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Clin. Vaccine Immunol. doi:10.1128/CVI.00334-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Detection of Mycobacterium avium subsp. paratuberculosis using Surface-Enhanced Raman Scattering: Part I – Sonicate Immunoassay

Departments of Chemistry and Chemical and Biological Engineering, Ames Laboratory-USDOE, and Institute for Combinatorial Discovery, Iowa State University, Ames, IA 50011; USDA/ARS/National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, Ames, IA 50010

^* To whom correspondence should be addressed. Email: marc.porter{at}utah.edu.

	Abstract

A sandwich immunoassay is developed for the rapid, low-level detection of Mycobacterium avium subsp. paratuberculosis (MAP). MAP 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 MAP lysate that is based on surface-enhanced Raman scattering (SERS). There are two key components of the assay: (1) an immobilized layer of monoclonal antibodies that target a surface protein on the microorganism; and (2) extrinsic Raman labels (ERLs) that are designed to selectively bind to captured proteins and produce large SERS signals. By correlating the number of MAP bacilli present prior to sonication and 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 at 500 and 1000 MAP/ml) for sonicate spiked in phosphate buffer and in whole milk, respectively. Moreover, the time required to complete the assay, which includes sample preparation, antigen extraction, ERL incubation, and read out, is less than 24 h. The potential for incorporation of this novel assay into diagnostic laboratories is also briefly discussed.

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]