Fractionation of IgG, IgG2a, IgG2b and IgG3 immunoglobulins from mouse serum by protein A-sepharose column chromatography.
Normal mouse serum was fractionated on protein A-Sepharose column by the elution with citrate buffer of different pH. At pH 8.0 IgG of all subclasses were retained on the column and main portion of IgG1, IgG2a, IgG3, and IgG2b were eluted at pH 6.0, 5.0, 4.0 and 3.0, respectively, as assessed by Ouchterlony immunodiffusion. Pure IgG1 and IgG2b were obtained by elution at pH 6.0 and 3.0, respectively, from normal serum. The identity of IgG1 globulin was confirmed by its antibody activity in chromatography fraction of anti-sheep red blood cell antiserum and also by its inability to fix complement. IgG3 globulin was eluted at pH 4.0 and also at pH 5.0. The identity of IgG3 was confirmed by the formation of M-bow-like precipitin line in gamma region of immuno-electrophoresis. The affinity of IgG3 to protein A was reduced by the treatment with 2-mercaptoethanol (2-ME). From 2-ME treated normal serum, therefore, pure IgG2a could be obtained at pH 4.0, although the recovery was low. Otherwise, IgG2a was always eluted with other subclass, IgG1 or IgG3.
D000818:Animals; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004912:Erythrocytes / Q000276:immunology; D005260:Female; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis*; D008070:Lipopolysaccharides / Q000032:analysis; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice / Q000276:immunology*; D008815:Mice, Inbred Strains / Q000276:immunology; D012756:Sheep / Q000276:immunology; D013205:Staphylococcal Protein A
D007106:Immune Sera; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D013205:Staphylococcal Protein A; D008623:Mercaptoethanol