Progress 01/01/04 to 09/30/06
Outputs Rapid immunoassay in sandwich ELISA format has been developed for specific detection of Salmonella. The detecting antibodies were labeled with biotin. Compatible antibodies as a pair of capture and detection reagents were selected by epitope mapping and the optimal concentration of capturing antibody and detecting antibody were determined by cross titration. Standard curves to mimic performance characteristics of the sample matrix were constructed and the functional assay protocol within the target sensitivity range has been evaluated. The rapid assay was validated on lettuce and tomato inoculated with three levels of Salmonella. Performance of the assay was compared with two commercial immunoassays. The sensitivity of the rapid assay was compatible with commercial immunoassays. An accuracy of 98% was achieved when the rapid assay was applied to measure the recovery of inoculated samples. The overall intra-assay and inter-assay variations of the rapid assay were 8% and
12%, respectively.
Impacts The use of recombinant antibodies for detection of foodborne pathogens offers advantages over the traditional detection reagents. These bio-engineered molecules provide solutions to improve and potentially replace the conventional antibodies. The recombinant antibodies developed in this project are important in developing advanced detection technologies, such as biosensors.
Publications
- Chen, F-C., Nahashon, S. N., Zhou, S., Kilonzo-Nthenge, A., and Bridgman, R. C. 2006. Immunochemical characterization of flagellar antigens of Salmonella typhimurium. Book of Abstract/ARD Biennial Research Symposium, Abstract P133.
- Kilonzo-Nthenge, A., Chen, F-C., Godwin, S. L. 2006. Pathogenic Enterobacteriaceae and aerobic bacteria isolates from domestic refrigerators. Book of Abstract/IAFP Annual Meeting, Abstract P1-58.
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Progress 01/01/05 to 12/31/05
Outputs Antibodies to Salmonella surface antigens have been produced. Specificity of the antibodies has been characterized by enzyme-linked immunosorbent assay and 2-D gel electrophoresis followed by Western blot. The antibodies bound specifically to the flagellar strain of S. Typhimurium. Epitopes of flagella proteins were compared; the antibodies recognized different epitopes present on a group of proteins migrating between 56 and 58 kD, with an isoelectric point (pI) of 5.2. These epitopes were further characterized by limited proteolysis and competitive mapping. Findings suggested that at least three epitope regions were shared among the antibodies.
Impacts These antibodies provide sensitive structural probes that are valuable for identification of this important pathogen. Immunoassays utilizing these antibodies have been developed for rapid detection of Salmonella in foods. The immunoassays will be used for screening of food contamination to reduce the risk of consumer exposure to Salmonella.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs The DNA encoding immunoglobulin G (IgG) heavy and light chain has been cloned and sequenced. Several sequences exhibited high homology with several sites of the IgG variable region gene. These DNA fragments will be introduced into a phagemid vector and expressed on the surface of the bacteriophage for the detection of Salmonella antigens.
Impacts The techniques to be developed, including the recombinant antibodies and the detection methodology, provide advanced techniques to food producers, processors and distributors, as well as federal and state regulatory agencies for identifying microbial safety problems.
Publications
- No publications reported this period
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