Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium bovis

<p>This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate<em> Mycobacterium bovis</em> cells from lymph node tissues. Gamma-irradiated whole <em>M. bovis</em> AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce <em>M. bovis</em>-speci?c polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate <em>M. bovis</em>-speci?c peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially,<em> M. bovis</em> capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10⁵ CFU/ml) was evaluated by IMS combined with an <em>M. bovis</em>-speci?c touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for <em>M. bovis</em> in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other <em>Mycobacterium</em> spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various <em>M. bovis</em>-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of <em>M. bovis</em> from lymph nodes and hence the diagnosis of bovine tuberculosis.</p>