Analysis of immune complexes by two-dimensional gel electrophoresis |
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| Authors: | S R Per J L Abruzzo R Heimer |
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| Affiliation: | 1. Department of Biochemistry, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 USA;2. Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 USA;1. Department of Surge ry, University of Florida Health, Gainesville, Florida;2. Departments of Biomedical Engineering, Computer and Information Science and Engineering, and Electrical and Computer Engineering, University of Florida, Gainesville, Florida;3. Departments of Anesthesiology, Orthopedics, and Information Systems/Operations Management, University of Florida Health, Gainesville, Florida;4. Department of Medicine, University of Florida Health, Gainesville, Florida;1. Department of otology and neurotology, CHU de Lille, 59000 Lille, France;2. Inserm U1008 – controlled drug delivery systems and biomaterials, université de Lille 2, CHU de Lille, 59000 Lille, France;1. Department of Biological Sciences, Southwestern Oklahoma State University, Weatherford, OK, U.S.A.;2. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.;3. Darrin Fresh Water Institute, Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, U.S.A.;1. Department of Mechanical and Aeronautical Engineering, 8 Clarkson Ave, Postdam, NY, 13699, United States of America;2. Aerothermodynamics Branch, 17 Victory St, Hampton, VA, 23681, United States of America |
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| Abstract: | 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. |
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