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Clinical and Vaccine Immunology, February 2008, p. 227-234, Vol. 15, No. 2
1071-412X/08/$08.00+0     doi:10.1128/CVI.00334-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Detection of Mycobacterium avium subsp. paratuberculosis by a Sonicate Immunoassay Based on Surface-Enhanced Raman Scattering{triangledown}

Betsy Jean Yakes,1,{dagger} Robert J. Lipert,1 John P. Bannantine,2 and Marc D. Porter1*

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 13 August 2007/ Returned for modification 5 September 2007/ Accepted 2 November 2007

A sandwich immunoassay for the rapid, low-level detection of Mycobacterium avium subsp. paratuberculosis has been developed. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and one of the major obstacles in controlling the spread of this disease is the inability to rapidly detect small amounts of bacteria or other diagnostic markers shed during the subclinical stage of infection. This paper details the development and performance of an assay for sonicated M. avium subsp. paratuberculosis lysate that is based on surface-enhanced Raman scattering (SERS). There are two key components of the assay: (i) an immobilized layer of monoclonal antibodies that target a surface protein on the microorganism; and (ii) extrinsic Raman labels (ERLs) that are designed to selectively bind to captured proteins and produce large SERS signals. By correlating the number of M. avium subsp. paratuberculosis bacilli present prior to sonication to the amount of total protein in the resulting sonicate, the detection limit determined for total protein can be translated to the microorganism concentration. These findings yield detection limits of 100 and 200 ng/ml (estimated to be 500 and 1,000 M. avium subsp. paratuberculosis bacilli/ml) for sonicate spiked in phosphate buffer and sonicate spiked in whole milk, respectively. Moreover, the time required to complete the assay, which includes sample preparation, antigen extraction, ERL incubation, and readout, is less than 24 h. The potential for incorporation of this novel assay into diagnostic laboratories is also briefly discussed.

* Corresponding author. Present address: Department of Chemistry, University of Utah, Salt Lake City, UT 84108. Phone: (801) 587-1505. Fax: (801) 585-0575. E-mail: marc.porter{at}utah.edu

{triangledown} Published ahead of print on 12 December 2007.

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

Clinical and Vaccine Immunology, February 2008, p. 227-234, Vol. 15, No. 2
1071-412X/08/$08.00+0     doi:10.1128/CVI.00334-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yakes, B. J., Lipert, R. J., Bannantine, J. P., Porter, M. D. (2008). Impact of Protein Shedding on Detection of Mycobacterium avium subsp. paratuberculosis by a Whole-Cell Immunoassay Incorporating Surface-Enhanced Raman Scattering. CVI 15: 235-242 [Abstract] [Full Text]  


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