Characterization of a monoclonal antibody MoAb bH6 reacting with a neoepitope of human C3 expressed on C3b, iC3b, and C3c.
Activation products of the complement cascade contain neoepitopes that are not present in the individual native components. Monoclonal antibodies detecting neoepitopes have been used for direct quantification of activation at different steps in the cascade. These methods are suggested to be more sensitive and reliable than conventional complement activation tests, which are hampered by precipitation or fractionation procedures. The present study describes production screening and characterization of a monoclonal antibody (MoAb) bH6. MoAb bH6 exhibited a significantly higher binding capacity to ELISA plates coated with zymosan-activated human serum than to plates coated with EDTA plasma. When fixed to the enzyme-linked immunosorbent assay (ELISA) plates, MoAb bH6 retained material from zymosan-activated serum that only reacted with anti-C3 antibodies. Crossed immunoelectrophoresis performed on zymosan-activated serum demonstrated that MoAb bH6 co-precipitated with anti-C3c antibodies. In experiments using highly purified cell-bound fragments MoAb bH6 showed reactivity with C3b and iC3b, but not with C3d. MoAb bH6 reacted in ELISA with purified C3c, but not with C3dg, both as capture antibody and in tests with the fragments absorbed to the solid phase. Thus, MoAb bH6 is highly specific for a neoepitope of human C3 expressed on the cleavage fragments of C3b, iC3b, and C3c.
D000911:Antibodies, Monoclonal / Q000032:analysis*; D000937:Antigen-Antibody Reactions; D002850:Chromatography, Gel; D003167:Complement Activation; D003176:Complement C3 / Q000276:immunology* / Q000378:metabolism; D003179:Complement C3b / Q000276:immunology / Q000378:metabolism; D003180:Complement C3b Inactivator Proteins / Q000276:immunology / Q000378:metabolism; D015932:Complement C3c; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007132:Immunoglobulin Isotypes / Q000032:analysis; D012397:Rosette Formation; D015054:Zymosan
D000911:Antibodies, Monoclonal; D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D000939:Epitopes; D007132:Immunoglobulin Isotypes; D003179:Complement C3b; D015932:Complement C3c; D015054:Zymosan