An Avidin-Biotin Conjugation Technique for Presenting Target Antigens on Mycobacterium bovis BCG

1. Biotinylation of BCG Cell Surface

1. Grow BCG in Middlebrook 7H9 broth with 10% OADC (oleic acid, albumin and dextrose solution) and 0.05% Tween 80 at 37 °C on a shaker platform at 50 rpm until OD = 0.5-1.
   NOTE: BCG is a biosafety level 2 pathogen and all experiments concerning BCG should be done in a biosafety cabinet with proper personal protective equipment.
2. Wash 10⁹ BCG 3 times with 500 µL of ice-cold endotoxin free PBS plus 0.1% Tween80 (PBST) (pH 8.0). Suspend BCG pellet in sterile endotoxin free PBS.
3. Immediately before use, prepare 1 mL of 10 mM Sulfo-NHS SS biotin in sterile filtered water.​
   1. Incubate bacteria with 1 mL of 0.5 mM of Sulfo-NHS SS biotin at room temperature for 30 min.
4. Wash labeled bacteria 3 times with 500 µL of iced cold PBST to remove non-reacted biotinylation reagent.
   1. Re-suspend pellet in 1 mL of PBST.

2. Binding of Monomeric Avidin-Fusion Protein to Biotinylated BCG Surface

1. Mix 5 x 10⁸ biotinylated BCG with Avi-protein (10 μg/mL final in PBS-T) for 1 h at room temperature on shaker platform.
2. Wash bacteria 3 times with 500 µL of iced cold PBST.