Isolation of circulating immune complexes by conglutinin and separation of antigen from dissociated complexes by immobilized protein A.
A method for the isolation of complement-fixing immune complexes from human serum and the separation of antigen from antibody is described. In order to isolate the complexes, we used soluble bovine conglutinin in a three-step procedure: (1) serum containing immune complexes is reacted with conglutinin in the presence of 10 mM calcium; (2) the conglutinin-bound immune complexes are precipitated by anti-conglutinin rabbit serum; (3) the precipitate is washed and the complexes are eluted from the precipitate by EDTA (pH 7.5) which chelates calcium and releases C3-associated immune complexes from conglutinin. To separate the antigen from the antibody, the isolated complexes are acid-dissociated (pH 3.0), and the antibody is absorbed to staphylococcal protein A conjugated to Sepharose leaving the antigen in solution. The antibody bound to Sepharose-protein A is recovered by elution with 3.5 M magnesium chloride. This procedure permitted the isolation of immune complexes from sera of hepatitis B surface antigen (HBsAg) positive chronic active hepatitis. In addition, immune complexes were isolated from sera of patients with rheumatoid arthritis, systemic lupus erythematosus and primary biliary cirrhosis. The isolated immune complexes contained IgG, IgM, C3 and albumin. Specific antibodies such as rheumatoid factors, anti-nuclear antibodies and antimitochondrial antibodies in varying titres have been found to be present in the isolated immune complexes. The conglutinin method has proven to be a useful technique for the isolation of immune complexes and for the identification of antibody and could be applied to the identification of the antigen in immune complexes.
D000936:Antigen-Antibody Complex / Q000302:isolation & purification*; D000941:Antigens / Q000302:isolation & purification*; D003095:Collagen Diseases / Q000276:immunology; D037222:Collectins; D003168:Complement Fixation Tests; D006514:Hepatitis B Surface Antigens / Q000302:isolation & purification*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007164:Immunosorbents; D008105:Liver Cirrhosis, Biliary / Q000276:immunology; D008722:Methods; D012712:Serum Globulins; D013205:Staphylococcal Protein A
D000936:Antigen-Antibody Complex; D000941:Antigens; D037222:Collectins; D006514:Hepatitis B Surface Antigens; D007074:Immunoglobulin G; D007164:Immunosorbents; D012712:Serum Globulins; D013205:Staphylococcal Protein A; C024014:conglutinin