Biological preparation of highly effective immunomagnetic beads for the separation, concentration, and detection of pathogenic bacteria in milk

• A novel approach to synthesize magnetic bead using enzymatic reaction was proposed.
• A one-pot reaction is possible with a simple and inexpensive substrate.
• A fusion protein, MBP-SPG, facilitated the oriented immobilization of antibodies.
• Effective immuno-magnetic bead for the separation of target bacteria was developed.
• This system can be applied to a various targets such as viruses and proteins.

We introduce a system for the efficient separation and concentration of pathogenic bacteria using biologically prepared immunomagnetic beads. Amylose magnetic beads (AMBs) were synthesized by an enzymatic reaction of amylosucrase from Deinococcus geothermalis (DGAS). The simple and rapid conjugation of AMBs and antibodies was achieved by the MBP-SPG fusion protein. MBP (maltose binding protein) binds to the surface of an AMB owing to its intrinsic affinity to the di-glucose in the AMB. SPG (streptococcal protein G) fused to the MBP has specific affinity to the Fc region of the antibody. Anti-Escherichia coli O157 antibodies were conjugated to the AMBs through a MBP-SPG linker without any physical and chemical treatments. The efficiency of separation and concentration of the target E. coli O157:H7 by the functionalized AMBs was revealed by plating counting, conventional polymerase chain reaction (PCR), and real-time RCR analysis. The immuno-AMBs effectively separated and concentrated the target bacteria from a commercial milk sample spiked with known number of bacteria, which was then analyzed by PCR to a detection limit of 10 CFU/mL. On the other hand, no PCR product was produced when milk was introduced directly to a PCR reaction. These results show that MBP-SPG is an effective linker and the resulting immuno-AMBs are capable of separating and concentrating the target bacteria from a food matrix.

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