Development of Listeria monocytogenes-specific immunomagnetic beads using a single-chain antibody fragment.
A method for coupling single-chain antibody fragments (scFvs) to immunomagnetic beads (IMBs) was developed and evaluated using scFvs specific for Listeria monocytogenes. A plasmid vector, pBAD380, was constructed that allowed the expression of histidine-tagged biotinylated scFvs in Escherichia coli. The gene encoding a scFv specific for L. monocytogenes was cloned into pBAD380 and the 6-histidine-tagged biotinylated anti-L. monocytogenes scFvs were coupled to streptavidin-coated IMBs. The ability of the anti-L. monocytogenes scFv-IMBs to capture L. monocytogenes and other Listeria species was evaluated in comparison to commercially available anti-Listeria IMBs. The anti-L. monocytogenes scFv-IMBs displayed higher efficiencies of capture (1.38-19.04%) for most strains of L. monocytogenes than were observed for the anti-Listeria IMBs (0.05-3.35%); also, the anti-L. monocytogenes scFv-IMBs exhibited improved specificity for L. monocytogenes as determined by cell capture efficiency in pure and mixed cultures.
Paoli
George C
GC
Microbial Biophysics and Residue Chemistry Research Unit, United States Department of Agriculture, Agricultural Research Service, Wyndmoor, PA 19038-8598, USA. george.paoli@ars.usda.gov
Kleina
Lynn G
LG
Brewster
Jeffrey D
JD
D000907:Antibodies, Bacterial; D000918:Antibody Specificity; D001710:Biotin; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics*; D018189:Immunomagnetic Separation / Q000379:methods*; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D010957:Plasmids; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin
D000907:Antibodies, Bacterial; D007135:Immunoglobulin Variable Region; D001710:Biotin; D019809:Streptavidin