LukAB is secreted by S.
aureus as a heterodimer, with oligomerization and pore formationoccurring on the surface of phagocytes at the time of cellular destruction. Our recent data indicate that neutralizing anti-LukAB human mAbs achievetoxin neutralization by distinct mechanisms. Based on these findings, wehypothesize that an oligoclonal mixture of anti-toxin mAbs with high affinityand distinct properties will have increased potency in the blockage ofLukAB-mediated cytotoxicity. To assessthis, we propose a series of related experiments: In vitro toxin neutralization with distinctmAb combinations.
We willassess potentiation of activity (i. e., combination effect) in a series ofexperiments measuring mAb neutralization of LukAB-mediated neutrophil killing. Methods: Anti-LukAB mAb preparations (or isotypecontrols) will be incubated in the presence of LukAB for 30 minutes, prior tothe addition of neutrophil-like HL-60 cells. After a one-hour intoxication, Cell Titer® Aqueous One dye will be added, and neutralization of cytotoxicitywill be measured as a function of the percentage of cells that remain alivefollowing intoxication relative to controls. Experiments will be performed intriplicate with cells from independent vials.
