Fluorescence correlation or cross-correlation spectroscopy (FCS or FCCS), a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. However, the inherent drawback of FCS is that species with similar molecular weight could not be differentiated. Although FCCS could resolve this through cross-correlation, it suffers from nonideal confocal volume overlap and spectral cross-talk which limits its application. In this work, we demonstrate for the first time the applicability of fluorescence lifetime correlation spectroscopy (FLCS) to monitor the interaction of an antagonist antibody with the epidermal growth factor receptor (EGFR) in live cells. As a proof of concept, we demonstrate the interaction of Cy5 labeled IgG and Alexa633 labeled anti-IgG using a single laser source (636 nm excitation) in vitro. The autocorrelation functions were separated based on their respective lifetime with a single detector and their K d value was determined to be 11 ± 3 nM. An in vivo application constituting the interaction of EGFR neutralizing antibody labeled with Alexa488 and EGFR-GFP in live HEK293 cells was successfully demonstrated. The binding specificity of EGFR neutralizing antibody was confirmed by fluorescence lifetime cross-correlation measurements and fluorescence lifetime imaging (FLIM). The dissociation constant of this complex was found to be 9.2 ± 2.7 nM. A quantitative assessment of receptor density calculations show that the density of EGFR significantly decreased, from 540 ± 64 receptors/μm 2 to 38 ± 7 receptors/μm 2 upon addition of the neutralizing EGFR antibody, indicating that the antagonist could induce receptor internalization. The demonstrated work not only opens up new opportunities in studying protein-protein interactions in solutions and in live cells but also provide new insights in biology to understand how the antagonists influence EGFR through live cell quantification and imaging.
ASJC Scopus subject areas
- Analytical Chemistry