The objective is to examine and further characterize the ability of antibody directed against gram-negative bacterial lipopolysaccharide (LPS, endotoxin) to afford protection during experimental gram-negative bacillary sepsis. The core LPS-lipid A portion of endotoxin represents an antigenic determinant common to many common gram-negative microorganisms. This region of LPS is expressed extensively on the cell surface of rough mutants of Escherichia coli and Salmonella minnesota. These organisms or their derived outer membrane LPS thus represent suitable immunogens for cross-reactive antibody production. Although we have demonstrated protection in vivo utilizing murine monoclonal antibodies directed against the core LPS-lipid A region, the exact binding site specificity and antibody class which will maximize protection has not been established. Thus, IgG and IgM monoclonal antibodies of similar binding specificity, and monoclonal and polyclonal preparations have not been directly compared. The major thrust of this work will be to develop and test both monoclonal and polyclonal antibody preparations which bind to different portions of LPS. Different classes of monoclonal antibodies with similar binding specificity will be developed. These antibody preparations will be tested in vitro for: 1) antibody titer (enzyme-linked immunosorbent assay), 2) binding specificity (Western blot analysis, immunofluorescent bacterial binding), and opsonophagocytic character (acridine orange slide phagocytosis assay). Initial in vivo testing will establish protective capacity by exact LD50 calculations in three murine sepsis models: 1) IV bacteremia, 2) ip hemoglobin peritonitis, and 3) iv endotoxemia. Further in vivo testing will center around maximizing the protective effect of suitable antibody preparations. The ability of antibody to promote survival as a therapeutic agent administered after the bacterial challenge, either alone or in conjunction with antimicrobial agents and the immunoenhancing agent muramyl dipeptide will be examined in the above models and a chronic peritonitis-sepsis model.
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