Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/18/2008
Publication Date: 4/18/2008
Citation: Yakes, B.J., Lipert, R.J., Bannantine, J.P., Porter, M.D. 2008. Detection of Mycobacterium avium subsp. paratuberculosis by a Heterogeneous Surface-Enhanced Raman Scattering Immunoassay [abstract]. Johne's Disease Integrated Program. p. 21. Interpretive Summary:
Technical Abstract: The etiological agent of Johne’s disease in cattle is Mycobacterium avium subsp. paratuberculosis (MAP). Controlling the spread of this disease is hindered by the lack of sensitive selective, and rapid detection methods for MAP. This presentation details the development and performance of an assay for MAP 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 selectively target a surface protein on the microorganism; and (ii) extrinsic Raman labels (ERLs) that are designed to selectively bind to captured MAP and provide large SERS signals. The time required to complete the assay, which includes sample preparation, antigen extraction, ERL incubation, and readout, is less than 24 h. Through the optimization of this sandwich immunoassay technique,a detection limit of ~MAP/ml is achieved in MAP-spiked phosphate-buffered saline and whole milk samples. Surprisingly, this level of performance is about three 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 selective assays for other cells and viruses. The potential for incorporation of this novel assay into diagnostic laboratories is also briefly discussed.