BOUND TRIS CONFOUNDS THE IDENTIFICATION OF BINDING-SITE RESIDUES IN APARAQUAT SINGLE-CHAIN ANTIBODY

We produced an anti-paraquat single chain antibody (scFv) to investiga te its potential use in immunotherapy for paraquat poisoning, However, this scFv was expressed in an insoluble form and only displayed moder ate binding affinity. An earlier examination of the pH dependence of a ntigen binding by the parent paraquat-specific mAb (7D7-3) suggested t hat the electrostatic effects of a tyrosine residue were important, Th e aims of the current study were to obtain expression of a soluble scF v (D10) and to increase its binding affinity. The former was achieved by expression in a phagemid vector, Site-directed mutagenesis of tyros ine residues in CDR H3 did not result in improved affinity for paraqua t, suggesting that the original pH dependence required re-examination, Nuclear magnetic resonance studies of 7D7-3 Fab revealed that the ori ginal observation of the ps-dependent paraquat binding with a mid-poin t of similar to pH 8.9 was due to tightly bound Tris, It appears that as Tris is titrated to a neutral species the energetically unfavourabl e juxtaposition of its positive charge with that of paraquat is reduce d, These findings have broad implications in the interpretation of the pH or salt dependence of any antibody-antigen interaction which shoul d be made cautiously and with regard to the possible interference of b uffer components introduced during the preparation of the antibody.