Fig. 1
Mycobacterium bovis cells captured by monoclonal antibody and biotinylated 12-mer peptide-coated Dynabeads® MyOne™ Tosylactivated—a large clump of M. bovis cells and an individual cell are clearly seen attached to the beads. Bead suspension after IMS was stained by auramine O fluorescent acid-fast stain
2 Materials
1.
Purified mouse anti-Mycobacterium bovis IgM monoclonal antibody (11G3) (see Note 1).
2.
N-terminally biotinylated 12-mer peptide (EEA302) with amino acid sequence NFRVSIDVVKSR (10 mg/ml in sterile distilled water), custom synthesized by any peptide synthesis company.
3.
Dynabeads® MyOne™ Tosylactivated (Life Technologies): bead diameter 1.08 μm, bead concentration 100 mg beads/ml (approx. 1012 beads/g), surface area 8 m2/g (see Note 2).
4.
Coating buffer: 0.1 M sodium borate buffer pH 9.5, prepared by dissolving 6.183 g H3BO3 (MW 61.83) in 800 ml distilled water, adjusting pH to 9.5 using 5 M NaOH and then adjusting volume to 1,000 ml with distilled water.
5.
3 M ammonium sulfate: prepared in coating buffer by dissolving 39.6 g (NH4)2SO4 (MW 132.1) in 0.1 M sodium borate (pH 9.5), adjusting pH (if necessary) and then adjusting volume to 100 ml.
6.
Phosphate buffered saline pH 7.4 (PBS): 0.01 M phosphate buffered saline composed of 0.138 M sodium chloride and 0.0027 M potassium chloride, pH adjusted to 7.4.
7.
Washing and storage buffer for beads (PBS/BSA/T20): phosphate buffered saline pH 7.4 (PBS) with 0.1 % (w/v) bovine serum albumin and 0.05 % (w/v) Tween 20 added.
8.
Wash buffer for IMS (PBS/T20): phosphate buffered saline pH 7.4 (PBS) with 0.05 % (w/v) Tween 20 added.
9.
Tris-EDTA buffer (pH 8.0): 10 mM Tris–HCl and 1 mM disodium EDTA, pH adjusted to 8.0.
10.
Reagents for conventional PCR reactions (for post-IMS detection of M. bovis by PCR): DNA polymerase enzyme and respective 10× buffer, primers INS1 (5′-cgt gag ggc atc gag gtg gc-3′) and INS2 (5′-gcg tag gcg tcg gtg aca aa-3′) [11], MgCl2 (25 mM), dNTPs (25 mM), and distilled PCR-grade water. In this protocol, a PCR SuperMix is employed for PCR reactions (Platinum® Blue PCR SuperMix, Life Technologies).
11.
BBL™ MGIT™ culture tubes for M. bovis previously supplemented with BBL™ MGIT™ OADC and BBL™ MGIT™ PANTA supplements (Becton Dickinson) (for post-IMS detection of M. bovis by culture).
12.
Equipment and other supplies: centrifuge; magnetic rack; Stuart rotator mixer (or similar); Dynal BeadRetriever™ and respective tube and tip strips (Life Technologies) (not necessary if using the manual IMS procedure); thermal cycler, electrophoresis apparatus, and UV transilluminator (for post-IMS detection of M. bovis by PCR); MGIT 960 instrument (Becton Dickinson) (for post-IMS detection of M. bovis by culture); sterile mortar, sterile sand, and pestle.
3 Methods
3.1 Coating Dynabeads® MyOne™ Tosylactivated with Phage Display-Derived Peptide and Monoclonal IgM Antibody
This coating procedure is essentially as described in the pack insert accompanying Dynabeads® MyOne™ Tosylactivated (see Note 2).
1.
Transfer 250 μl of uncoated Dynabeads® MyOne™ Tosylactivated to a sterile microcentrifuge tube.
2.
Wash beads twice with 1 ml of coating buffer, separating on a magnetic rack for 2 min between washes.
3.
Resuspend beads in 100 μl of coating buffer and vortex thoroughly.
4.
Premix 50 μl of N-terminally biotinylated 12-mer peptide and 50 μl of purified mouse anti-M. bovis monoclonal IgM antibody (see Notes 1 and 3); add to activated beads and mix by vortexing.
5.
Add a further 735 μl of coating buffer and mix again by vortexing.
6.
Add 415 μl of 3 M ammonium sulfate and mix by vortexing.
7.
Incubate overnight at 37 °C with “end-to-end” mixing on a Stuart rotator mixer (or similar) at 10 rpm.
8.
Wash freshly coated beads twice with 1 ml of washing buffer, separating on a magnetic rack for 2 min between washes.
9.
Resuspend coated beads in 500 μl storage buffer, which is twice the original volume of uncoated beads used. This final volume is sufficient for 50 IMS reactions when 10 μl of coated beads are used per 1 ml test sample.
10.
The Dynabeads® MyOne™ Tosylactivated are now coated and ready for use. They should be stored at 2–8 °C until required (see Note 4).
3.2 Lymph Node Preparation for IMS
For health and safety reasons all procedures involving bovine tissues containing, or potentially containing, M. bovis must be carried out in a class 1 biological safety cabinet located in a containment level 3 laboratory facility.
1.
Transfer approx. 3 g of cubed lymph node material to a sterile mortar, add sterile sand, and grind thoroughly with pestle.
2.
Add 4.5 ml of sterile phosphate buffered saline pH 7.4 (PBS) and grind further (=1:1.5 dilution).
3.
Transfer sample to centrifuge tube and centrifuge at 300 × g for 3 min to sediment sand and tissue particulates.
4.
Make a 1:10 dilution of clarified lymph node tissue supernatant in sterile PBS (see Note 5) and use 1 ml of this dilution (=1:15 dilution of original lymph node sample) for immunomagnetic separation (IMS).
3.3 Immunomagnetic Separation
3.3.1 Automated Immunomagnetic Separation (IMS) Using Dynal BeadRetriever™
1.
Add 10 μl of dually coated (Mab 11G3 and biotinylated peptide EEA302) Dynabeads® MyOne™ Tosylactivated to first well of BeadRetriever™ tube strip, 1 ml of PBS/T20 buffer in the next 2 wells, and an appropriate volume of buffer, depending on subsequent endpoint detection method (PCR or culture; see later), to 4th well.
2.
Transfer 1 ml of the 1:10 dilution of the clarified lymph node tissue supernatant to the 1st well of the BeadRetriever™ tube strips. Fifteen samples can be processed at the same time.
3.
Transfer tube rack containing samples to BeadRetriever™ machine.
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