Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The primary goal of this proposal is to identify novel and specific antigens for future use in developing a diagnostic test for Johne�s disease. Approach (from AD-416) To accomplish this goal, three specific aims are proposed. In aim #1, we will construct a 384-spot protein array, the largest of its kind in the world. Aim #2 will focus on using the array to screen sera from infected cattle and aim #3 will evaluate a selection of proteins in a different immunological assay entitled the gamma interferon assay. An additional 92 MAP proteins were evaluated during this fiscal year. Humoral immune responses against these proteins were analyzed for fifteen dairy cows at various stages of infection with Johne�s disease (control, subclinical, and clinical). Some proteins generated significantly higher responses than other proteins in clinical cows, indicating specific proteins that could be useful in developing improved diagnostic tests for Johne�s disease in animals at mid to late stages of disease. Among these are MAP0184c, MAP0392c, MAP0016c and MAP1272c. 11 of the 92 proteins in this project had high responses in both protein immunoblots and interferon-gamma (IFN-g) responses, and are promising as potential diagnostic test candidates. Further analysis of these proteins in additional animals is needed to strengthen the statistics and conclusions about their antigenicity. Monitoring of progress and activities were via quarterly conference calls, weekly to monthly email exchanges, and occasional face to face visits at national meetings. Final project summary The project is now completed and over 400 proteins were evaluated for antibody responses using a protein array platform. While the 384-well array initially proposed failed because of the nature of protein solutions and small bore hole on the custom-made 384-well array mold, we were able to overcome this with several 96-well arrays. Furthermore, a set of 92 proteins were evaluated by IFN-g responses in the final year of the project. A few of the best candidate proteins were evaluated in competition with a commercially available ELISA test, but our ELISA test performed slightly better (Bannantine et al., 2011). In conclusion, while solid candidate antigens were uncovered in this project, thus far, no single antigen is better than what is commercially available. Nonetheless, there is another 3,000-plus candidate proteins that could still provide antigens for development of better diagnostic tests.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The primary goal of this proposal is to identify novel and specific antigens for future use in developing a diagnostic test for Johne�s disease. Approach (from AD-416) To accomplish this goal, three specific aims are proposed. In aim #1, we will construct a 384-spot protein array, the largest of its kind in the world. Aim #2 will focus on using the array to screen sera from infected cattle and aim #3 will evaluate a selection of proteins in a different immunological assay entitled the gamma interferon assay. A custom-made 384-well vacuum manifold was produced in order to arrange proteins on a solid nitrocellose support; however, pilot studies using this equipment failed due to the inability to obtain reproducible spotting of the proteins. The problem was discovered when evaluating the spotting process. Tiny bubbles formed in the small wells, blocking the delivery of solution containing defined amounts of protein. These bubbles also resisted vacuum forces to apply the solution to the solid support. Therefore, a commercially available 96-well vacuum manifold with a wider bore was used in order to obtain some data for the project. A set of 96 recombinant proteins, which have not been previously analyzed, were spotted and probed with Johne�s disease cattle sera obtained from a standardized sample repository at United States Department of Agriculture- National Veterinary Services Laboratory (NVSL), (a Johne's Disease Integrated Program core function). This serum set is used in proficiency testing by the NVSL. Three proteins were identified from this set to be excellent antigen candidates. More proteins remain to be evaluated using the same approach. Progress and activities were reported through a single web conference to the funding body. This web conference enabled the sharing of data and results in a seminar-based format.
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