Monoclonal Antibody to Human Cross-Linked Fibrin

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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-NSl/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 > 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 > 80 kDa (γ-γ dimer), Mr > 45 kDa (β chain of fragment D) and Mr > 16 kDa (α 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.

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