Biotherapy

, Volume 8, Issue 1, pp 69–83

Semi-synthesis of polymyxin-B conjugated ovalbumin: Evaluation of lipopolysaccharide binding avidity and neutralization of induced tnf-α synthesis

Authors

  • C. P. Coyne
    • Veterinary Research ProgramVeterinary Pharmacology Research Laboratory
    • College of Veterinary Medicine, Wise CenterMississippi State University
  • J. T. Moritz
    • Veterinary Research ProgramVeterinary Pharmacology Research Laboratory
    • College of Veterinary Medicine, Wise CenterMississippi State University
  • V. Cory Langston
    • Veterinary Research ProgramVeterinary Pharmacology Research Laboratory
    • College of Veterinary Medicine, Wise CenterMississippi State University
Article

DOI: 10.1007/BF01878123

Cite this article as:
Coyne, C.P., Moritz, J.T. & Langston, V.C. Biotherapy (1994) 8: 69. doi:10.1007/BF01878123

Abstract

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 labeledE. coli (055:B5) LPS fractions (FITC-LPS). Efficacy of PSO to inhibitin vitro LPS-induced synthesis of tumor necrosis factor-alpha (TNF-α) was assessed with a tissue culture based biological assay system. In this context, semi-synthetic conjugates of PSO (0.349μg/ml) effectively inhibitedSalmonella minnesota (RS) LPS (2.5 ng/ml well) induced TNF-a synthesis and corresponding cytoprotection (100%) to WEHI 164 clone 13 cell populations.

Key words

Polymyxin B lipid A-core lipopoly saccharide LPS TNF-α HPTLC HPCAE

Copyright information

© Kluwer Academic Publishers 1995