Human antibodies against spores of the genusBacillus: A model study for detection of and protectionagainst anthrax and the bioterrorist threat

摘要

A naïve, human single-chain Fv (scFv) phage-display library was used in bio-panning against live, native spores of Bacillus subtilis IFO 3336 suspended in solution. A direct in vitro panning and enzyme-linked immunosorbent assay-based selection afforded a panel of nine scFv-phage clones of which two, 5B and 7E, were chosen for further study. These two clones differed in their relative specificity and affinity for spores of B. subtilis IFO 3336 vs. a panel of spores from 11 other Bacillus species/strains. A variety of enzyme-linked immunosorbent assay protocols indicated these scFv-phage clones recognized different spore epitopes. Notably, some spore epitopes markedly changed between the free and microtiter-plate immobilized state as revealed by antibody-phage binding. An additional library selection procedure also was examined by constructing a Fab chain-shuffled sublibrary from the nine positive clones and by using a subtractive panning strategy to remove crossreactivity with B. licheniformis 5A24. The Fab-phage clone 52 was improvedcompared with 5B and was comparable to 7E in binding B.subtilis IFO 3336 vs. B. licheniformis 5A24, yetshowed a distinctive crossreactivity pattern with other spores. We alsodeveloped a method to directly detect individual spores by usingfluorescently labeled antibody-phage. Finally, a variety of“powders” that might be used in deploying spores of B.anthracis were examined for antibody-phage binding. Thestrategies described provide a foundation to discover human antibodiesspecific for native spores of B. anthracis that can bedeveloped as diagnostic and therapeutic reagents.