scholarly journals Membrane and Cytoplasmic Proteins of Mycobacterium avium subspecies paratuberculosis that Bind to Novel Monoclonal Antibodies

2018 ◽  
Vol 6 (4) ◽  
pp. 127 ◽  
Author(s):  
John Bannantine ◽  
Judith Stabel ◽  
John Lippolis ◽  
Timothy Reinhardt
Keyword(s):  
Monoclonal Antibodies ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Cell Extract ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Mass Spectrometry Analysis ◽  
Lambda Phage ◽  
Expression Library ◽  
Spectrometry Analysis

Monoclonal antibodies against Mycobacterium avium subspecies paratuberculosis (Map) proteins are important tools in Johne’s disease research and diagnostics. Johne’s disease is a chronic inflammatory intestinal disease of cattle, sheep, and other ruminant animals. We have previously generated multiple sets of monoclonal antibodies (mAbs) in different studies; however, because many were generated and screened against a whole-cell extract of Map, the antigens that bind to these antibodies remained unknown. In this study, we used three different approaches to identify the corresponding Map antigens for 14 mAbs that could not be identified previously. In the first approach, a new Map-lambda phage expression library was screened to identify corresponding antigens for 11 mAbs. This approach revealed that mAbs 7C8, 9H3, 12E4, 3G5, and 11B8 all detect MAP_3404 encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, while mAbs 7A6, 11F8, and 10C12 detect the GroEL2 chaperonin (MAP_3936), 6C9 detects electron transfer flavoprotein (MAP_3060c), and 14G11 detects MAP_3976, a lipoprotein anchoring transpeptidase. The epitopes to a selection of these mAbs were also defined. In a second approach, MAP_2698c bound monoclonal antibody (mAb) 14D4 as determined using protein arrays. When both of these approaches failed to identify the antigen for mAb 12C9, immunoprecipitation, mass spectrometry analysis, and codon optimization was used to identify the membrane protein, MAP_4145, as the reacting antigen. Characterized antibodies were used to quickly interrogate mycobacterial proteomic preps. We conclude by providing a complete catalog of available mAbs to Map proteins, along with their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes.

scholarly journals Development and Characterization of Monoclonal Antibodies and Aptamers against Major Antigens of Mycobacterium avium subsp. paratuberculosis

2007 ◽  
Vol 14 (5) ◽  
pp. 518-526 ◽  
Author(s):  
John P. Bannantine ◽  
Thomas J. Radosevich ◽  
Judith R. Stabel ◽  
Srinand Sreevatsan ◽  
Vivek Kapur ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Mycobacterium Avium ◽  
Isocitrate Lyase ◽  
Hypothetical Protein ◽  
Cell Extract ◽  
Cross Reactivity ◽  
Cell Protein ◽  
Lambda Phage ◽  
Expression Library ◽  
Whole Cell

ABSTRACT Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. avium subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.

Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

2020 ◽  
Author(s):  
Manju Singh ◽  
Saurabh Gupta ◽  
Shoor Vir Singh ◽  
Gururaj Kumaresan ◽  
Deepansh Sharma ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Lamp Assay ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Isothermal Amplification ◽  
Assay Method ◽  
Loop Mediated Isothermal Amplification ◽  
Is900 Pcr

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

scholarly journals Mycobacterium avium subspecies paratuberculosis: An Emerging Bacterial Disease of Global Public Health Significance

2016 ◽  
Vol 4 (1) ◽  
pp. 4-13
Author(s):  
Mahendra Pal ◽  
Md Tanvir Rahman
Keyword(s):  
Public Health ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Economic Losses ◽  
Public Health Significance ◽  
Health Significance

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of chronic enteric disease of ruminant known as paratuberculosis (Johne's disease). The disease causes considerable economic losses worldwide due to reduced milk production and eventually, diarrhoea, weight loss and death. Johne's disease (JD) has some pathological similarities with Crohn's disease (CD) in humans, and the role of MAP in the causation of CD has been under investigation for last 100 years. Animals infected with JD shed viable MAP in the blood, and tissues. Consequently, transmission to humans may occur via consumption of animal derived foods. In developing countries, limited information is available on the occurrence of MAP infection in animals and humans. MAP infection has been established in animals and humans may get the MAP exposure through food chain or contaminated environment. Presently, MAP is of great public health significance because it is speculated to be involved in Crohn's disease in humans. The present review summarizes the information primarily on the nature of MAP in animals and humans, economic losses and morbidity and mortality due to JD and CD at global level. Current concept on the possible relationship between MAP and Crohn's disease has also been reviewed.Microbes and Health, January 2015. 4(1): 4-13

Sign in / Sign up

Export Citation Format

Share Document