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 rabbit immunized with guinea pig complement protein C3. By means of the phage display technology we selected guinea pig C3 specific single chain Fv (scFv) antibodies from each of the libraries. None of the scFv antibodies cross reacted 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 higher diversity than clones, obtained from blood, as determined by fingerprint analysis with the restriction enzyme AluI. Dissociation rate constants for all scFvs were similar, indicating that the source of the scFvs had no influence on affinities. The antibody fragments were used to analyze complement activation during xenotransplantation. Several blood or bone marrow derived scFvs bound to C3 located on rat liver endothelium after hyperacute rejection of a heterotopically transplanted rat liver into guinea pig. These data demonstrate that monoclonal rabbit scFvs can be easily generated from recombinant phage display libraries, constructed from spleen, blood or bone marrow. The selected guinea pig C3 specific scFvs appear to be useful to detect complement activation during xenotransplantation in guinea pigs.
Hawlisch
H
H
Institute of Medical Microbiology, Medical School Hannover, Hannover, Germany.
Meyer zu Vilsendorf
A
A
Bautsch
W
W
Klos
A
A
Köhl
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000910:Antibodies, Heterophile / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D000916:Antibody Diversity; D000918:Antibody Specificity; D001483:Base Sequence; D001853:Bone Marrow / Q000276:immunology; D003001:Cloning, Molecular; D003176:Complement C3 / Q000276:immunology*; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D016031:Liver Transplantation / Q000276:immunology / Q000473:pathology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000276:immunology; D014183:Transplantation, Heterologous
D000910:Antibodies, Heterophile; D000911:Antibodies, Monoclonal; D003176:Complement C3; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv