Detection of Mycobacterium avium subsp. paratuberculosis using Surface-Enhanced Raman Scattering: Part II – Whole Cell Immunoassay and Impact of Protein Shedding

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

The etiological agent of Johne's disease is Mycobacterium avium subspecies paratuberculosis (MAP). Controlling the spread of this disease, however, is hindered by the lack of sensitive, selective, and rapid detection methods for MAP. By using a recently optimized sandwich immunoassay (B. J. Yakes, R. J. Lipert, J. P. Bannantine, M. D. Porter, Clin. Vaccine Immunol., in press), which incorporates a new monoclonal antibody for the selective capture and labeling of MAP and surface-enhanced Raman scattering (SERS) for sensitive readout, detection limits of 630 and 740 MAP/ml are respectively achieved in phosphate buffered saline and whole milk samples after spiking with heat-treated MAP. Surprisingly, these detection limits are three orders of magnitude lower than expected based 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 functions as a facile amplification strategy for lowering the limit of detection for MAP that may also be applicable to the design of a wide range of highly sensitive assays for other cells and viruses.