JoVE Encyclopedia of Experiments
Immunology
0 views • 3:12 min • July 8th, 2025
Begin with a suspension of Mycobacterium bovis BCG, treated with a non-ionic surfactant, to prevent bacterial clumping.
Next, add a biotinylation reagent and incubate.
This biotinylation reagent, a complex of biotin complexed to a functional linker, facilitates binding biotin molecules to bacterial surface proteins, labeling bacteria.
Post-incubation, centrifuge the biotin-labeled bacterial suspension to remove unreacted biotinylation reagent.
Resuspend the bacteria in a buffer. Add a solution of avidin-fusion protein, a recombinant protein comprising a target antigen domain fused to monomeric avidin.
During incubation, avidin binds to biotin, facilitating bacterial surface modification with target antigens.
Centrifuge the suspension to remove unbound fusion proteins.
Resuspend the antigen-coated bacteria in a buffer containing a non-ionic surfactant. The antigen coating on the bacteria can enhance its property to trigger immune responses.
Work in a biosafety cabinet using personal protective equipment because BCG is a level II pathogen.
To biotinylate the surface of BCG cells, first, grow the cells on a shaker platform to the proper density. Collect about 1 billion cells, and wash
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