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anti-FITC_Final.pdf (4.98 MB)

Antibody-Inspired Affinity Measurements of Fluorescein-Linked Small Molecule Ligands

submitted on 28.05.2020 and posted on 29.05.2020 by Marco Catalano, Sebastian Oehler, Luca Prati, Nicholas Favalli, Gabriele Bassi, Jörg Scheuermann, Dario Neri
The availability of reliable methods for the characterization of the binding of small molecule ligands to protein targets is crucially important for Drug Discovery. We have adapted a method, routinely used for the characterization of monoclonal antibodies (Enzyme-linked immunosorbent assay, or “ELISA”), to small molecule ligands, using fluorescein conjugates and anti-fluorescein antibodies as detection reagents. The new small molecule-ELISA methodology was tested using a panel of binders specific to carbonic anhydrase II, with dissociation constants ranging between 6 uM and 14 nM. An excellent agreement was found between ELISA measurements and fluorescence polarization results. The methodology was also extended to BIAcore measurements and implemented for ligands coupled to oligonucleotides. Small molecule-ELISA procedures are particularly useful in the context of DNA-encoded libraries, for which hit validation procedures need to be performed on dozens of candidate molecules and hit compounds can be conveniently resynthesized on DNA.


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Email Address of Submitting Author


Swiss Federal Institute of Technology (ETH Zürich)



ORCID For Submitting Author


Declaration of Conflict of Interest

D.N. is a cofounder and shareholder of Philochem AG.