Complexation with a Cognate Antibody Fragment Facilitates Affinity Measurements of Fluorescein-Linked Small Molecule Ligands
Abstract
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 antifluorescein 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 mu M 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. Show more
Publication status
publishedExternal links
Journal / series
Analytical ChemistryVolume
Pages / Article No.
Publisher
American Chemical SocietyOrganisational unit
03463 - Neri, Dario (ehemalig) / Neri, Dario (former)
03463 - Neri, Dario (ehemalig) / Neri, Dario (former)
Funding
670603 - Fulfilling Paul Ehrlich’s Dream: therapeutics with activation on demand (EC)
182003 - Understanding and Exploiting the Molecular Targeting of Tumor Neo-vasculature (SNF)
Related publications and datasets
Is part of: https://doi.org/10.3929/ethz-b-000483530
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