Complexation with a Cognate Antibody Fragment Facilitates Affinity Measurements of Fluorescein-Linked Small Molecule Ligands
Authors: Gabriele Bassi
Nicholas Favalli
Marco Catalano
Dario Neri
Sebastian Oehler
Luca Prati
Jörg Scheuermann
Journal: Analytical Chemistry (2020)
Discipline: CHEMISTRY
Keywords: binding characterization; small molecule ligands; ELISA; fluorescein conjugates; antifluorescein antibodies
Affiliations: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland, Philochem AG, Libernstrasse 3, 8112 Otelfingen, Switzerland

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 μM 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. 

Retracted: No

PubPeer comments:
 Below there are two parts copied from the commented article in which it is described that the u201cDEL technologyu201d is based on covalent attachment of small organic compounds to DNA strands. In fact there is no u201cDEL technologyu201d. What is used is the u201cSplit and Mix Technologyu201d of Furka et al. modified with DNA encoding. Furka, u00c1., Sebestyu00e9n, F., Asgedom, M., Dibu00f3, G. CORNUCOPIA OF PEPTIDES BY SYNTHESIS (1988) . In Highlights of Modern Biochemistry, Proceedings of the 14th International Congress of Biochemistry, Vol. 5, p. 47, VSP.Utrecht. Furka u00c1., Sebestyu00e9n F., Asgedom M., Dibu00f3 G. General method for rapid synthesis of multicomponent peptide mixtures Int. J. Pepiide Protein Res. 37, 1991, 487-493. The Spit and Pool Technology makes it possible to synthesize million or even trillion component chemical libraries. This method is realized in stepwise cycles. Each cycle comprised three operations: 1.tSplitting compound mixtures into equal portions 2.tCouple a building block (BB) to each portion 3.tThoroughly mix the portions. After having this, may come into the picture the DNA encoding of BBs. In Operation 2 an additional coupling is executed in which the encoding DNA is elongated with the DNA oligomer encoding the BB. This clearly shows that the use of the split and pool method in DNA encoding is unavoidable and so citation of the method is normally inevitable. In this paper however, citation of the split and pool method is completely omitted. The proper citation is replaced by irrelevant publications in Ref. 12-14. ![file] ![file] ,