A diazophenylthio-ether-IgG probe for analysing fibrin- and fibrinogen-related antigens

A technique for identifying and quantitating fibrin- and fibrinogen-related antigens (FRAs) in serum is described. Panspecific antifibrinogen IgG is bound covalently to diazotized aminophenylthio-ether cellulose paper discs. Under conditions of antibody excess, a disc probe extracts 95% of all fibrinogen-related antigen from appropriately diluted serum samples. Bound antigen is then eluted from the washed probe and electrophoresed on 4-11% gradient SDS-polyacrylamide gels. Derivatives of cross-linked fibrin, non-cross-linked fibrin and fibrinogen may thus be identified by their molecular weights, and their relative concentrations determined by densitometric analysis. The total amount of FRA is estimated by a radio-immunoassay which uses labelled D-dimer as the competitive antigen. The probes can extract as little as 0.20 and 0.15 microgram/ml of D-dimer and D-monomer, respectively. Specific FRA binding was confirmed with Western blotting. Potential interfering substances do not influence antigen binding to the discs and, since complete elution of all antigens is achieved, discs may be re-used. This procedure provides estimates of the serum levels of fragments YY, DY, X, DD, Y and D. The procedure is simple to perform and, as a research tool, may help to delineate some of the features of clotting disturbances in clinical states.