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

作者：Pu, J.; Hu, X. L.; Liao, J.; Li, Y. W.; Qin, J. L.; Xie, Y. L.; Zhan, C. -G.; Yang, X. L.^*; Liao, F.^*

来源：RSC Advances, 2016, 6(112): 110858-110865.

DOI：10.1039/c6ra06426d

• TRYPTOPHAN
• PROTEINS
• AFFINITY
• SERUM

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 Forster 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 mM probe plus 0.50 mu M streptavidin, there was a linear decrease of fluorescence at 430 nm for biotin concentrations ranging from similar to 36 to similar 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.

• 出版日期2016
• 单位重庆医科大学