Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

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.

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk

“…1 , 2 , 3 and 4 should clearly demonstrate our decision-making in relation to these parameters, on the basis of conventional IS900 PCR testing immediately after PhMS. We had previously used this subjective evaluation approach to successfully optimise MS methods for MAP (Foddai et al 2010b ; O’Brien et al 2016 ) and Mycobacterium bovis (Stewart et al 2012 ). Our objective was to achieve similar or better MAP capture capability and detection sensitivity with the D29 phage-coated BcMag Tosylactivated beads as we had previously with biotinylated peptide-coated Tosylactivated Dynabeads, and we have done this (Figs.…”

Diagnostic potential of the peptide-mediated magnetic separation (PMS)-phage assay and PMS-culture to detectMycobacterium aviumsubsp.paratuberculosisin bovine milk samples

“…Previously published studies have detected 10–10 3 MAP /50 ml milk by decontamination and culture (Ayele, Svastova, Roubal, Bartos, & Pavlik, ; Giese & Ahrens, ; Sweeney, Whitlock, & Rosenberger, ) and 10 2 –10 4 MAP /50 ml milk by qPCR (Slana et al., ) or IS 900 PCR (Stabel, Bradner, Robbe‐Austerman, & Beitz, ). The PMS‐phage assay and PMS‐culture have sufficient analytical sensitivity (10–10 2 pfu/ml and 10 2 –10 3 cfu/ml, respectively; O'Brien et al., ) to be able to detect these low numbers of MAP in cow's milk. However, it is documented that MAP shedding into milk can be intermittent (Fecteau & Whitlock, ), and the number of MAP shed can vary by stage of infection, with clinical animals shedding higher numbers of MAP in their milk than subclinical animals (Stabel et al., ; Sweeney et al., ).…”

“…The PMS‐based methods employ paramagnetic beads coated with MAP ‐specific biotinylated aMp3 and aMptD peptides (Foddai, Elliot, & Grant, ) to selectively capture and concentrate low numbers of MAP cells from milk samples. The analytical specificity of the PMS assay applied to milk was previously determined to be >98%, with an analytical sensitivity for the PMS‐phage and PMS‐culture assays of 10–10 2 pfu/ml and 10 2 –10 3 cfu/ml, respectively (O'Brien, Stewart, Strain, & Grant, ). These methods have principally been developed and optimized for detection of MAP in milk.…”

Systems medicine approaches for peptide array-based protein kinase profiling: progress and prospects

“…It should, for instance, be possible to incubate different substrate peptides with clinical samples and upon subsequent extraction of the substrate peptides from the mixture (e.g. by coupling peptides to magnetic beads and subsequent magnetic separation, compare e.g [73].) the unphoshorylated levels of these peptides can be compared with the phosphorylated levels of these peptides.…”