Pbs tw 20

Saliva samples, before and after treatment with PNGase F (New England Biolabs, US) were run on a 12.5% polyacrylamide gel under standard conditions, transferred onto a PVDF membrane (Fisher Scientific, UK), and blocked with 1% BSA (Sigma, St. Louis, US) in PBS-Tw 20 (Sigma, St. Louis, US) overnight at 4 °C. Membrane was incubated with 1 µg/ml biotinylated Concanavalin A (ConA) lectin (Vector Labs, Peterborough, UK) for 1 h at room temperature. After washing, the membrane was incubated with 1:100,000 streptavidin-HRP (Vector Labs, Peterborough, UK). SuperSignal West Pico Chemiluminescent substrate (ThermoFisher, Massachusetts, US) was used to detect the bands. Egg albumin (Sigma, St. Louis, US), a highly mannosylated N-linked glycoprotein^{68 (link)}, was used as positive control.

Bacterial suspensions used to spike broths and milk samples tested in this study were prepared as previously described by Foddai and Grant (2015 (link)). Briefly, glass vials containing stationary MAP broth cultures were processed through ultrasonication, applied at 37 kHz for 4 min on ice in an Ultrasonic PH 30 (Fisher Scientific Ltd., Loughborough, UK) in order to disperse clumps of mycobacteria. The purity of de-clumped MAP suspensions was then verified by Ziehl-Neelsen (ZN) staining in order to ensure presence of only red acid-fast cells. The number of MAP cells per ml of broth was estimated by measuring the optical density at 600 nm (OD₆₀₀) using a WPA CO8000 cell density meter (SISLAB, Cornaredo, Italy). For each sample, optical density was adjusted to OD₆₀₀ 0·1 (approximately 10⁶–10⁷ MAP cells per ml) followed by serial dilution of cultures in phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBS–TW20, Sigma). Four spiking levels (10⁴–10³, 10³–10², 10²–10, and approximately 10 MAP per ml) were finally used to prepare the artificially contaminated broth and milk samples tested in this study to assess recovery rates of MAP by phagomagnetic separation and the new phagomagnetic separation (PhMS)-qPCR test.