Aims: The objectives of this study were to produce Salmonella-specific peptide ligands by phage display biopanning and evaluate their use for magnetic separation (MS). Methods and Results: Four-phage display biopanning rounds were performed, and the peptides expressed by the two most Salmonella-specific (on the basis of phage-binding ELISA results) phage clones, MSal020401 and MSal020417, were chemically synthesized and coupled to MyOne . MS + Greenlight TM gave a more comprehensive picture of capture capability than MS + plate counts and showed that Peptide MSal020417-coated beads exhibited at least similar, if not better, capture capability to anti-Salmonella Dynabeads â (mean capture values of 36Á0 AE 18Á2 and 31Á2 AE 20Á1%, respectively, over Salmonella spp. concentration range 3 9 10 1 -3 9 10 6 CFU ml À1 ) with cross-reactivity of 1Á9% to three other foodborne pathogens: Escherichia coli, Listeria monocytogenes and Campylobacter jejuni. Conclusions: One of the phage display-derived peptide ligands was demonstrated by MS + Greenlight TM to be a viable antibody alternative for MS of Salmonella spp. Significance and Impact of the Study: This study demonstrates an antibodyfree approach to Salmonella detection and opens substantial possibilities for more rapid tests for this bacterium.
The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens ( Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries.
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