Molecular Recognition of a Monoclonal Antibody (AC1106) Cross-Reactive for Derivatives of Ru(bpy)₃²⁺ and Ru(phen)₃²⁺

The characterization of a monoclonal antibody (AC1106) elicited via immunization with a Co(dmbpy)(bpy)₂³⁺−methyl viologen hapten (1) is described. AC1106 was found cross-reactive for a variety of luminescent ruthenium(II) metal complexes which served as useful probes to investigate the molecular recognition properties of this antibody. AC1106 was found to be specific for methylated derivatives of Ru(bpy)₃²⁺ and Ru(phen)₃²⁺ in the order of Ru(dmbpy)₃²⁺ > Ru(dmbpy)(bpy)₂²⁺ > Ru(dmphen)₃²⁺ > Ru(bpy)₃²⁺ ≫ Ru(phen)₃²⁺. The affinities of AC1106 for these metal complexes were found to range from ≥ 5 × 10⁷ to ≤ 1 × 10³ M^-1. When bound (>98%) by AC1106, the luminescence decay traces for the racemic Ru(dmbpy)₃²⁺ and Ru(dmbpy)(bpy)₂²⁺ gave a satisfactory fit to a single-exponential decay process. Furthermore, D₂O/H₂O experiments with Ru(dmbpy)₃²⁺ indicate that AC1106 protects approximately 70% of the antibody-bound Ru(dmbpy)₃²⁺ from excited state deactivation by the solvent. Competition ELISA data indicate that both the metal center and the methyl viologen moiety present in a Ru(bpy)₃²⁺−methyl viologen conjugate ([Ru(mv²⁺-bpy)(bpy)₂]⁴⁺) are important recognition elements for AC1106. Despite the apparent affinity of AC1106 for methyl viologen, no evidence for simultaneous binding of methyl viologen and Ru(dmbpy)(bpy)₂²⁺ inside the binding pocket of AC1106 could be found. Rather, the addition of methyl viologen was found to result in the displacement of AC1106-bound Ru(dmbpy)(bpy)₂²⁺ from the antibody binding site.