Preparation and characterization of 4-azido-2-nitrophenyl human serum albumin as an antigen for covalent cross-linking of immune complexes.

Human serum albumin (HSA) was conjugated with 4-fluoro-3-nitrophenyl azide to yield varying density of the 4-azido-2-nitrophenyl haptenic group, useful for covalent cross-linking in the antibody-combining site. The epitope density of the antigen influenced several examined biologic properties. Precipitation in gel diffusion occurred when the average epitope density was 13 or above. Complement (C) activation was not found by incubation with guinea pig C, by binding to human Clq, or by conversion of the electrophoretic mobility of human C3 with epitope densities up to 13. Upon i.v. injection, rapid removal of the conjugated HSA occurred when more than seven 4-azido-2-nitrophenyl groups were present. This rapid removal was in part due to hepatic uptake. These studies point out the epitope density-dependent alterations of biologic properties of an antigen useful for preparation of immune complexes covalently cross-linked in the antibody-combining site.