Detection of Mycobacterium avium subsp. paratuberculosis by a Sonicate Immunoassay Based on Surface-Enhanced Raman Scattering

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.

Paratuberculose em ruminantes no Brasil

“…É importante salientar que os testes sorológicos para diagnóstico da paratuberculose bovina podem revelar resultados falso positivos, especialmente na presença de tuberculose bovina e aviária (Timms et al 2011). Além disso, deve-se considerar as possibilidades de reações cruzadas com outros agentes como Escherichia coli (Gilot & Missone 1994apud Gomes et al 2002, Mycobacterium spp, Corynebacterium pseudotuberculosis e Nocardia spp (Yakes et al 2008, Behr & Collins 2010, devido à presença de determinantes antigênicos comuns entre os gêneros. Componentes da parede celular especialmente as glicoproteínas e lipoarabinomannan (LAM) têm sido es-tudados e utilizados como antígenos para aumentar a especificidade do teste ELISA, assim como para a elaboração de vacinas contra a paratuberculose, devido ao seu papel no processo patogênico (Stevenson & Sharp 1997, Jolly et al 2011.…”

Microspectroscopic SERS detection of interleukin-6 with rationally designed gold/silver nanoshells

“…[17][18][19][20] The benets of using MEG-OH and TEG-CO 2 H as hydrophilic spacers attached to aromatic Raman labels include the increased stability of the SERS labels due to the EG termini and the controllable bioconjugation via the MEG-OH/TEG-CO 2 H stoichiometry, together with the advantages of a SAM (maximum surface coverage with Raman reporters, uniform molecular orientation, and minimal coadsorption of other molecules from the surrounding onto the metal surface). SERS immunoassays have been reported for the detection of cell surface antigens, 21 immunoglobulin G (IgG), 8,13 protein A,…”

Design, Characterization, and Integration of Nanometric Objects with Chip-scale Platforms for Disease Diagnosis

“…The detection limit determined for total protein can thus be translated to the microorganism concentration. These findings, summarized in Figure 6, yielded an LOD of 1000 MAP/mL for sonicate spiked in whole milk, which approaches the levels measured in the subclinical stage of the disease (56). Moreover, the assay time, which includes sample preparation, antigen extraction, ERL incubation, and read out, was under 24 h.…”

“…paratuberculosis (MAP) (56), the causative agent of Johne's disease in cattle. This disease is responsible for the annual loss of several hundred million dollars worldwide in animal production.…”