FRACTIONATION OF IGG1, IGG2A, IGG2B AND IGG3 IMMUNOGLOBULINS FROM MOUSE SERUM BY PROTEIN A-SEPHAROSE COLUMN CHROMATOGRAPHY

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  • 1 January 1981
    • journal article
    • research article
    • Vol. 51  (1) , 65-70
Abstract
Normal mouse serum was fractionated on staphylococcal 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 the 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 IgG1 globulin identity was confirmed by its antibody activity in chromatographic 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. IgG3 identity was confirmed by the formation of M-bow-like precipitin line in the .gamma.-region of the immunoelectrophoresis. IgG3 affinity to protein A was reduced by the treatment with 2-mercaptoethanol (2-ME). From 2-ME treated normal serum, 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.