Achievement of linear response for competitive bioaffinity assays of ligands: criteria of optimized interaction systems

For a linear response in a competitive bioaffinity assay of a ligand, an optimized system requires CRT greater than 3-fold of CPT, CPT greater than 50-fold of KdR, and KdR greater than 260-fold of KdX, based on chemometrics for bioaffinity interactions (CRT and CPT are the concentrations of the probe and the biomacromolecule and KdX and KdR are the dissociation constants of complexes with the ligand and the probe, respectively). These criteria were tested for a competitive bioaffinity assay of biotin. The probe was a conjugate of monomethyl-poly-(ethylene glycol)-5000, 1-naphthyl-ethylenediamine and biotin. The complex of the probe with streptavidin was quantified by the fluorescence at 430 nm based on Forster resonance energy transfer with tryptophan residues as the intrinsic donors. By fluorometric titration, KdR of the probe was 5.4 ± 1.4 nM (n = 4). At 1.5 μM probe plus 0.50 μM streptavidin, there was a linear decrease of fluorescence at 430 nm for biotin concentrations ranging from ∼36 to ∼500 nM; the linear response slope was consistent with that for the fluorescence at 430 nm to the concentration of the complex of streptavidin and the probe. Biotin at 81 and 414 nM was estimated with variation coefficients below 7%. These proposed criteria may be universally applicable for linear responses in competitive assays of ligands.