Surface plasmon resonance for rapid screening of uranyl affine proteins.
A sensitive immunoassay based on SPR analysis was developed to measure uranyl cation (UO(2)(2+)) affinity for any protein in a free state under physiological conditions. The technique involves immobilization of a specific monoclonal antibody (mAb) raised against UO(2)(2+) and 1,10-phenanthroline-2,9-dicarboxylic acid (DCP) used as a probe of UO(2)(2+) captured by the mAb. Calibration curves were established for accurate determination of UO(2)(2+) concentrations with a detection limit of 7 nM. The remaining free UO(2)(2+) could be accurately quantified from the different protein-metal equilibrium and a dose-response curve established for K(D) determination. This generic method was applied not only to proteins such as transferrin and albumin but also to small phosphonated ligands. Its robustness allows the fast UO(2)(2+) K(D) determination of any kind of macromolecules and small ligands using very few amount of compounds, thus opening new prospects in the field of uranium toxicity.
Service de Biochimie et de Toxicologie Nucléaire/LEPC, DSV/iBEB, CEA Marcoule, BP 17 171, F-30207 Bagnols sur Cèze, France.
Olivier
O
Averseng
Agnès
A
Hagège
Frédéric
F
Taran
Claude
C
Vidaud
D055910:Antibodies, Immobilized / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D007118:Immunoassay / Q000379:methods; D007700:Kinetics; D010618:Phenanthrolines / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000379:methods*; D017974:Uranium Compounds / Q000032:analysis*
C519844:1,10-phenanthroline-2,9-dicarboxylic acid; D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D010618:Phenanthrolines; D011506:Proteins; D017974:Uranium Compounds; C047385:uranium oxide