Characterization of fibrinogen/fibrin derivatives isolated from normal and fibrinaemic plasma and serum using paramagnetic particles coated with a monoclonal antibody (mAb) to D-dimer.
Paramagnetic particles coated with a monoclonal antibody to D-dimer (mAb S4) were used to isolate and concentrate D-dimer and D-dimer-containing complexes in plasma and serum. Antibody-captured material was eluted with SDS-urea buffer and examined by either SDS-polyacrylamide or submerged SDS-agarose gel electrophoresis followed by Western blotting. The protein pattern was visualized by either polyclonal antibodies to human fibrinogen or monoclonal antibodies specific for fibrinogen derivatives containing fibrinopeptide A (FpA; mAb Y18), the N-terminus of the beta-chain in fibrin (mAb 59D8) or the gamma-chains (mAb J88B). The results obtained show that paramagnetic particles coated with mAb S4 catch intact D-dimer as well as a variety of cross-linked fibrin molecules of high-molecular-weight (HMW) in plasma. The existence of HMW derivatives in fibrinaemic serum indicates that some of the HMW fibrin related material in such plasma is not clottable. Fibrinogen/fibrin monomers and some of the fibrinogen/fibrin related derivatives found in the eluates were probably non-covalently bound to the complexes caught by mAb S4 coated particles. The present technique combines the selective concentrating power of immunoparticles and the sensitivity of immunovisualization and allows rapid and direct identification of minute amounts of circulating immunoreactive fibrinogen/fibrin derivatives.
Grøn
B
B
Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Norway.
Bennick
A
A
Filion-Myklebust
C
C
Brosstad
F
F
D000911:Antibodies, Monoclonal / Q000276:immunology*; D005337:Fibrin / Q000302:isolation & purification*; D005338:Fibrin Fibrinogen Degradation Products / Q000276:immunology / Q000302:isolation & purification*; D005340:Fibrinogen / Q000302:isolation & purification*; D006801:Humans; D008280:Magnetics; D008863:Microspheres
D000911:Antibodies, Monoclonal; D005338:Fibrin Fibrinogen Degradation Products; C036309:fibrin fragment D; D005337:Fibrin; D005340:Fibrinogen