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ABSTRACTThe etiological agent of Johne's disease is Mycobacterium aviumsubsp. paratuberculosis. Controlling the spread of this disease is hindered by the lack of sensitive, selective, and rapid detection methods for M. aviumsubsp. 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. aviumsubsp. paratuberculosisand surface-enhanced Raman scattering for sensitive readout, detection limits of ~630 and ~740 M. aviumsubsp. paratuberculosiscells/ml are achieved in phosphate-buffered saline and whole milk samples, respectively, after spiking with heat-treated M. aviumsubsp. 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. aviumsubsp. paratuberculosisthat may also be applicable to the design of a wide range of highly sensitive assays for other cells and viruses.

ABSTRACTA sandwich immunoassay for the rapid, low-level detection of Mycobacterium aviumsubsp. paratuberculosishas been developed. M. aviumsubsp. paratuberculosisis 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. aviumsubsp. paratuberculosislysate 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. aviumsubsp. paratuberculosisbacilli 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. aviumsubsp. paratuberculosisbacilli/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.