Impact of Protein Shedding on Detection of
            <i>Mycobacterium avium</i>
            subsp.
            <i>paratuberculosis</i>
            by a Whole-Cell Immunoassay Incorporating Surface-Enhanced Raman Scattering

Yakes, Betsy Jean; Lipert, Robert J.; Bannantine, John P.; Porter, Marc D.

doi:10.1128/cvi.00335-07

Cited by 25 publications

(17 citation statements)

References 58 publications

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“…It is difficult to extrapolate these results to the single-cell level for mycoplasmas, which tend to clump and for which CFU and plating efficiency are poorly defined [3] . Furthermore, we also cannot rule out the possibility that intact cells are not essential, and the ability to detect M. pneumoniae spectra may fall below the single-cell level due to recognition by SERS of cell components in cell lysates, as was reported recently for mycobacteria [41] .…”

Section: Resultsmentioning

confidence: 83%

Detection of Mycoplasma pneumoniae in Simulated and True Clinical Throat Swab Specimens by Nanorod Array-Surface-Enhanced Raman Spectroscopy

et al. 2010

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The prokaryote Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The limitations of existing options for mycoplasma diagnosis highlight a critical need for a new detection platform with high sensitivity, specificity, and expediency. Here we evaluated silver nanorod arrays (NA) as a biosensing platform for detection and differentiation of M. pneumoniae in culture and in spiked and true clinical throat swab samples by surface-enhanced Raman spectroscopy (SERS). Three M. pneumoniae strains were reproducibly differentiated by NA-SERS with 95%–100% specificity and 94–100% sensitivity, and with a lower detection limit exceeding standard PCR. Analysis of throat swab samples spiked with M. pneumoniae yielded detection in a complex, clinically relevant background with >90% accuracy and high sensitivity. In addition, NA-SERS correctly classified with >97% accuracy, ten true clinical throat swab samples previously established by real-time PCR and culture to be positive or negative for M. pneumoniae. Our findings suggest that the unique biochemical specificity of Raman spectroscopy, combined with reproducible spectral enhancement by silver NA, holds great promise as a superior platform for rapid and sensitive detection and identification of M. pneumoniae, with potential for point-of-care application.

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Section: Resultsmentioning

confidence: 83%

Detection of Mycoplasma pneumoniae in Simulated and True Clinical Throat Swab Specimens by Nanorod Array-Surface-Enhanced Raman Spectroscopy

et al. 2010

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“…paratuberculosis 35,36 ), and small molecules (i.e., a Vitamin D metabolite). 37 This paper describes the development of SERS-based sandwich immunoassays for the detection of MUC4 and CA19-9 and their application to the first ever measurement of MUC4 in patient sera.…”

mentioning

confidence: 99%

Detection of the Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering

Wang¹,

Lipert²,

Jain³

et al. 2011

Anal. Chem.

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227

159

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Pancreatic cancer (PC) is one of the most lethal malignancies. It has a 5-year survival rate of only 6%, owing in part to the lack of a reliable tumor marker for early diagnosis. Recent research has shown that the mucin protein MUC4 is aberrantly expressed in pancreatic adenocarcinoma cell lines and tissues but is undetectable in normal pancreas and chronic pancreatitis. Thus, the level of MUC4 in patient sera has the potential to function as a diagnostic and prognostic marker for PC. However, the measurement of MUC4 in sera using conventional test platforms (e.g., enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)) has been unsuccessful. This has prevented the assessment of the utility of this protein as a possible PC marker in sera. In addressing this obstacle, the work herein examines the potential to create a simple diagnostic test for MUC4 through the development of a surface-enhanced Raman scattering (SERS)-based immunoassay, which was then used to demonstrate the first ever detection of MUC4 in cancer patient serum samples. Importantly, these measurements showed that sera from patients with PC produced a significantly higher SERS response for MUC4 compared to sera from healthy individuals and from patients with benign diseases. These results indicate that a SERS-based immunoassay can monitor MUC4 levels in patient sera, representing a much needed first step toward assessing the potential of this protein to serve as a serum marker for the early stage diagnosis of PC. This paper details these and other findings (i.e., the detection of the mucin protein CA19-9), which demonstrate that our SERS assay outperforms conventional assays (i.e., RIA and ELISA) with respect to limits of detection, readout time, and required sample volume.

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“…While it has previously been indicated that MMP contains a 30 amino acid hydrophobic domain near the C-terminal end suggestive of a membrane protein, the protein lacks a discernible N-terminal signal sequence (Bannantine et al, 2003 ). Moreover, it has already been experimentally demonstrated that MMP is both surface exposed and actively shed from live MAP bacilli (Yakes et al, 2008 ), suggesting that MMP is translocated in a non-classical manner similar to other mycobacterial proteins (Pallen, 2002 ; Bendtsen et al, 2005 ).…”

Section: Resultsmentioning

confidence: 99%

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

Johnston

Bannantine

Govender

et al. 2014

Front. Cell. Infect. Microbiol.

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It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

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Impact of Protein Shedding on Detection of Mycobacterium avium subsp. paratuberculosis by a Whole-Cell Immunoassay Incorporating Surface-Enhanced Raman Scattering

Cited by 25 publications

References 58 publications

Detection of Mycoplasma pneumoniae in Simulated and True Clinical Throat Swab Specimens by Nanorod Array-Surface-Enhanced Raman Spectroscopy

Detection of Mycoplasma pneumoniae in Simulated and True Clinical Throat Swab Specimens by Nanorod Array-Surface-Enhanced Raman Spectroscopy

Detection of the Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

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