Novel Monoclonal Antibodies with Specificity for Chelated Uranium(VI): Isolation and Binding Properties

Abstract

A derivative of 1,10-phenanthroline that binds to UO₂²⁺ with nanomolar affinity was found to be a very effective immunogen for the generation of antibodies directed toward chelated complexes of hexavalent uranium. This study describes the synthesis of 5-isothiocyanato-1,10-phenanthroline-2,9-dicarboxylic acid and its use in the generation and functional characterization of a group of monoclonal antibodies that recognize the most soluble and toxic form of uranium, the hexavalent uranyl ion (UO₂²⁺). Three different monoclonal antibodies (8A11, 10A3, and 12F6) that recognize the 1:1 complex between UO₂²⁺ and 2,9-dicarboxy-1,10-phenanthroline (DCP) were produced by the injection of BALB/c mice with DCP−UO₂²⁺ covalently coupled to a carrier protein. Equilibrium dissociation constants for the binding of DCP−UO₂²⁺ to antibodies 8A11, 10A3, and 12F6 were 5.5, 2.4, and 0.9 nM, respectively. All three antibodies bound the metal-free DCP with roughly 1000-fold lower affinity. The second-order rate constants for the bimolecular association of each antibody with soluble DCP−UO₂²⁺ were in the range of 1 to 2 × 10⁷ M^-1 s^-1. Binding studies conducted with structurally related chelators and 21 metal ions demonstrated that each of these three antibodies was highly specific for the soluble DCP−UO₂²⁺ complex. Detailed equilibrium binding studies conducted with three other derivatives of DCP, either complexed with UO₂²⁺ or metal-free, suggested that the antigen binding sites on the three antibodies have significant functional and structural similarities. Biomolecules that bind specifically to uranium will be at the heart of any new biotechnology developed to monitor and control uranium contamination. The three antibodies described herein possess sufficient affinity and specificity to support the development of immunoassays for hexavalent uranium in environmental and clinical samples.