title: Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7.
abstract: Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.
authors.affiliation: Department of Biology, Adrian Building, University of Leicester, University Rd., Leicester LE1 7RH, Leicestershire, United Kingdom.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kühne
authors.forename: William S
authors.initials: WS
authors.lastname: Hawes
authors.forename: Roberto M
authors.initials: RM
authors.lastname: La Ragione
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Woodward
authors.forename: Garry C
authors.initials: GC
authors.lastname: Whitelam
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Gough
mesh_terms: D018829:Adhesins, Bacterial / Q000276:immunology*; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification*; D001483:Base Sequence; D002352:Carrier Proteins / Q000276:immunology*; D019453:Escherichia coli O157 / Q000276:immunology* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D037521:Virulence Factors / Q000032:analysis
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D037521:Virulence Factors; C055966:immunoglobulin Fv; C073819:eaeA protein, E coli