This report describes the characterization of a monoclonal antibody (mAb) synthesized by a hybridoma (5B2) that was isolated by injection of Balb/c mice with Pb(II)-benzyl isothiocyano diethylenetriamine pentaacetic acid (DTPA) conjugated to keyhole limpet hemocyanin (KLH). The binding characteristics of the purified mAb (mAb 5B2) were investigated using a KinExA 3000 immunoassay instrument. Binding studies demonstrated that while mAb 5B2 also recognized DTPA complexes of Ca(II), Sr(II), and In(III), it bound the Pb(II)-DTPA complex with highest affinity (Kd = 3.9 +/- 0.7 x 10-7 M). The structure of the DTPA chelator used to generate the Pb(II)-chelate was found to modulate the antibody's affinity for the Pb(II) ion. The addition of a cyclohexyl ring to the DTPA backbone (CHX-DTPA) resulted in a modest increase in the mAb's affinity for the Pb(II) (Kd = 2.3 +/- 0.5 x 10-7 M) relative to its affinity for Pb(II) in the context of DTPA. However, mAb 5B2's affinity increased eighty-five fold (Kd = 4.6 +/- 0.7 x 10-9 M) when p-nitrobenzyl DTPA, a chelator that bears the benzyl moiety of the original immunogen, was used to generate the Pb(II)-chelate complex. Binding studies of a monovalent Fab fragment derived from the native 5B2 mAb demonstrated that the removal of the Fc region resulted in consistent six- to nine-fold decreases in the antibody's affinity for these same Pb(II)-chelate ligands. Molecular modeling and covalent modification of lysine residues with the primary amine-specific reagent trinitrobenzenesulfonic acid (TNBS) suggested that Lys58 in the third framework region of the heavy chain variable domain was important in the antibody's recognition of the Pb(II)-chelate ligand. The 5B2 antibody's ligand-binding domain was expressed in COS-1 cells and mutation of Lys58 to Ile or Ala resulted in 1.8- and 3.2-fold decreases, respectively, in the antibody's Pb(II)-DTPA-benzyl-BSA binding activity. These data demonstrate that the Lys58 side chain is involved in ion pair and/or hydrogen bond formation to the chelate portion of the Pb(II)-chelate complex. Further, the differential effects of the mutations suggest that side chain length at residue 58 in the heavy chain plays an important role in the antibody's Pb(II)-chelate recognition