Progress 02/15/19 to 02/14/24
Outputs
Target Audience:The aim of developing the biosensor in this project was to streamline the detection of Campylobacter contamination in both processing facilities and final food products. The intended users for this biosensor technology were professionals in the food industry and regulatory testing laboratories, all of whom prioritize food safety. Moreover, students and faculty at TSU acquired valuable knowledge and skills in biosensors for agricultural research through workshops and training sessions. Additionally, industrial partners were informed about the potential for innovative technological advancements. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training to two graduate students in the detection and identification of Campylobacter in foods, employing both cultural and molecular methods. One student graduated with a Ph.D. degree, while the other obtained an M.S. degree. These students showcased the project findings at professional conferences and research forums. Notably, one graduate student received the 3rd place poster award in the 4th Annual State-Wide Competition for Food Safety Modernization Act, Food Safety, and Food Science Students, and the 1st place poster award in the 130th Annual Meeting of the Tennessee Academy. How have the results been disseminated to communities of interest? The findings from this project have been shared with food industry experts through three conferences and have been published in scientific journals. We have actively disseminated these new technological advancements to interested industrial partners. The outcomes of this project were presented at professional conferences attended by food safety experts, as well as companies involved in food production, processing, and distribution. Moreover, public health agencies at local, state, and national levels, which require Campylobacter detection technologies to ensure product safety and regulatory compliance, have shown a keen interest in our research. This research project on the development of a biosensor for rapid detection of Campylobacter was featured in the "Latest Research behind Campylobacter" section of Food Quality and Safety. These detection technologies play a crucial role in identifying and preventing contaminated food products from reaching consumers, leading to a reduction in foodborne illnesses and ensuring safer food for the public. Consumers indirectly benefit from access to safer food products, a decrease in foodborne illnesses, and greater transparency in food labeling. Essentially, Campylobacter detection technologies are pivotal in safeguarding public health, reducing healthcare costs, and fostering trust in the food supply. They contribute to healthier communities, stronger economies, and increased transparency in the food industry. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project, utilizing immunological capturing with two sets of monoclonal antibodies, has devised a robust and efficient technique for concentrating Campylobacter from contaminated food samples. By selectively capturing and concentrating Campylobacter cells, this method enhances the sensitivity and reliability of Campylobacter detection. Two sets of monoclonal antibodies (Group I and V), comprising seven clones, were individually tested in Western blots to ascertain specific binding patterns with naturally expressed flagellin proteins. The results revealed distinct binding patterns between Groups I and V monoclonal antibodies. Across all tested C. jejuni strains, a major band with a molecular weight of 65 kDa and a minor band of 45 kDa were consistently observed. Notably, the binding patterns were found to be unique for different strains, showcasing the potential of these monoclonal antibodies in strain identification and immunological tests. Leveraging the specificity of antibodies and the signal enhancement provided by magnetic nanoparticles (MNPs), a surface plasmon resonance (SPR) biosensor protocol was formulated. The efficacy of the immunomagnetic capturing and concentration methods was validated in three separate studies. Chicken samples deliberately contaminated with various levels of Campylobacter jejuni were subjected to deionized water washes. Antigens of Campylobacter were then extracted from the chicken rinse sediments post-centrifugation. Immunomagnetic nanoparticles functionalized with monoclonal antibody (MAb 5A6) were incubated with the sample extract. Following incubation, the antigen-antibody nanoparticle complexes formed were concentrated and purified using a magnetic column. Subsequently, the purified antigen-antibody nanoparticle complexes were assessed by an SPR biosensor immobilized with a monoclonal antibody (MAb 6H12) to facilitate a sandwich assay. The SPR biosensor exhibited high sensitivity in detecting Campylobacter and could be completed within 2.5 hours. The log-linear correlation of the assay for Campylobacter jejuni concentration was observed within the range of 102 to 105 CFU/g, with a detection limit of 102 CFU/g. These results underscore the need for further optimization and validation studies to transition the biosensor into practical applications in food industry settings. Nonetheless, the developed MNPs-enhanced SPR biosensor holds promise as a technology for sensitive detection of Campylobacter in food samples.
