Immunochemical Studies on Human Fibrinogen and Its Fragments

A radioimmunoassay has been developed for the NH 2 ‐terminal portion of human fibrinogen (N‐DSK) as well as for the Bβ chain fragment (residues 1–115) of N‐DSK. This assay has proved valuable in the estimation of the amount of cross‐reaction between N‐DSK and related fragments. The effective range for each assay described was found to be between 1 nmol and 1 pmol. The first system studied was 125 I‐labeled N‐DSK–anti‐N‐DSK (System 1). Reduced and alkylated ( S ‐carboxymethylated) N‐DSK was found to be a strong competitor in this system. The inhibition plateau with the latter was significantly lower than that obtained with unlabeled N‐DSK at equivalent concentrations. This suggests that intact antisera contain a population of antibodies which are elicited to conformational determinants of N‐DSK. However, this antisera must also contain antibodies to altered N‐DSK since a displacement at identical inhibition levels was observed at lower concentrations by S ‐carboxymethylated N‐DSK as poposed to intact N‐DSK. These results indicate that 125 I‐labeled N‐DSK is in fact “immunologically damaged” by the labeling procedure and consequently reacts with antibodies elicited to structurally altered N‐DSK. Fibrinogen was found to inhibit system 1 to only a very small degree at the concentration levels investigated. Using another N‐DSK antisera and 125 I‐labeled N‐DSK (System 1 A), both unlabeled N‐DSK and the NH 2 ‐terminal portion of plasmic fragment E (Aα 1–51, Bβ 54–115, γ 1–58) 2 , obtained by cleavage with CNBr, were shown to be strong competitors. However, the parent molecule, fragment E itself, (Aα 1–86, Bβ 54–119, γ1–58) 2 , was shown to be a poor competitor. The significance of these findings is discussed. The 125 I‐labeled Bβ fragment–anti‐Bβ fragment assay System (system 2) demonstrated that the latter is a highly immunogenic structure of intact N‐DSK. This was established by a comparison of the inhibition curves obtained in this system using N‐DSK and unlabeled Bβ fragment. In addition, low levels of inhibition were also shown by two fragments of the Bβ chain of N‐DSK. It would appear that the latter fragments, obtained by digestion of Bβ fragment with reptilase and arvin respectively, have few, if any, antigenic determinants of intact Bβ fragment. Fragment E was shown to be a poor competitor in System 2.