Maximizing capture efficiency and specificity of magnetic separation for Mycobacterium avium subsp. paratuberculosis cells

Abstract

In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (<1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 10(3) to 10(4) CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD(50)) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h.

FIG. 1.

Initial evaluation of the performance of six different in-house-coated or commercially available paramagnetic beads for magnetic separation applied to 1-ml aliquots of Middlebrook 7H9-OADC broth containing 10³ to 10⁴ CFU Mycobacterium sp./ml in terms of the mean efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis (A) and the mean percentage of nonspecific recovery of other Mycobacterium spp. (B). Methods A and B, M280 sheep anti-rabbit IgG Dynabeads coated with polyclonal antibody S624 and a 1:10 dilution of polyclonal antibody S624, respectively; method C, Pathatrix PM50 beads coated with a polyclonal antibody (Matrix Microscience, Newmarket, United Kingdom); method D, AnDiaTec beads coated with a monoclonal antibody (AnDiaTec GmbH, Kornwestheim, Germany); methods E and F, Pierce MagnaBind carboxyl derivatized beads carbodiimide linked with aMp3 and aMptD, respectively; method G, uncoated Pierce MagnaBind carboxyl derivatized beads.

FIG. 2.

Improved mean capture efficiency of M. avium subsp. paratuberculosis achieved by using additional paramagnetic bead-coating antigen combinations for magnetic separation applied to 1-ml aliquots of Middlebrook 7H9 broth containing 10³ to 10⁴ CFU/ml. Beads 1 to 3, amine-coated magnetic hollow glass beads coated with polyclonal antibody S624, aMp3, and aMptD, respectively; beads 4 to 6, MyOne carboxylic acid-activated Dynabeads coated with polyclonal antibody S624, aMp3, and aMptD, respectively; beads 7 and 8, MyOne streptavidin T1 Dynabeads coated with biotinylated aMp3 and biotinylated aMptD, respectively; beads 9 and 10, M280 streptavidin Dynabeads coated with biotinylated aMp3 and biotinylated aMptD, respectively; beads 11 to 14, MyOne Tosylactivated Dynabeads coated with polyclonal antibody S624, aMp3, biotinylated aMp3, and biotinylated aMptD, respectively; bead 15, a 50:50 mixture of beads 13 and 14.

FIG. 3.

Schematic illustrating the predicted binding between the tosyl group on the surfaces of MyOne Tosylactivated Dynabeads and the M. avium subsp. paratuberculosis-specific peptides (aMp3, biotinylated aMp3, and biotinylated aMptD). Dashed lines indicate the supposed areas of interaction for binding with M. avium subsp. paratuberculosis in broth or milk samples.