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 displayed 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) inhibited the activity of H. pylori urease with inhibitory constants (K(i)) of 7 and 2 microM, respectively. Their affinity (K(d)) for H. pylori urease as determined 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 demonstrated 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) displayed 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 urease recognized by these human scFv antibody fragments.
Houimel
M
M
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Corthesy-Theulaz
I
I
Fisch
I
I
Wong
C
C
Corthesy
B
B
Mach
J
J
Finnern
R
R
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D001428:Bacterial Vaccines; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000276:immunology / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D016480:Helicobacter pylori / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D019151:Peptide Library; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D014510:Urease / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004791:Enzyme Inhibitors; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv; D014510:Urease