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

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 them three times using 500 microliters of ice-cold endotoxin-free PBST. Then, spin down the cells, and suspend them in sterile endotoxin-free PBS. Next, prepare fresh 10-millimolar Sulfo-NHS SS biotin in sterile filtered water. Then, incubate bacteria in 1 milliliter of broth containing 0.5 millimolar of Sulfo-NHS SS biotin. Perform the incubation at room temperature for 30 minutes.

Next, wash the now labeled bacteria three times with 500 microliters of ice-cold PBST, to remove the unreacted reagent. Then, resuspend the pellet in 1 milliliter of PBST.

Mix 500 million biotinylated BCGs with avidin fusion protein, for a final concentration of 10 micrograms of protein per milliliter. Let this reaction go for an hour at room temperature on a shaker. Next, wash the bacteria three times with 500 microliters of ice-cold PBST.