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 C_RT greater than 3-fold of C_PT, C_PT greater than 50-fold of K_dR, and K_dR greater than 260-fold of K_dX, based on chemometrics for bioaffinity interactions (C_RT and C_PT are the concentrations of the probe and the biomacromolecule and K_dX and K_dR 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 Förster resonance energy transfer with tryptophan residues as the intrinsic donors. By fluorometric titration, K_dR 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.