This application note was produced in collaboration with the Coleman Lab at Lawrence Livermore National Laboratory.
In this application note, we demonstrate how fluorescence correlation spectroscopy (FCS)
can be performed using the EI-FLEX to assess antibody-antigen binding affinities and generate Kd values for candidate molecules.
Nikfarjam et al. used the EI-FLEX to assess the binding of single-chain variable fragments (scFvs) and antibody fragments (Fabs) against the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. A homebuilt system was used to perform FCS on full-length antibodies.
Overview of this application note:
- FCS can determine Kd values for a range of binders, such as monoclonal antibodies, scFvs and Fabs
- High-throughput data acquisition and analysis permit rapid assessment of binding affinity for molecules produced by different methods, such as in cell-free systems
- Different labels, such as FLAG-tags, can impact antibody-antigen binding
Figure 1 – Calculation of Kd values for antibody-RBD binding from FCS correlation curves and diffusion time
Top) FCS correlation curves for various antibody concentrations, indicating slower diffusion time with increasing antibody concentration
Bottom left ) Diffusion time plotted against antibody concentration
Bottom right) Fitting of bound RBD protein against antibody concentration to derive a Kd value
Data was collected on a homebuilt system.
