Characterization of a monoclonal antibody, D73H, that maps to a highly conserved region on fibrinogen Bbeta chain.
The primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bbeta chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bbeta chain of plasmin fragment D, localizing its epitope to Bbeta134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bbeta243-253, further localizing the epitope to Bbeta243-305. In silico analysis indicated that Bbeta243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bbeta252-258, Bbeta262-269, and Bbeta279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bbeta262-269 (FGRKWDPY) is predominantly alpha-helical and located on the surface of the molecule adjacent to a bend imposed in the beta chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bbeta261-272 competitively inhibited the binding of D73H to the Bbeta chain of denatured intact fibrinogen and reduced and denatured Bbeta chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bbeta chain residues Bbeta261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.
University of Rochester School of Medicine and Dentistry, Department of Pathology, USA.
B J
BJ
Rybarczyk
M
M
Pereira
P J
PJ
Simpson-Haidaris
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002417:Cattle; D003429:Cross Reactions; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D019143:Evolution, Molecular; D005340:Fibrinogen / Q000737:chemistry / Q000276:immunology*; D006168:Guinea Pigs; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000911:Antibodies, Monoclonal; C115499:BBeta fibrinogen; D000939:Epitopes; D021122:Protein Subunits; D005340:Fibrinogen