Overview
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.
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Materials
Name | Company | Catalog Number | Comments |
Endotoxin-free RPMI 1640 | StemCell Technologies | 36750 | |
Sulfo-NHS SS biotin | Thermo Fisher | 21328 | |
Middlebrook 7H9 broth | BD Diagnostic Systems | 271310 | |
OADC | BD Diagnostic Systems | B11886 | |
Tween 80 | Sigma-Aldrich | P1379 |