Publications
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Progress 02/15/22 to 02/14/23
Outputs
Target Audience:The purpose of developing the biosensor in this project was to simplify the process of detecting Campylobacter contamination in processing facilities and final food products. The target audiences for this biosensor were professionals in the food industry and regulatory testing laboratories concerned with food safety. As a result of this project, students and faculty at TSU gained valuable knowledge and skills in the field of biosensors for agricultural research. Industrial partners who were interested were informed of the opportunity to take advantage of the new technology developments. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Starting in the Fall semester of 2022, a newly recruited graduate student began working on this project. The student has received training in both cultural and molecular methods for detecting and identifying Campylobacter in foods. Currently, the student is assisting the PD in developing SPR biosensor protocols. How have the results been disseminated to communities of interest?The PD in coordination with collaborators has conducted a hand-on workshop for faculty and graduate students at TSU on biosensor and molecular detection methods. The workshop included an in-depth experience with the SPR technique, practical hands-on applications, and techniques that further enable participants to confidently execute SPR experiments. What do you plan to do during the next reporting period to accomplish the goals?PD will continue to validate the performance of the developed assay and SPR biosensor with collaborators. Samples then will be independently analyzed, and the results will be compared with culture methods and between the laboratories. This project is expected to produce a process of rapid isolation and concentration of Campylobacter from foods and an automated SPR biosensor analysis. The improved time-saving concentration process coupled to the automated SPR biosensor analysis provides a rapid alternative to the current culture methods for efficient isolation and identification of Campylobacter in food products.
Impacts
What was accomplished under these goals?
To evaluate the efficacy of the immunomagnetic separation methods that were developed, validation studies were carried out. In these studies, chicken drumsticks were deliberately contaminated with various levels of Campylobacter jejuni (BAA 1153, ATCC 49943, and ATCC 33560) and then washed with deionized water. Antigens were extracted from the chicken rinse sediments after centrifugation, and then, monoclonal antibody (MAb 5A6) functionalized immunomagnetic nanoparticles were prepared and incubated with the sample extract. After incubation, the antigen-antibody nanoparticle complexes that formed were concentrated and purified using a magnetic column. Subsequently, the purified antigen-antibody nanoparticle complexes were reacted with another biotin-labeled MAb 6H12 and detected using an avidin-enzyme conjugate in a sandwich immunoassay format. The assay, which was found to be highly sensitive, could be carried out within 2.5 hours. The log-linear correlation of the assay for the concentration of Campylobacter jejuni ranged from 103-106 CFU/g. The assay's detection limit was determined to be 103 CFU/g. These results indicate that the assay could be further developed into small, portable measuring devices to facilitate preliminary screening tests.
Publications
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Progress 02/15/21 to 02/14/22
Outputs
Target Audience:The biosensor developed in this project is intended to facilitate the identification of Campylobacter contamination in the processing facilities and final products. The target audiences were food safety professionals in food industry and regulatory testing laboratories. The students and faculty at TSU gained knowledge and skills of biosensors in agricultural research. Interested industrial partners were informed about the opportunity to avail themselves of the new technology developments. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has trained one doctoral graduate student in the detection and identification of Campylobacter in foods using cultural and molecular methods. The student has completed a dissertation entitled "Characterization of Campylobacter flagellin using a panel of monoclonal antibodies and their application in direct detection using Surface Plasmon Resonance" and graduated with a Ph.D. degree, December 2021. Major advisor: Fur-Chi Chen A new graduate student has been recruited and will continue to work on this project starting Fall semester, 2022. How have the results been disseminated to communities of interest?One research paper was presented at professional conferences. Shreya Singh Hamal and Fur-Chi Chen. Kinetics characterization of specific monoclonal antibodies targeting campylobacter flagellin protein using surface plasmon resonance. 43rd Annual Research Symposium, Tennessee State University, March 22, 2021, Nashville, TN What do you plan to do during the next reporting period to accomplish the goals?This project will continue to develop an immunomagnetic separation method to concentrate Campylobacter from contaminated food samples and to incorporate the magnetic nanoparticle enhancement in SPR to produce a sensitive detection technology.
Impacts
What was accomplished under these goals?
