The preparation and characterization of monoclonal antibodies to human complement component C8 and their use in purification of C8 and C8 subunits.
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.
Abraha
A
A
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Morgan
B P
BP
Luzio
J P
JP
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
D000911:Antibodies, Monoclonal; D003185:Complement C8; D000939:Epitopes