Abstract
The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.
Highlights
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent for Johne’s disease, a highly-infectious wasting disease that affects a range of domestic ruminants including cattle, sheep, goats and deer [1]
Mice immunised with MAP whole cell antigens (WCA) and extracted antigens (EEA) elicited immune responses after just two immunisations, evidenced by binding of whole MAP cells by direct and competitive ELISA
Monoclonal antibody cell lines 6G11 from the WCA fusion and 15D10 from the EEA fusion were selected for further evaluation based on their competitive ELISA results, with ~18% and ~66% binding inhibition respectively (% binding inhibition was calculated by subtracting the % binding obtained in the presence of ‘free MAP cells’ from 100% binding observed in the negative control wells; % binding refers to antibody bound to attached MAP cells on the well surface)
Summary
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent for Johne’s disease, a highly-infectious wasting disease that affects a range of domestic ruminants including cattle, sheep, goats and deer [1]. Some infected animals may be asymptomatic and not show any clinical signs of infection, both symptomatic and asymptomatic animals can shed MAP in their milk and faeces, thereby constituting an infectious risk to susceptible animals which typically acquire infection through the ingestion of MAP or MAP contaminated material [3, 4, 5] Often, these sub-clinical animals outnumber clinically infected animals within a herd, and so their rapid identification is key to controlling within-herd transmission of Johne’s disease [4]. In order to avoid this, molecular-based methods have commonly been employed for the detection of MAP [7, 10, 11] While these methods are much more rapid and often more sensitive, they are typically unable to assess the viability of the MAP cells, which is important when identifying the infection status of an animal or herd
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