Guinea pig C3 specific rabbit single chain Fv antibodies from bone marrow,spleen and blood derived phage libraries

We constructed combinatorial immunoglobulin libraries from the whole rabbit antibody repertoire of bone marrow, spleen and peripheral blood of a rabbi t immunized with guinea pig complement protein C3. By means of the phage di splay technology we selected guinea pig C3 specific single chain Fv (scFv) antibodies from each of the libraries. None of the scFv antibodies cross re acted with guinea pig C3a, human C3 or rat C3. The frequency of bone marrow derived C3 positive clones was much higher as compared to blood or spleen derived clones. Additionally bone marrow and spleen derived clones show hig her diversity than clones, obtained from blood, as determined by fingerprin t analysis with the restriction enzyme AluI. Dissociation rate constants fo r all scFvs were similar, indicating that the source of the scFvs had no in fluence on affinities. The antibody fragments were used to analyze compleme nt activation during xenotransplantation. Several blood or bone marrow deri ved scFvs bound to C3 located on rat liver endothelium after hyperacute rej ection of a heterotopically transplanted rat liver into guinea pig. These d ata demonstrate that monoclonal rabbit scFvs can be easily generated from r ecombinant phage display libraries, constructed from spleen, blood or bone marrow. The selected guinea pig C3 specific scFvs appear to be useful to de tect complement activation during xenotranplantation in guinea pigs. (C) 20 00 Elsevier Science B.V. All rights reserved.