Biological preparation of highly effective immunomagnetic beads for the separation, concentration, and detection of pathogenic bacteria in milk.
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 10CFU/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.
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea; Korea Food Research Institute, Seongnam, Republic of Korea.
Min-Cheol
MC
Lim
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.
Gwan-Hyung
GH
Lee
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.
Duyen Thi Ngoc
DTN
Huynh
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.
Chae-Eun
CE
Hong
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.
Soo-Yeon
SY
Park
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.
Jong-Yun
JY
Jung
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.
Cheon-Seok
CS
Park
Department of Biotechnology, CHA University, Seongnam, Republic of Korea. Electronic address: shko7@cha.ac.kr.
Sungho
S
Ko
Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea. Electronic address: youngkim@khu.ac.kr.
Young-Rok
YR
Kim
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D019453:Escherichia coli O157 / Q000302:isolation & purification; D005516:Food Microbiology; D018189:Immunomagnetic Separation / Q000379:methods*; D058619:Maltose-Binding Proteins / Q000737:chemistry; D008892:Milk / Q000382:microbiology*
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D058619:Maltose-Binding Proteins
Amylose; E. coli O157:H7; Food; Immunomagnetic separation; Magnetic bead; Maltose binding protein; Protein G