits a reduction of the band intensity accompanied with a slight red-shift following its association with IgG; recovery of the initial band features was observed following IgG surface-dissociation. Direct detection of changes in hapten conformation following IgG association at an interface marks a major innovation in the characterization of interfacial biorecognition reactions. Having established our ability to detect specific protein binding accompanied with detectable changes in ligand state following association, we plan to calibrate the response of the hapten to directly study how hapten accessibility changes as a function of IgG density.

Confocal-Raman microscopy investigations of antibody association with ligands immobilized at supported-lipid-bilayer interfaces

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.