Alpha chain crosslinking of human fibrin: Purification and radioimmunoassay development for two Aα chain regions involved in crosslinking

Rapid and efficient purification methods that include hydrophobic chromatography on Phenyl Sepharose CL-4B have been developed for the peptides that span residues 241–476 (CNBr VIII), and 518–584 (CNBr X) in the Aα chain of human fibrinogen. Amino acid analysis, NH₂-terminal sequence determination, and SDS-PAGE indicated that greater than 95% purity of each peptide was achieved. Sensitive and specific radioimmunoassays capable of detecting antigen in the range of 0.1–10.0 pmol/ml have also been developed for CNBr VIII and CNBr X. These assays have been characterized and successfully applied in studies designed to localize the two COOH-terminal Aα chain regions in column effluents of CNBr-digested fibrinogen and crosslinked fibrin. When these immunoassays were used to study purified preparations of a high molecular weight, crosslink-containing CNBr derivative of α polymer, the data provided immunologic confirmation for the involvement of CNBr VIII and CNBr X in α chain crosslinking.