This application note was produced in collaboration with the Coleman Lab at Lawrence Livermore National Laboratory.
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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.
can be performed using the EI-FLEX to assess antibody-antigen binding affinities and generate Kd values for candidate molecules.
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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.
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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.
