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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
Development of an improved ELISA test for detection of paratuberculosis infection in body fluids in cattle and other ruminants.

1b.Approach (from AD-416)
Eleven (11) of the most promising recombinant M. paratuberculosis proteins (MAP0865, MAP0857c, MAP3817c, MAP3155c, MAP2077c, MAP3761c, MAP4014, MAP2166c, MAP3735c, MAP0900 and MAP1272c) will be evaluated by ELISA using a set of well-defined Johne’s disease-infected cattle sera as well as sera from control cattle. Additional recombinant proteins may be selected and screened based on preliminary data on antigenicity and specificity. Methodologies that will be used in this project include genomic and bioinformatics analysis techniques, immunological assays (Western blot and ELISA), and molecular cloning, as well as recombinant protein expression and purification, diagnostic test development and optimization. Proprietary PRIONICS ELISA technology will be used in this project, as well as a process for mass production of recombinant proteins developed by ARS.

3.Progress Report

The sole objective of this CRADA is to develop an improved ELISA for detection of paratuberculosis infection in body fluids of cattle and other ruminants. To accomplish this objective, eleven of the most promising Mycobacterium avium subspecies paratuberculosis recombinant proteins, developed by Agriculture Research Services, were transferred to Prionics for testing using their proprietary ELISA technology. Sera from known negative (healthy) cows as well as sera from Johne’s disease infected cows were used to test the specificity and sensitivity of the proteins. The data from Prionics show that while MAP1272c is the best of the eleven antigens, originally selected from 96 proteins, it still is not quite as good as the antigen used in Prionics proprietary antigen cocktail. ARS has repeated Prionics findings. The project was closed prior to completing the testing of additional proteins. Progress was monitored by emails exchanging data and interpretation of data, five teleconferences and one face-to-face meeting in Minneapolis, Minnesota.

Last Modified: 1/30/2015
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