Affiliation:
1. Departments of Chemistry and Chemical and Biological Engineering, Ames Laboratory-USDOE, and Institute for Combinatorial Discovery, Iowa State University, Ames, Iowa 50011
2. USDA/ARS/National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, Ames, Iowa 50010
Abstract
ABSTRACT
A sandwich immunoassay for the rapid, low-level detection of
Mycobacterium avium
subsp.
paratuberculosis
has been developed.
M. avium
subsp.
paratuberculosis
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
M. avium
subsp.
paratuberculosis
lysate 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. avium
subsp.
paratuberculosis
bacilli 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. avium
subsp.
paratuberculosis
bacilli/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.
Publisher
American Society for Microbiology
Subject
Microbiology (medical),Clinical Biochemistry,Immunology,Immunology and Allergy
Cited by
73 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献