Selection, characterization and X-ray structure of anti-ampicillin single-chain Fv fragments from phage-displayed murine antibody libraries

Single-chain Fv (scFv) antibody libraries were constructed from mice immuni zed with an ampicillin-bovine serum albumin conjugate. Several antibodies w ith specificity for intact ampicillin were selected by phage display and ch aracterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determi ned the X-ray structures of two crystal forms of w.t. aL2, which differ mai nly in the side-chain conformation of Trp H109 (according to a new consensu s nomenclature Kabat residue number H95) in the extremely short (three resi dues) CDR H3 and the presence or absence of a well-resolved molecule of 2-m ethyl-pentane-2,4-diol in the bottom of the binding pocket. Attempts to co- crystallize aL2 with its antigen or to diffuse ampicillin into the wild-typ e aL2 crystals were unsuccessful, since crystal contacts obstruct the bindi ng pocket. However, a mutant with two point mutations near the N terminus ( Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen-binding. Although the mutations affect t he conformation of framework I, the conformations of the binding pocket of the uncomplexed wild-type aL2 and of the mutant complex were almost identic al. The structure explains the specificity of the antibody for intact ampic illin and the degree of cross-reactivity of aL2 with a wide variety of ampi cillin analogs. This antibody system will be very useful as a diagnostic re agent for antibiotics use and abuse, as a model for the effect of expressio n of antibiotic binding molecules in Escherichia coli, and for directed evo lution towards high antibiotic resistance. (C) 2001 Academic Press.