Semi-synthesis of polymyxin-B conjugated ovalbumin: evaluation of lipopolysaccharide binding avidity and neutralization of induced tnf-alpha synthesis.

A method is described in these investigations for the semi-synthetic production of polymyxin-B conjugated ovalbumin in the form of polymyxin-B.Sulfo-SMCC.ovalbumin (PSO). The heterobifunctional "cross-linking" agent, Sulfo-SMCC was first reacted with polymyxin-B to produce a relatively pure reactive intermediate in the form of polymyxin-B.Sulfo-SMCC. Highly purified ovalbumin was then combined with the polymyxin-B.Sulfo-SMCC reactive intermediate and contaminants removed from the final PSO end product by exhaustive microdialysis. Purity of PSO was established with by high-performance cellulose acetate electrophoresis (HPCAE), and high-performance thin layer chromatography (HPTLC) analyses. Verification of polymyxin-B.Sulfo-SMCC.ovalbumin binding avidity for lipopolysaccharide (LPS) was determined by DotBLot analysis applying fluorescein isothiocyanate labeled E. coli (055:B5) LPS fractions (FITC-LPS). Efficacy of PSO to inhibit in vitro LPS-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed with a tissue culture based biological assay system. In this context, semi-synthetic conjugates of PSO (0.349 microgram/ml) effectively inhibited Salmonella minnesota (RS) LPS (2.5 ng/ml well) induced TNF-alpha synthesis and corresponding cytoprotection (100%) to WEHI 164 clone 13 cell populations.