Surface Plasmon Resonance (SPR) biosensors are sensitive methods for detection of Campylobacter in foods. This project continues to develop a rapid SPR screening method by comparing the binding characteristics for the selection of five monoclonal antibodies. Experiments were conducted to compare the binding responses of antigen from three different strains of Campylobacter jejuni (BAA 1153, ATCC 49943 and ATCC 33560) using five different immobilized monoclonal antibodies (MAb-5A6, MAb-5B5, MAb-5D7, MAb-5E4, and MAb-6H12). The results from SPR showed that the highest response was obtained at 3X107 CFU/ml for the strain BAA 1153 on immobilized MAb-5A6. The results also indicated that there were significant differences (p<0.05) among binding responses of the same strain of Campylobacter jejuni on five different immobilized antibodies. All three strains showed higher binding responses toward two immobilizations (MAb-5A6 and MAb-6H12) as comparing to other immobilizations (MAb-5D7, MAb-5E4 and MAb-5B5). Therefore, MAb-5A6 and MAb-6H12 were selected for magnetic nanoparticle enhancement in SPR.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Hamal, S. S. (2021) Characterization of Campylobacter flagellin using a panel of monoclonal antibodies and their application in direct detection using Surface Plasmon Resonance (Accession No.) [Doctoral dissertation, Tennessee State University, Nashville, TN]. ProQuest Dissertations Publishing.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Shreya Singh Hamal and Fur-Chi Chen. Kinetics characterization of specific monoclonal antibodies targeting campylobacter flagellin protein using surface plasmon resonance. 43rd Annual Research Symposium, Tennessee State University, March 22, 2021, Nashville, TN
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Progress 02/15/20 to 02/14/21
Outputs
Target Audience:The biosensor developed in this project is intended to facilitate the identification of Campylobacter contamination in the processing facilities and final products. The target audiences were food safety professionals in food industry and regulatory testing laboratories. The students and faculty at TSU gained knowledge and skills of biosensors in agricultural research. Interested industrial partners were informed about the opportunity to avail themselfs of the new technology developments. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has trained one doctoral graduate student in the detection and identification of Campylobacter in foods using cultural and molecular methods. The graduate student has presented the project findings in professional conferences and research forums. This student won the 3rd place poster award in the 4th Annual State-Wide Competition for Food Safety Modernization Act, Food Safety, and Food Science Students on September 8, 2020, and the 1st place poster award inthe 130th Annual Meeting of the Tennessee Academy of Science on November 21, 2020. How have the results been disseminated to communities of interest?Three research papers were presented at professional conferences. Shreya Singh Hamal and Fur-Chi Chen. Kinetics Characterization of Campylobacter Flagellin Protein by Specific Monoclonal Antibodies Using Surface Plasmon Resonance (SPR). IFT-Volunteer Section Meeting, September 8, 2020. (Virtual Conference) Shreya Singh Hamal and Fur-Chi Chen. Characterization of Campylobacter flagellin Protein Specific Monoclonal Antibodies and Evaluation of Their Binding Affinities Using Surface Plasmon Resonance. International Association for Food Protection (IAFP) Annual Meeting, October 27, 2020. (Virtual Conference) Shreya Singh Hamal and Fur-Chi Chen. Comparison and Screening of Polyclonal Antibodies for Detection of Campylobacter Flagellin Protein using Surface Plasmon Resonance (SPR). The 130th Annual Meeting of the Tennessee Academy of Science, November 21, 2020. (Virtual Conference) What do you plan to do during the next reporting period to accomplish the goals?This project will continue to incorporate magnetic nanoparticles enhanced biosensor with the immunological separation and biochemical extraction to concentrate Campylobacter from contaminated food samples and to produce a sensitive detection technology.
Impacts
What was accomplished under these goals?
