Analysis of immune complexes by two-dimensional gel electrophoresis.
High resolution two-dimensional (2D) gel electrophoresis is useful for analysis of constituents of immune complexes (IC) in serum, provided that the samples for the analysis are prepared by a standardized and effective purification protocol. The details of the protocol, which involve gel permeation chromatography and adsorption with protein A-Sepharose, were worked out with a model system of radiolabeled antigen bound to antibody. With this protocol one can attain an over 50% recovery of the antigen, in a protein preparation purified over 1000-fold with respect to starting amounts in an initial 0.5 ml serum. With silver staining of the 2D gel, the model antigen was detectable at levels of 100 ng initial input. The analysis of eight normal sera and eight sera of patients with systemic lupus erythematosus (SLE) showed no clearly demonstrable differences, suggesting that the latter sera did not contain homogeneous antigens exceeding 100 ng within IC. In addition to IgG, albumin was seen in all preparations, probably due to complexing with immunoglobulin. Trace amounts of other constituents of the samples appeared in some gels, and the presence of C3 related material was detectable only by Western blotting.
D000936:Antigen-Antibody Complex / Q000302:isolation & purification*; D005782:Gels; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007074:Immunoglobulin G / Q000276:immunology; D007525:Isoelectric Focusing; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D008832:Microchemistry; D012685:Sepharose; D012709:Serum Albumin / Q000276:immunology; D013205:Staphylococcal Protein A
D000936:Antigen-Antibody Complex; D005782:Gels; D007074:Immunoglobulin G; D012709:Serum Albumin; D013205:Staphylococcal Protein A; D012685:Sepharose