Production of monoclonal antibodies against the human anaphylatoxin C5a des Arg and their application in the neoepitope-specific sandwich-ELISA for the quantification of C5a des Arg in plasma.
A panel of ten murine monoclonal antibodies (MAbs) was raised against the human anaphylatoxin C5a des Arg. The MAbs were shown to abrogate or significantly inhibit the chemotactic activity in zymosan activated serum. MAb 4A2E10E2 and MAb 3G3C4 were used as capture and detecting antibody, respectively, in a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of C5a des Arg. This ELISA was shown to be very sensitive (detection limit 20 pg/ml) and could be applied directly to plasma/serum samples. The lack of interference by plasma components, in particular C5, suggested specificity for an epitope on C5a (des Arg) which is concealed in native C5 and exposed on the activation fragment only, i.e., a 'neoepitope'. The mean C5a des Arg level in EDTA-plasma from 25 healthy individuals assessed at a 1/20 dilution was 11.2 ng/ml (SD 3.4; range 6.4-16.8 ng/ml). The applicability of the assay was investigated in patients treated with haemodialysis using different membranes. Markedly elevated plasma levels of C5a des Arg were found in blood returning from the dialyzer following contact with cuprophane membranes.
Bergh
K
K
Department of Microbiology, Regional and University Hospital, Trondheim, Norway.
Iversen
O J
OJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D002634:Chemotaxis, Leukocyte; D015937:Complement C5a, des-Arginine / Q000032:analysis / Q000276:immunology* / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D000939:Epitopes / Q000097:blood*; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000276:immunology
D000911:Antibodies, Monoclonal; D015937:Complement C5a, des-Arginine; D000939:Epitopes