title: The preparation and characterization of monoclonal antibodies to human complement component C8 and their use in purification of C8 and C8 subunits.
abstract: 1. Ten mouse monoclonal antibodies to human complement component C8 were prepared. It was found that six of these antibodies reacted with the alpha-subunit, two with the beta-subunit and two with the gamma-subunit, when assessed by immunoblotting after separation of C8 subunits by SDS/polyacrylamide-gel electrophoresis. 2. Epitope analysis of the ten monoclonal antibodies in a competitive binding assay showed that the six antibodies to the alpha-subunit could be classified in four overlapping epitope groups. The antibodies to the beta- and gamma-subunits bound to a single antigenic site on each, but also cross-reacted with the antigenic sites on the alpha-subunit. 3. Monoclonal anti-C8 immunoaffinity columns were used to purify C8 from fresh human plasma and to prepare C8-depleted serum. Immunoaffinity purified C8 was biologically active when assessed by using haemolysis assays of sheep and rabbit erythrocytes. 4. Salt elution was used to purify either alpha gamma- or beta-subunits when C8 was respectively bound to an anti-beta or anti-alpha immunoaffinity column. The purified subunits reconstituted C8-depleted serum when added together in a haemolysis assay.
authors: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
authors: A
authors: A
authors: Abraha
authors: B P
authors: BP
authors: Morgan
authors: J P
authors: JP
authors: Luzio
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D003185:Complement C8 / Q000276:immunology* / Q000302:isolation & purification; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D007122:Immunoelectrophoresis
chemical_list: D000911:Antibodies, Monoclonal; D003185:Complement C8; D000939:Epitopes