Monoclonal Antibody to Human Cross-Linked Fibrin

Abstract

Cross-linked Fibrin II was prepared using Kabi grade (L) fibrinogen. Fibrin plasmic digest was separated on Sepharose CL-6B. Fragments Mr 135-300 kDa were used to immunize 6-9 weeks old female BALB-c mice. A stable hybridoma secreting monoclonal antibody (MAb) TD-1 (IgG 2a, Kappa) was prepared by fusion using myeloma cells (P3-NS1/1-Ag4-1) and immunized cells. Fibrinogen and plasmin digest of fibrinogen in serial dilutions did not compete with the immunizing antigen. To prove that TD-1 binds specifically to cross-linked fibrin, immunoprecipitation with S. aureus and affinity chromatography were performed. In both experiments, we demonstrated that TD-1 binds specifically to a protein Mr greater than 200 kDa which is found in XL-fibrin and not fibrinogen. Reduced samples showed the antibody bands (heavy and light chains) and three protein bands, Mr greater than 80 kDa (gamma-gamma dimer), Mr greater than 45 kDa (beta chain of fragment D) and Mr greater than 16 kDa (alpha chain from fragment D) were present. TD-1 reacted strongly with HPLC fraction of the immunizing antigen Mr 220 kDa (probably DD/E complex). Affinity binding constants (Scatchard Plot Analysis) were determined. The highest affinity was obtained with XL-fibrin fraction Mr 220 kDa, KD = 1.39 X 10(-8) and high molecular weight XL-fibrin fragments, KD = 1.6 X 10(-7). Fragment DD had KD of 2.8 X 10(-6). These results suggest that TD-1 is specific for the DD region of human cross-linked Fibrin II.