Purification of Pasteurella multocida antigens by ultracentrifugation and isoelectrofocusing

A procedure was developed to purify Pasteurella multocida X-731 antigens extracted by potassium thiocyanate. The crude extract was centrifuged at 105 000 x g; the antigens were then separated into a particulate (40p) fraction and a soluble (40s) fraction consisting of proteins and polysaccharides. These fractions were antigenically different. The ultracentrifuged antigens were resolved further by preparative isoelectrofocusing. The 40p antigens focused in a pH range of 3.0 to 6.0; distinctive proteins focused at pH's of 3.5, 3.6, and 3.8. The electrofocused 40p antigens were antigenically similar. The 40s antigens were initially electrofocused in a broad pH range and were found within a pH range of 4.6 to 9.0. The process was repeated with a narrower pH range and antigens that were focused in a narrower pH range could be separated and unique antigenic activities identified. Specific antigens from defined pH ranges were pooled and examined further by immunoelectrophoresis, analytical electrofocusing, and sodium dodecyl sulphate--polyacrylamide gel electrophoresis.