Affiliation:
1. National Animal Disease Center, USDA-ARS, Ames, Iowa
2. Veterinary Population Medicine Department
3. Departments of Microbiology and Biomedical Genomics, University of Minnesota, St. Paul, Minnesota
Abstract
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.
Publisher
American Society for Microbiology
Subject
Microbiology (medical),Clinical Biochemistry,Immunology,Immunology and Allergy
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