Detection of preexisting immunity against lysostaphin and LysK binding domains in human serum.

Blots for detection of antibody response in human serum were prepared by separating 250 ng of each protein on 10% SDS-PAGE and transferring the resulting gel to a polyvinylidene difluoride membrane. Blots were blocked with PBS supplemented with 5% nonfat dry milk and 0.1% Tween 20 (PBS-DT), washed with Tris-buffered saline, and incubated overnight with 10 ml PBS-DT containing 100 μl human serum. Blots were washed 3 times and incubated with goat anti-human Fc horseradish peroxidase conjugate diluted 1:5,000 in PBS-DT. Blots were developed using SuperSignal West Pico chemiluminescent substrate (Pierce).