Rapid purification of a 110-kilodalton hemolysin of Actinobacillus pleuropneumoniae by monoclonal antibody-affinity chromatography

Jianneng Ma From the Veterinary Microbiology Research Laboratories, Department of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

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 DVM, MS
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Thomas J. Inzana From the Veterinary Microbiology Research Laboratories, Department of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

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Summary

An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actmobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.

Summary

An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actmobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.

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