Magnetic Separation Methods for the Detection of Mycobacterium avium subsp. paratuberculosis in Various Types of Matrices: A Review

Table 1

Magnetic separation methods used for the detection of Mycobacterium avium subsp. paratuberculosis cells in milk.

Method	Type of beads	Coating ligand	Type of milk	Artificially contaminated	Preparation of sample	Volume of sample [mL]	Initial number of MAP	Limit of Detection	Capture efficiency (CE) Sensitivity (S)	Reference
IMS	Dynabeads M-280 sheep anti-rabbit IgG	Polyclonal rabbit anti-MAP antibodies	Bovine raw and pasteurized	Yes	Aliquots serially diluted, cultured prior to IMS, or centrifuged and resuspended in PBS before IMS	1	10^6 CFU/mL	10^4 CFU/mL	37% (CE)	[29]
			Bovine raw and pasteurized	Yes	No preparation	1, 5, 10, and 50	10^6 CFU/mL	10^3 CFU/50 mL	100% (S)	[13]
			Sheep raw	No	No preparation	1	n. a.	n. a.	n. a.	[13]
			Goat raw	No	Milk centrifuged, pellet resuspended in PBS containing 0.05% Tween 20	50	n. a.	n. a.	n. a.	[34]
			Bovine unpasteurised	Yes	MAP suspended in 220 mL milk and diluted 10-fold	50	50 × 10^6 CFU	1 CFU/mL	n. a.	[34]
								0.1 CFU/mL
	BioMag goat anti-rabbit IgG	Polyclonal rabbit anti- MAP antibodies	Bovine	Yes	Milk centrifuged, pellet resuspended in PBS	2	10^6 to 10^0 organisms/mL	10 or fewer MAP organisms	n. a.	[35]
	ParaTub-S and ParaTub-SL	Monoclonal antibody	Bovine BTM	No	No preparation	1	10^6–10^0 organisms/mL	5–10 bacteria/mL 10–20 cells/mL	96% (S)	[30]
	Goat anti-mouse IgG magnetic beads	Monoclonal anti-MAP antibody 1A6E10 (mAb); polyclonal anti-MAP antiserum (pAb)	Bovine	No	Bacterial clumps broken up by passage through needle, samples heated 15 min at 50°C and centrifuged, pellets resuspended in 1 mL of PBS, and 10 µL of coated beads added	10	10^12 CFU/mL	10^1 CFU/mL	n. a.	[36]
PMS	MagneSphere	Biotinylated aMp3 peptide, fMp3 phage	Bovine	Yes	Milk incubated with aMp3 peptide or fMp3 phage coated beads	1	10^0 to 10^5 PFU/mL	10^1 PFU/mL (aMp3);	n. a.	[32]
								10^2 PFU/mL (fMp3 phage)
	aMptD peptide paramagnetic beads	aMptD peptide	BTM, pasteurized	Yes	No preparation	1	5 × 10^2 and 5 × 10^3 CFU/mL	5 × 10^2 MAP/mL	5 × 10^2 CFU/mL (S)	[33]
	MyOne Tosylactivated Dynabeads	Biotinylated peptides (aMp3, aMptD)	UHT	Yes	50 : 50, 5 µL of both types of beads added separately per reaction to 1 mL MB-OADC with MAP	1	10^3 to 10^5 CFU	n. a.	(CE)	[24]
			Bovine BTM	No	Milk centrifuged, pellet resuspended in 1 mL of MB with 10% OADC	50	n. a.	1 to 110 PFU/50 mL	n. a.	[19]
			Bovine UHT	Yes	Serial 10-fold dilutions of MAP in MB with 2 mmol/L CaCl2 added (100 µL) to 900 µL aliquots of milk	1		n. a.	n. a.	[17]
			Pasteurized	Yes	100 µL of MAP aliquots added to 900 µL of milk, vortexed, and centrifuged, pellets resuspended in 1 mL of 0.05% PBS-Tween20	1	10^4 CFU/mL	5 × 10^2 CFU/mL	n. a.	[38]

Dynabeads M-280 sheep anti-rabbit IgG (Dynal, Oslo, Norway); almost 100% when 10² to 10⁴ of MAP/mL used; EtBr staining; Dot Blot; BioMag goat anti-rabbit IgG with rabbit polyclonal anti-MAP antibodies (Polysciences, Inc., Warrington, Pennsylvania, USA); the ParaTub-SLand ParaTub-S kits (AnDiaTec GmbH and Co. KG, Kornwestheim, Germany); artificially contaminated milk was used to set the Limit of Detection; IMS-real-time PCR; automated IMS-PCR-ELISA; ParaTub-S; goat anti-mouse IgG magnetic beads (New England BioLabs Inc., Ipswich, MA, USA); artificially contaminated milk was used for standardization of IMS-IS1 PCR; MagneSphere streptavidin paramagnetic particles (Promega, Madison, Wisconsin, USA); aMptD peptide coupled directly by carbodiimide method to paramagnetic beads (Chemicell, Berlin, Germany); the mean LOD₅₀ = 14,4 PFU/50 mL equivalent to 0,3 PFU/mL; the number of PFU mL⁻¹ used to spike the samples was determined by subjecting the 10⁻³, 10⁻⁴, and 10⁻⁵ spiking dilutions to the optimized phage assay and counting the plaques produced. In the final assay format, a consistent number of D29 seed phages (10⁸ PFU mL⁻¹) was added to the samples; dynamic range of the assay was 3 × 10²–6 × 10⁸ phages/mL; BTM = bulk tank milk; CE = capture efficiency; CFU = colony forming units; ELISA = enzyme-linked immunosorbent assay; HEYM = Herrold’s egg yolk medium; IMS = immunomagnetic separation; LOD = Limit of Detection; LOD₅₀ = 50% Limit of Detection; MAP = Mycobacterium avium subsp. paratuberculosis; MB = Middlebrook 7H9 broth; n. a. = not available; OADC = oleic acid-albumin-dextrose-catalase; PBS = phosphate-buffered saline; PFU = plaque-forming units; PMS = peptide-mediated magnetic separation; S = sensitivity; UHT = ultra-heat-treated milk.