title: Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads.
abstract: Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 10(3) E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 10(2) E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 10(1) E. coli O104:H4 initial load). The specificity was 100%.
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo Italy.
authors.forename: Mirella
authors.initials: M
authors.lastname: Luciani
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Di Febo
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo Italy.
authors.forename: Katiuscia
authors.initials: K
authors.lastname: Zilli
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Di Giannatale
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo Italy.
authors.forename: Gisella
authors.initials: G
authors.lastname: Armillotta
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Manna
authors.affiliation: EU Reference Laboratory for E. coli, Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Minelli
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Tittarelli
authors.affiliation: EU Reference Laboratory for E. coli, Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome Italy.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Caprioli
keywords: Escherichia coli O104:H4; detection; immuno-magnetic separation; milk; monoclonal antibodies