Source: TENNESSEE STATE UNIVERSITY submitted to
INNOVATIVE METHODS FOR RAPID DETECTION OF FOODBORNE PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0198964
Grant No.
(N/A)
Project No.
TENX-0406-FSHNT23
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jan 1, 2004
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Project Director
Chen, F.
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
FAMILY AND CONSUMER SCIENCES
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
10%
Applied
40%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010104030%
7124010109040%
7124010110030%
Goals / Objectives
The overall objective of this project is to develop innovative methods utilizing phage-displayed recombinant antibodies for rapid detection and identification of Salmonella in foods. The specific objectives of this project are to: (1) Construct cDNA antibody libraries from murine lymphocytes immunologically challenged with surface antigens of Salmonella, (2) Characterize and screen the libraries for antibodies with diverse specificity to surface antigens of Salmonella, (3) Develop rapid immunochemical techniques utilizing phage-displayed recombinant antibodies for sensitive detection of Salmonella in foods, and (4) Validate performance of the developed assays for detection and identification of Salmonella in various food products.
Project Methods
The experiments will be implemented in four phases. 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. 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. 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. 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.

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.


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


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