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The primary goal of this project is to apply proteomic approaches to develop an innovative protein-based fingerprinting system for detection and identification of Campylobacter. The secondary goal of this project is to increase proteomic research capacity in agricultural sciences at TSU.<P> Objective 1: To complete a proteomic mapping of Campylobacter surface antigens using phage displayed recombinant antibodies PD will apply the proteomic tools to screen a phage displayed antibody library for recombinant antibodies to Campylobacter surface antigens. It is anticipated 10-15 protein markers will be identified, and recombinant monoclonal antibodies specific to these protein markers will be developed for immunochemical fingerprinting of Campylobacter spp. The fingerprints would serve as the groundwork for further development of advanced detection system, such protein microarrays and biosensors for identification of Campylobacter spp. <P>Objective 2: To establish collaborative partnerships and stimulate exploration of proteomic approaches in agricultural research at TSU In addition to complete the work in Objective 1, this project will build a synergy in proteomic research by bringing together existing expertise from several disciplines within TSU, and establish collaborative partnerships with leading research institutions and industrial laboratories to expand capability in protein mass spectrometry. These efforts will produce an online resource that will allow other researchers at TSU to access to the needed information and technical supports. This project will promote the infrastructure by conducting a series of seminars and hand-on workshops to faculty and students on campus.
Non-Technical Summary: The incidences of foodborne illness have prompted great public health concerns. There is an urgent need to explore new detection methods to facilitate implementation of preventive measurements and intervention strategies. This project will develop an innovative method utilizing phage displayed recombinant antibodies for rapid detection and identification of Campylobacter, the leading cause of bacterial diarrheal illness in the United States. We will apply proteomic approaches to study Campylobacter surface antigens, to screen a phage displayed antibody library, and to develop an innovative technology for immunochemical fingerprinting of Campylobacter. In addition, we plan to conduct seminars on proteomic applications and hand-on workshops on laboratory techniques for faculty and graduate students. Results from this project will enable PD to secure support funding from interested industrial partners and to continue research activities to validate the developed technology and to incorporate it into microarray, biosensor and other advanced instrumentations to improve current detection methodology. <P> Approach: Work Plan 1.1: Identification of Campylobacter surface antigens Campylobacter cells will be homogenized. The cell lysates will be labeled with the cyanine dyes Cy2, Cy3, and Cy5. The Cy3 and Cy5 labeling reactions from two different strains of Campylobacter will be mixed and run on the same gels with an equal amount of Cy2-labeled standard. Gels will be imaged using the Typhoon 9400 Variable Mode Imager. Images will be analyzed using DeCyder software. Mass spectrometry and database interrogation will be used for protein identification. Work Plan 1.2: Selection of phage displayed antibodies to major surface antigens Ten to fifteen Campylobacter surface antigens will be selected for panning specific recombinant monoclonal antibodies. A phage displayed antibody library constructed in the PD's laboratory will be used for selection of recombinant monoclonal antibodies against various surface antigens of Campylobacter. Antigen-binding fragment (Fab) of immunoglobulin molecule will be expressed on the surface of the bacteriaphage. Fab will be purified using an affinity column. Purified Fab will be used for SDS-PAGE and subsequent immunoblot detection. Work Plan 1.3: Immunochemical fingerprinting of Campylobacter Campylobacter will be isolated from samples collected from poultry processing plants using standard methods. A collection of Campylobacter stains will be established from the isolates and from cultures obtained from ATCC. Cell lysates of Campylobacter will be separated by SDS-PAGE. For immunoblot, proteins on the gel will be transferred to nitrocellulose membrane. Colorimetric detection will be performed using combinations of two to three recombinant antibodies. The immunochemical fingerprints derived from different strains of Campylobacter will be analyzed by Kodak Gel Logic 100 Imaging System. Work Plan 2.1: Enhance research capacity through collaboration and integration of existing expertise and resources This project will compile a directory of currently available equipment for proteomic research. The directory will contain information regarding the equipment capability, availability, and operation instructions. Standard laboratory protocols for proteomic analysis will be developed. A directory of academic institutions and industrial laboratories capable of providing mass spectrometry services will be complied and information regarding their facilities and capability will be listed. This information will be available online to students and faculty. Work Plan 2.2: Stimulate exploration of proteomic approaches in agricultural research and student training Two graduate students will be recruited to work on project. Students will gain knowledge and experience in proteomic techniques. A total of six seminars will be conducted during the three-year project period. At least two of the seminars will be presented by invited speakers from collaborative institutions or industrial laboratories. PD and Co-PDs will conduct a hand-on workshop for faculty and students at TSU on protein separations using difference gel electrophoresis. A workshop on protein mass spectrometry will be arranged with one of the partner institutions.