Campylobacteriosis is an important worldwide foodborne disease caused by Campylobacter. Optical based Surface Plasmon Resonance (SPR) biosensors are sensitive methods for detection of Campylobacter in foods. This project continues to develop a rapid screening method by comparing the binding characteristics for the selection of three polyclonal antibodies using SPR. This work used a SPR biosensor for the selection of three polyclonal antibodies (Invitrogen, Biorad and Prosci) against the surface antigens of three different strains of C. jejuni. Individual polyclonal antibody was immobilized on the sensor surface and the evaluation was based on the binding characteristics against the surface antigens of C. jejuni. The SPR biosensor showed the highest binding signal for Biorad polyclonal antibody against antigens of three different strains of C. jejuni in comparison to Invitrogen and Prosci polyclonal antibodies. These differences were statistically significant (p<0.05). The application of SPR in this study provided a rapid and accurate selection of fine performing antibodies for Campylobacter detection.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Shreya Singh Hamal and Fur-Chi Chen. Characterization of Campylobacter Flagellin Protein Specific Monoclonal Antibodies and Evaluation of Their Binding Affinities Using Surface Plasmon Resonance. International Association for Food Protection (IAFP) Annual Meeting, October 27, 2020. (Virtual Conference)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Shreya Singh Hamal and Fur-Chi Chen. Comparison and Screening of Polyclonal Antibodies for Detection of Campylobacter Flagellin Protein using Surface Plasmon Resonance (SPR). The 130th Annual Meeting of the Tennessee Academy of Science, November 21, 2020. (Virtual Conference)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Shreya Singh Hamal and Fur-Chi Chen. Kinetics Characterization of Campylobacter Flagellin Protein by Specific Monoclonal Antibodies Using Surface Plasmon Resonance (SPR). IFT-Volunteer Section Meeting, September 8, 2020. (Virtual Conference)
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Progress 02/15/19 to 02/14/20
Outputs
Target Audience:Students participated in the project acquired knowledge and skills of biosensors in agricultural research. Food safety professionals in the food industry and government laboratories learned the opportunity of the new technology developments. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has trained one doctoral graduate student in detection and identification of Campylobacter in foods using cultural and molecular methods. The graduate student has presented the project findings in professional conferences and research forums. How have the results been disseminated to communities of interest?The biosensor method to be developed is intended to be used by food industry and regulatory testing laboratories to facilitate identification of Campylobacter in the processing facilities and final products. The results from this project have been presented to food industry interested in the advanced detection technologies at International Association for Food Protection Annual Meeting, Louisville, KY, July 21-24, 2019. What do you plan to do during the next reporting period to accomplish the goals?This project will design a process to incorporate immunological separation and biochemical extraction as a means to concentrate Campylobacter from contaminated food samples.
Impacts
What was accomplished under these goals?
This project has identified common epitope binding sites and molecular variations of flagellin among Campylobacter species using two sets of monoclonal antibodies (MAbs). Ten strains of Campylobacter from three species namely Campylobacter jejuni, Campylobacter coli and Campylobacter fetus were cultured in Campylobacter blood-free agar (CCDA). Flagellin proteins were extracted from the cell surfaces and separated by SDS-Polyacrylamide Gel Electrophoresis. Two sets of MAbs (Groups I and V), composed of seven clones of MAbs, were used in Western blot to detect the binding patterns with respect to naturally expressed flagellin proteins. Both groups recognized flagellin proteins with molecular weights around 64 kDa and divers of flagellin fragments with molecular weights between 20 and 55 kDa. The results showed that two distinct binding patterns between Groups I and V MAbs with all tested strains. Within the same group of MAbs, the differences were also observed among the tested strains with respect to the molecular weights and relative binding intensities of flagellin fragments. The diverse patterns of Western blots from distinct strains of Campylobacter were compared using image tool analysis. The result showed that the binding patterns were unique for different strains and consistent in the subsequent cultures from the same stains. These monoclonal antibodies therefore are valuable in strain identification and immunological tests.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Shreya Singh Hamal and Fur-Chi Chen. Characterization and Analysis of Campylobacter Flagellin Protein Using a Panel of Monoclonal Antibodies. P3-62. International Association for Food Protection Annual Meeting, Louisville, KY, July 21-24, 2019
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Shreya Hamal and Fur-Chi Chen. Antigenic Diversity of Flagellin Protein of Campylobacter Species Characterized by Monoclonal Antibodies. Association of 1890 Research Directors (ARD) Research Symposium, Hyatt Regency Jacksonville, FL, March 30 - April 3, 2019
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