Selection of human single chain Fv antibody fragments binding and inhibiting Helicobacter pylori urease

Single chain Fv antibody fragments (scFv) binding to purified Helicobacter pylori urease were selected from a nonimmune human antibody repertoire disp layed on filamentous phage, After three rounds of screening on solid phase urease, 44 clones were found to bind the enzyme and four distinct scFv were identified by sequencing their heavy and light chain variable region genes (V-H and V-L). Two of the selected human scFv (scFv B4 and scFv D9) inhibi ted the activity of H, pylori urease with inhibitory constants (Ki) of 7 an d 2 muM, respectively, Their affinity (Kd) for H pylori urease as determine d by surface plasmon resonance ranged from 17 to 42 nM Both scFv were able to bind to urease present on the surface of living H, pylori organisms as d emonstrated by flow cytometry analysis, The binding sites of scFv B4 and D9 were mapped by the use of two random hexapeptide libraries (X6 and CX6C) d isplayed on filamentous bacteriophage, The selected peptide sequences were shown to inhibit scFv binding to H, pylori urease and thus could be used in a vaccination strategy as epitopes mimicking (mimotopes) the region of ure ase recognized by these human scFv antibody fragments, Copyright (C) 2000 S . Karger AG, Basel.