Radioimmunoassay Of Bβ 15-42 Peptide From Human Fibrinogen

Early plasmin attack of fibrinogen or fibrin results in the removal of C00H- and NH2-terminal portions of the Aα and 3β chains, respectively. This paper describes a radioimmunoassay (RIA) for measurement, in clinical blood samples, of peptides containing the Bβ 15-42 sequence. Bβ 15-42 was prepared from the NH₂-terminal CNB_r fragment of human fibrinogen (N-DSK) by sequential proteolysis with Batroxobin and thrombin. Antiserum was obtained by immunizing rabbits with Bβ 15-42 peptide and BSA. The peptide was labeled with ¹²⁵I by the chloramine-T method. A double antibody RIA was subsequently made.

Radiolabeled Bβ 15-42 (tracer) was bound by antiserum and binding was inhibited by excess cold peptide. Free tracer can be separated from bound by second antibody or by charcoal. Fibrinogen and N-DSK completely cross-react. However, the slope of the inhibition curve is different to that of free Bβ 15-42. Plasma, with Trasylol and heparin,must be treated with ethanol in order to precipitate fibrinogen prior to its use for RIA. The level of peptides with Bp 1542 sequence in treated plasma from normal men and women (20-43 year old) showed a mean value of 1.71±0.59 pmol/ml. Some patient plasma (e.g. uremics) showed 10-20 times higher values.

The immunoreactive material isolated by gel filtration from normal and patient plasma, as well as from streptokinase-plasmin digests of either plasma or purified fibrinogen, all,in gel chromatography, displayed the same molecular weight range.

This RIA is a rapid, specific and sensitive method to measure earliest stage of plasmin proteolysis and will be useful in clinical studies on all disease states associated with fibrino(geno)lysis.