White paper 6 | Version 2 | Kris Ver Donck, Dagmara Minczakiewicz, Filip Delport, Kim Stevens
Based on original publication: Knez et al. (2013) Analytical Chemistry 85, 10075 – 10082
ABSTRACT
Phage display in conjunction with biopanning is a frequently used strategy in the selection process for nanobodies and other expressed binding proteins with specificity to a target antigen. Phage are engineered to display a peptide of interest and be captured by conjugation to the desired target immobilized on a surface. Non specifically bound phage are washed off and the bound phage are eluted to obtain the desired high affinity clones which can then be used to create further display libraries.
Here we present an FO-SPR based approach that combines both fast kinetic characterization of phage binding and an efficient phage selection cycle in a single step. This technique presents a potential for phage selection based on the binding of molecules and interaction characterization, as it allows for real-time kinetics monitoring rather than the end point data from methods such as ELISA. Even avidity versus affinity binding can be assessed based on k on and k off behavior. Furthermore, it also avoids the risk of readout bias from affecting the binding of the secondary generic phage sandwich antibody needed in ELISA procedures.
The FO-SPR probes of the White FOx are ideally suited for this procedure. The user friendly dip in, fluidics free setup offers a simple protocol for the capture, washing, and elution steps, while the binding affinity, association, and dissociation kinetics of the phage can be measured in real time.