High-Affinity Uranyl-Specific Antibodies Suitable for Cellular Imaging

Abstract

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO2(2+)-DCP-BSA (DCP, 1,10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO2(2+)-DCP-casein, we selected two highly specific mAbs against uranyl-DCP ( K D 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO2(2+), Fe (3+), Zn2+, Cu2+, and Ca2+) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO2(2+) equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immunolabeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its paratopes can accommodate a wide variety of chelating groups.

Deciphering the Energy Landscape of the Interaction Uranyl-DCP with Antibodies Using Dynamic Force Spectroscopy

“…The weak interaction between Mab U08S and Cu-DCP, as observed using DFS, has also been observed from surface plasmon resonance experiments (5). However, no detectable interaction by surface plasmon resonance was observed for divalent metals complexed with Mab U04S, in agreement with our DFS results that show unbinding events between Mab U04S and Ni-DCP occurring rarely (,0.3% of total events; see Fig.…”

“…The very high apparent affinity of Mabs U04S and U08S toward UO 2 -DCP as measured in bulk solution (5), led us to postulate that such high affinity was gained from an enhanced association rate constant, probably driven by strong Coulombic interactions between uranyl and antibodies.…”

“…In aqueous solution, chelated uranyl ion (UO 21 2 ) is the most common species of uranium (2,3). To study biological targets of UO 21 2 ; such as proteins or DNA, the approach that at least two teams have adopted was to raise antibodies against the uranyl chelates (4,5) where UO 21 2 was attached to a dicarboxy-phenanthroline chelator (DCP) coupled to a protein carrier. Using this approach, our group has selected a set of monoclonal antibodies (Mabs) specific for binding with UO 2 -DCP (5), including U04S, U08S and PHE03S; Mab PHE03S was chosen also for its strong cross-reactivity with the chelator DCP.…”

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

“…Uranyl is expected to be coordinated by the side chains of two aspartates and two histidines at this site, while in the metal binding loop of calmodulin site 1, uranyl ligands may be provided by oxygen atoms from aspartate and/or peptide carbonyl groups. Although we cannot directly compare our results with those obtained by other measuring approaches, it is interesting to note that dissociation constants in the 10 −9 M to 10 −10 M range were reported for the interaction of monoclonal antibodies with uranyl complexed to dicarboxylic-phenanthroline derivatives [18], [22].…”

Rapid superparamagnetic‐beads‐based automated immunoseparation of Zn‐proteins from Staphylococcus aureus with nanogram yield

“…Heavy‐metal immunoassays utilized anti‐heavy‐metal antibodies that monitored binding environment pollutants, Pb(II) , Hg(II) , As(IV, V), Cd(II) , Cu(II) , U(VI) , and rare heavy metals Ru(II) and In(III) . Uranyl‐specific antibodies suitable for cellular imaging were developed based on these studies. Antibodies against Zn(II) were successfully prepared in previous investigations .…”