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

ABSTRACTThis study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium boviscells from lymph node tissues. Gamma-irradiated whole M. bovisAF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific 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, M. boviscapture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 105CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovisin 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 Mycobacteriumspp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-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 M. bovisfrom lymph nodes and hence the diagnosis of bovine tuberculosis.