On-bead binding assays

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

Nakamura, C. Inuyama, Y. Goto, H. Obataya, I. Kaneko, N. Nakamura, N. Santo, N. Miyake, J., Dioxin-binding pentapeptide for use in a high-sensitivity on-bead detection assay. Anal. Chem. 2005, 77, 7750-7757... 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

Evaluated from Aj values based on data from DHA-beads for [j2AR with Kcompetitive binding assays for FPR with of fMLFF-FITC set at 4.8 nM (for fMLFF, fMLF, and ML). 

OBOC peptide library can be screened on bead or in solution phase if the bound peptides are released via a cleaveable linker. The on-bead assays include binding and functional assays. In the binding assays, the target of interest could be a... 

Fig. 15.2 Flow injection set-up for optical sensing and screening library members and competitive binding assay for fructose developed on-bead using fluorescent labeled glucose...

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