A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma

Abstract

Background: Soluble fibrin (SF), composed of fibrin monomer (FM) and fibrinogen, is well known to exist in the circulating blood derived from patients with thrombotic diseases, and its quantification is useful to get some information on the state and degree of intravascular coagulation. However, there was no convenient method for the determination of SF. Methods: We prepared a novel monoclonal antibody (MoAb) (F405) to FM and SF using desAA-fibrin as the immunogen in the presence of anti-polymerant peptide (Gly-Pro-Arg-Pro, GPRP), and the characterization of the F405 was performed by Western blotting analysis and an enzyme-linked immunosorbent assay (ELISA). We also tried to detect SF in human plasma using an ELISA involving the immobilized F405 and horseradish peroxidase (POD)-labeled anti-fibrinogen polyclonal antibody. Results: The antibody reacted with the fibrin degradation products fragments X, Y and E, but not with fibrinogen or its fragments X, Y, D and E, or the fibrin D-dimer. The epitope recognized by F405 appeared to be the α-chain N-terminal region exposed upon removal of the A peptide from the Aα-chain because F405 was found to bind to the α-chain N-terminal oligo-peptide of fibrin (GPRVVERHQ). Since F405 reacted not only with FM in the presence of GPRP peptide, but also with the SF complex prepared by the addition of thrombin-treated FM to human fibrinogen, we attempted to detect SF in human plasma using ELISA. The analytical range of this method was 1–300 μg/ml. The assay detection limit was <0.5 μg/ml, and the results of intra- and inter-assay precision studies indicated that this method is accurate and yields reproducible results (<9.4% and <10%, respectively). When 56 samples of plasma from patients with disseminated intravascular coagulation (DIC) and 117 control samples from healthy individuals were tested, elevated levels of SF complex were detected in the DIC samples: the mean±S.D. of the SF concentration in the DIC and control samples were 63.4±65.3 μg/ml and 1.9±1.0 μg/ml, respectively. Conclusions: The ELISA using F405 is useful for the diagnosis of DIC.