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Hapten-induced dimerization of a single-domain VHH camelid antibody.

Antibodies that recognize small molecule ligands (haptens) provide unique insight into the immune response and frequently serve as biological reagents for the detection of small molecules. While conventional antibodies typically recognize haptens using two variable domains (VL and VH), much less is known regarding how antibodies with a single variable domain recognize small ligands. Here we investigate the binding thermodynamics for an anti-caffeine camelid (VHH) antibody. Surprisingly, a nonconventional binding stoichiometry was observed in which the final complex includes two VHH domains for every caffeine molecule. DeltaC(p) analysis and size exclusion chromatography support this unusual stoichiometry. An apparent consequence of ligand-induced dimerization is that a relatively high affinity (K(b,obs) = 7.1 x 10(7)) is obtained. The binding profiles of three caffeine metabolites, theophylline, theobromine, and paraxanthine, were also investigated. Each ligand maintains a 2:1 stoichiometry while displaying an approximately 50-fold range of observed binding affinities. These results suggest nonconventional mechanisms of hapten recognition are possible with single-domain antibodies.