Dually Labeled Biomolecules for Characterizing Biotinylated Species through Competitive Binding Reactions

The capability of biotinylated molecules to bind streptavidin may be a more functional measure of the success of target biotinylation than titration of total bound biotins per molecule. It was demonstrated that the binding capability could be assessed by a competitive assay, in which a biotinylated antibody (BA) (or protein, ligand, receptor etc.) of interest competed with a reference antibody (or a protein) dually labeled with biotin and electrochemiluminescence (ECL) moieties for the binding sites of streptavidin coated on the surface of magnetic beads. Inversely related to the ECL signal, the binding capability of a biotinylated antibody can be reproducibly evaluated by multiple sets of easily acquired data series rather than by a single measurement. This method can be employed in an ordinary laboratory with an automated ECL analyzer or other readout instruments for routine characterization of any biotinylated species, such as proteins, ligands, receptors, and polypeptides.