Composition of IgA immune complexes precipitated with polyethylene glycol. A model for isolation and analysis of immune complexes.
Covalently cross-linked large and intermediate-sized IgA oligomers, prepared with IgA anti-dinitrophenyl (DNP) and bis-DNP-pimelic acid ester, were used to examine the ability of different concentrations (3.5%, 5% and 7%, w/v) of polyethylene glycol (PEG) to precipitate IgA immune complexes (IgA-IC). The size of the IgA-IC precipitated with PEG was determined by gradient polyacrylamide gel electrophoresis and quantitative autoradiography. The standard concentration of 3.5% PEG precipitated only a minor fraction (20%) of the IgA-IC. In contrast, 5% and 7% PEG precipitated 45% and 79% of the complexes, respectively. To test the influence of the antigen on the PEG assay. IgA-IC prepared with IgA anti-DNP and DNP conjugates of either bovine serum albumin or Ficoll were also used. Approximately 38% of these IgA-IC were precipitated with 3.5%, PEG. By comparison, the concentration of 5% and 7% PEG precipitated 60% and 76% of the IgA-IC, respectively. Distilled water rather than the standard borate-buffered saline was shown to be the optimal solvent for resolubilization of the PEG precipitates. Serum samples from 22 IgA nephropathy patients and 12 normal donors were tested with 3.5%, 5% and 7% PEG. Only the 7% PEG assay showed a significant difference between patients and controls (P less than 0.001) in the IgA levels of precipitates. Thus, the use of 7% PEG is recommended for the detection, isolation and analysis of large- and intermediate-sized IgA-IC.
Imai
H
H
Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston 77225.
Chen
A
A
Wyatt
R J
RJ
Rifai
A
A
D000936:Antigen-Antibody Complex / Q000032:analysis*; D011232:Chemical Precipitation; D005922:Glomerulonephritis, IGA / Q000276:immunology; D006801:Humans; D007070:Immunoglobulin A / Q000032:analysis*; D008970:Molecular Weight; D011092:Polyethylene Glycols; D012995:Solubility; D013329:Structure-Activity Relationship
D000936:Antigen-Antibody Complex; D007070:Immunoglobulin A; D011092:Polyethylene Glycols