Skip to main content
ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Publications at this Location » Publication #215443
Title: Impact of Protein Shedding on Detection of Mycobacterium avium subsp. paratuberculosis by a Whole-Cell Immunoassay Incorporating Surface-Enhanced Raman Scattering

Author
item YAKES, BETSY - IOWA STATE UNIVERSITY
item LIPERT, ROBERT - IOWA STATE UNIVERSITY
item Bannantine, John
item PORTER, MARC - IOWA STATE UNIVERSITY

Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/29/2007
Publication Date: 2/1/2008
Citation: 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. Clinical and Vaccine Immunology. 15(2):235-242.

Interpretive Summary: This communication extends upon a novel way to detect Mycobacterium paratuberculosis, the bacterium that causes Johne’s disease. Raman labels are a part of this novel spectroscopy test which greatly enhances the signal obtained with a protein-antibody interaction. Using the new, very sensitive technique called surface-enhanced Raman scattering (SERS), we were actually able to detect the bacterium at levels below the theoretical threshold limit of the test itself! Upon further evaluation and experimentation, we discovered the reason for this paradox was that the bacterial protein was being shed from each bacterium, effectively amplifying the possible number of protein-antibody interactions and thus the signal as well. This novel finding has broad application to virtually all protein-antibody based tests.

Technical 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., submitted for publication) which incorporates a newly prepared monoclonal antibody for MAP and surface-enhanced Raman scattering (SERS) for sensitive readout, detection limits of ~630 and ~740 MAP/ml are achieved in phosphate buffered saline and whole milk samples, respectively, after spiking with heat-treated MAP. Surprisingly, these detection limits are much lower than that expected based on a simple theoretical model for the assay. Experiments reveal the presence of shed protein in this assay and indicate that this mechanism can be used for the design of a highly sensitive assay for MAP, which can potentially be applied to a wide range of other cell and virus assays.