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Impact of Protein Shedding on Detection of Mycobacterium avium subsp. paratuberculosis by a Whole-Cell Immunoassay Incorporating Surface-Enhanced Raman Scattering

Betsy Jean Yakes,^1, Robert J. Lipert,¹ John P. Bannantine,² and Marc D. Porter^1*

Departments of Chemistry and Chemical and Biological Engineering, Ames Laboratory-USDOE, and Institute for Combinatorial Discovery, Iowa State University, Ames, Iowa 50011,¹ USDA/ARS/National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, Ames, Iowa 50010²

Received 14 August 2007/ Returned for modification 11 October 2007/ Accepted 29 November 2007

The etiological agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis. Controlling the spread of this disease is hindered by the lack of sensitive, selective, and rapid detection methods for M. avium subsp. paratuberculosis. By using a recently optimized sandwich immunoassay (B. J. Yakes, R. J. Lipert, J. P. Bannantine, and M. D. Porter, Clin. Vaccine Immunol. 15:227-234, 2008), which incorporates a new monoclonal antibody for the selective capture and labeling of M. avium subsp. paratuberculosis and surface-enhanced Raman scattering for sensitive readout, detection limits of 630 and 740 M. avium subsp. paratuberculosis cells/ml are achieved in phosphate-buffered saline and whole milk samples, respectively, after spiking with heat-treated M. avium subsp. paratuberculosis. Surprisingly, these detection limits are 3 orders of magnitude lower than expected based on theoretical predictions. Experiments designed to determine the origin of the improvement revealed that the major membrane protein targeted by the monoclonal antibody was present in the sample suspensions as shed protein. This finding indicates that the capture and labeling of shed protein function as a facile amplification strategy for lowering the limit of detection for M. avium subsp. paratuberculosis that may also be applicable to the design of a wide range of highly sensitive assays for other cells and viruses.

Published ahead of print on 12 December 2007.

Present address: U.S. FDA-CFSAN, 5100 Paint Branch Parkway, College Park, MD 20740.

This article has been cited by other articles:

• Yakes, B. J., Lipert, R. J., Bannantine, J. P., Porter, M. D. (2008). Detection of Mycobacterium avium subsp. paratuberculosis by a Sonicate Immunoassay Based on Surface-Enhanced Raman Scattering. CVI 15: 227-234 [Abstract] [Full Text]