Innovative Methods for Rapid Detection of Foodborne Pathogens

NON-TECHNICAL SUMMARY: With the increase in reports of foodborne infections, great attention has been given to the development of new/improved methods for rapid detecting microbial pathogens. The purpose of this project is to develop innovative methods utilizing phage-displayed recombinant antibodies for rapid detection and identification of Salmonella in foods. <P>

APPROACH: The experiments will be implemented in four phases. <P>
Phase 1: Construction of cDNA Antibody Libraries - Antibody libraries comprised of a large pool of gene encoding antibodies with different antigen recognition sites are required to generate recombinant antibodies. Construction of cDNA libraries will enable us to generate antibodies specific to surface antigen of Salmonella. At the beginning of the project, the total messenger RNA (mRNA) will be extracted from murine B lymphocytes. Out of this mRNA, corresponding DNA chain will be deducted and synthesized by means of enzyme "reverse transcriptase." From this very complex mixture of cDNA chains, immunoglobulin fragment will be specifically amplified by polymerase chain reaction (PCR). In order to select immunoglobulin fragment specific for the target antigens, genes encoding for the light chain (L) and heavy chain (H) will be multiplied by PCR using primers that have been designed based on conserved nucleotide sequences of antibody gene extracted form the Kabat database. The PCR products will then will be introduced in a phagemid vector. <P>Phase 2: Characterization and Screening of the Libraries - Once libraries are generated, they will be used for selection of recombinant monoclonal antibodies against various surface antigens of Salmonella, by immobilizing the antigen of interest and applying various selection rounds with the appropriate phage display libraries. Antigen-binding fragment (Fab) of immunoglobulin molecule will be expressed on the surface of the bacteriaphage. This sublibrary will be subjected to further panning rounds or binding analysis by phage ELISA. <P>Phase 3: Development of Rapid Immunochemical Techniques - Two immunoassay formats will be developed for specific detection of Salmonella using the recombinant antibodies. (1) Sandwich ELISA: Antibodies will be labeled with biotin or enzyme for assay signal generation. Compatible antibodies to be used as pair of capture and detection reagents will be selected by mapping different antibodies, and (2) Immunochromatography: These assays, also called lateral flow tests or rapid strip tests are a logical extension of the technology used in latex agglutination tests. This format will be developed to test for Salmonella surface antigens. Species-specific anti-Salmonella antibodies conjugated on the microspheres are required and will be produced from the purified recombinant antibodies. The protocols for reagent application and stabilization will be optimized. <P>Phase 4: Validation of the Developed Assays - Both the rapid assays developed will be validated on selected vegetables and fruits inoculated with known level of Salmonella. Performance of the assays will be evaluated and compared with commercial rapid assays on sensitivity, specificity, accuracy, and precision.

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PROGRESS: 2004/01 TO 2006/09<BR>
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.
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IMPACT: 2004/01 TO 2006/09<BR>
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.