Biochemical analysis and biological activities of immunoaffinity purified natural murine gamma interferon (MuIFN-gamma).
A rapid one-step procedure for the purification of natural murine gamma interferon (MuIFN-gamma) by immunoaffinity chromatography has been developed. Crude MuIFN-gamma was passed through a column containing immobilized anti-MuIFN-gamma monoclonal antibody, and the column was washed with high salt and detergent. Active MuIFN-gamma was subsequently eluted with ammonium thiocyanate/glycerol (yields of 20-40%). Analysis of the immunopurified interferon by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions revealed biologically active bands of about 19.5, and 14 kd. Under nonreducing conditions, three active bands at 35, 19.5, and 14 kd could be seen. Gel filtration of the immunopurified MuIFN-gamma under reducing or nonreducing conditions showed a single major peak of antiviral activity corresponding to a molecular weight of 43 kd. The protein eluted from each band from the SDS-gel, in addition to possessing antiviral activity, was shown to activate macrophages in three assays: macrophage tumoricidal activity, hydrogen peroxide secretion, and expression of la antigen on the surface of exudate macrophages.
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006861:Hydrogen Peroxide / Q000378:metabolism; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D011489:Protein Denaturation; D014762:Viral Interference
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D007371:Interferon-gamma; D006861:Hydrogen Peroxide