The video demonstrates a technique for loading antigens on Mycobacterium bovis BCG to improve its immunogenic properties. The method uses the avidin-biotin system to coat the bacterial surface with exogenous antigens.
Protocol
1. Biotinylation of BCG Cell Surface
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.
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.
Immediately before use, prepare 1 mL of 10 mM Sulfo-NHS SS biotin in sterile filtered water.
Incubate bacteria with 1 mL of 0.5 mM of Sulfo-NHS SS biotin at room temperature for 30 min.
Wash labeled bacteria 3 times with 500 µL of iced cold PBST to remove non-reacted biotinylation reagent.
Re-suspend pellet in 1 mL of PBST.
2. Binding of Monomeric Avidin-Fusion Protein to Biotinylated BCG Surface
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.
Wash bacteria 3 times with 500 µL of iced cold PBST.
An Avidin-Biotin Conjugation Technique for Presenting Target Antigens on Mycobacterium bovis BCG. J. Vis. Exp. (Pending Publication), e22273, doi: (2024).