|Yakes, Betsy - IOWA STATE UNIVERSITY|
|Lipert, Robert - IOWA STATE UNIVERSITY|
|Porter, Marc - IOWA STATE UNIVERSITY|
Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 2, 2007
Publication Date: February 1, 2008
Citation: Yakes, B.J., Lipert, R.J., Bannantine, J.P., Porter, M.D. 2008. Detection of Mycobacterium avium subsp. paratuberculosis by a Sonicate Immunoassay Based on Surface-Enhanced Raman Scattering. Clinical and Vaccine Immunology. 15(2):227-234. Interpretive Summary: This communication describes a novel way to detect Mycobacterium paratuberculosis, the bacterium that causes Johne’s disease. The assay is similar to a protein-antibody reaction used in standard diagnostic tests such as the ELISA, but the detection of this protein-antibody interaction is much more sensitive than has been described for the ELISA or other such tests. This detection is by surface-enhanced Raman scattering (SERS), which is a physical chemistry technique that employs spectroscopy. Raman labels are used which greatly enhances the signal obtained with a protein-antibody interaction. While the majority of the manuscript describes and optimizes this new test, its performance was also evaluated using whole milk spiked with the bacterium. From these experiments, we were able to quantitatively determine the numbers of organisms in the milk, something that no other test can do. These findings have great impact on milk and dairy producers and scientists working in the field.
Technical 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 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.