CamPSF workflow: screening binders to produce best variant
Modular approach taken to enable multiple degree of flexibility tailored to the users requirements
Hit identification alternative 1: Screening monoclonal hits by ELISA/DELFIA
ELISA or alternatively DELFIA for increased sensitivity (added costs) can be performed on the selected antibody-expressing phage to assess relative binding.
Clones identified for tight binding will be sent for Sanger sequencing to identify the sequence of the binder - optional provision of binders to user
Hit identification alternative 2: Next generation sequencing (NGS) of output cells from selection rounds
Proteins encoded by the phagemid plasmids are sequenced and sequences and are clustered to identify the best binders: the most represented sequences are the specific binder sequences.
NGS also allows for selection quality control by checking sequence enrichment throughout the selection rounds.
NGS provides a wealth of sequencing data of the clones being selected that empowers advanced sequence analysis:
(i) Selecting CDR types where AI based approaches (e.g., Alphafold2) is good at predicting binding mode to antigen
(ii) Prioritising binders with superior biophysical attributes (solubility, stability, polyspecifcity, polyreactivity, developability) predicted by computational tools
There is optional provision of binders to user at this stage
SpyBLI kinetic secondary screening of hits identified by ELISA/DELFIA or NGS
The kinetics of binding are as important as the affinity as the off-rate (koff) defines the residence time bound to the receptor. Biolayer interferometry (BLI) enables the measurement of binding kinetics (kon, koff, and Kd). However, the cloning, expression and purification of individual variants is time-consuming and is a bottleneck to measuring large numbers of variants. To overcome this CamPSF and members of the Sormanni lab developed inhouse the SpyBLI approach.
SpyCatcher003 reacts at close to the diffusion limit with with SpyTag003 to produce irreversible covalent coupling. https://doi.org/10.1073/pnas.1909653116
SpyCatcher-SpyTag react across a wide-range of conditions and rapidly even at very low concentrations (e.g., 10 nM). SpyBLI allows SpyTagged proteins to be able to be be purified out of crude samples (e.g., cell free synthesis reactions or mammalian cell supernatants) by reaction with the site-specifically biotinylated SpyCatcher that has been directionally pre-immobilised on a Streptavidin BLI pin. SpyBLI enables higher quality screening of up to 50 selected variants identified by ELISA screening or NGS. Analytical throughput is increased by using single cycle kinetic analysis.
Predeina et al (2025) The SpyBLI cell-free pipeline for the rapid quantification of binding kinetics from crude samples RSC Chem Biol 6(8):1313-1327. https://doi.org/10.1039/D5CB00079C