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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%.

Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads