Production and Characterization of Monoclonal Antibodies against a Major Membrane Protein of<i>Mycobacterium avium</i>subsp.<i>paratuberculosis</i>

Bannantine, John P.; Radosevich, Thomas J.; Stabel, Judith R.; Berger, Sven; Griffin, J.F.T.; Paustian, Michael L.

doi:10.1128/cvi.00353-06

Cited by 15 publications

(25 citation statements)

References 29 publications

(26 reference statements)

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“…avium-infected cow 6137 (10 10 CFU of strain 3988 split between the tonsillar crypts) were also diluted 1:500 (Table 2). A monoclonal antibody to the MBP affinity tag (diluted 1:3,000) was developed at the National Animal Disease Center and has been used in previous studies (6).…”

Section: Mycobacterial Antigen Preparationmentioning

confidence: 99%

Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays

et al. 2008

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With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n ‫؍‬ 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n ‫؍‬ 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n ‫؍‬ 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis.

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Section: Mycobacterial Antigen Preparationmentioning

confidence: 99%

Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays

et al. 2008

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“…In this study, ELISA analysis was performed using two monoclonal antibodies specific to the MMP protein (8G2 and 13E1), which detected two distinct epitopes (Bannantine et al, 2007). …”

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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“…The lack of antibody binding to M. bovis may be surprising given the high similarity of NlpC/P60 domain-containing proteins in Table 1; however, this has been observed with other MAbs generated against M. avium subsp. paratuberculosis proteins (4) and there are 54 amino acid differences between these species for NlpC. Both of these antibodies demonstrated the variable size of the NlpC protein as expressed from mycobacterial species.…”

Section: Discussionmentioning

confidence: 99%

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

Bannantine

Lingle

Stabel

et al. 2012

Clin. Vaccine Immunol.

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The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.

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Production and Characterization of Monoclonal Antibodies against a Major Membrane Protein ofMycobacterium aviumsubsp.paratuberculosis

Cited by 15 publications

References 29 publications

Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays

Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays

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

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

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