Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7557788 | Analytical Biochemistry | 2016 | 30 Pages |
Abstract
Listeria monocytogenes (LM), one of the eight species belonging to the genus Listeria, is pathogenic for both humans and animals. In this study, two novel LM-specific clones, designated L5-78 and L5-79, were isolated from a phage display antibody library that was derived from the variable domain of heavy-chain antibodies (VHHs) of non-immunized alpaca. These two clones were expressed, purified, and characterized. Results showed that both isolated VHHs recognize three serotypes (1/2a, 1/2b, and 4b), which are responsible for more than 95% of documented human listeriosis cases. The recombinant VHHs possess high thermal stability, pH tolerance, and urea resistance. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the VHH clone L5-79 and a monoclonal antibody was developed to detect LM in pasteurized milk, with a detection limit of 1Â ÃÂ 104Â colony-forming units (CFU)/ml. These findings indicated that the species-specific VHHs could be directly isolated from the non-immunized library with a properly designed panning strategy and VHH could be a new source for possible diagnosis/detection of foodborne pathogens in food because it was shown to be highly specific and stable.
Keywords
PVDFPBSTIMACHRPOVAIPTGRTECDRTMBVHHmAbCFUPBSFR23,3′,5,5′-tetramethyl benzidineHKCBSAPBS containing 0.1% Tween 20SDS–PAGEbovine serum albuminReady-to-eatamino acidMonoclonal antibodyEnzyme-linked immunosorbent assay (ELISA)sodium dodecyl sulfate–polyacrylamide gel electrophoresisOvalbuminisopropyl β-D-1-thiogalactopyranosideELISAEnzyme-linked immunosorbent assaypolyvinylidine fluorideLodLuria brothListeria monocytogeneslimit of detectionPhosphate-buffered salinecomplementary-determining regionPhage displaycolony-forming unitsHorseradish peroxidaseimmobilized metal affinity chromatographynegative control
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Zhui Tu, Qi Chen, Yanping Li, Yonghua Xiong, Yang Xu, Na Hu, Yong Tao,