Fibrinogen is an abundant plasma protein (5-10uM) produced in the liver. The intact protein has a MW of 340kD. It is composed of 3 pairs of disulfide-bound polypeptide chains named Aalpha, Bbeta and gamma. Fibrinogen is a triglobular protein consisting of a central E domain and terminal D domains. Proteolysis by thrombin results in release of Fibrinopeptide A (FPA, Aalpha1-16) followed by Fibrinopeptide B (FPB, Bbeta1-14) and the fibrin monomers that result polymerize in a half-overlap fashion to form insoluble fibrin fibrils. The chains of fibrin are referred to as alpha, beta and gamma, due to the removal of FPA and FPB. The polymerized fibrin is subsequently stabilized by the transglutaminase activated Factor XIII that forms amide linkages between gamma chains and, to a lesser extent, alpha chains of the fibrin molecules. Proteolysis of fibrinogen by plasmin initially liberates C-terminal residues from the A-alpha chain to produce fragment X (intact D-E-D, which is still clottable). Fragment X is further degraded to non-clottable fragments Y (D-E) and D. Fragment Y can be digested into its constituent D and E fragments. Digestion of non-crosslinked fibrin with plasmin is very similar to the digestion of fibrinogen, which results in production of fragments D and E. Degradation of crosslinked fibrin by plasmin results in fragment DD (D-Dimer consisting of the D domains of 2 fibrin molecules crosslinked via the gamma chains), fragment E (central E domain) as well as DDE in which fragment E is non-covalently associated with DD. For human crosslinked fibrin, the relative weights of the cleavage fragments produced are: 184kD for fragment DD, 92kD for D, 50kD for E, 1.54kD for FPA and 1.57kD for FPB1-3.
Principle of the Assay:Affinity-purified polyclonal antibody to Fg is coated onto the wells of a microtiter plate. Any remaining binding sites on the plastic wells are blocked with bovine serum albumin. The plates are washed and plasma or other fluids containing Fg are applied. The coated antibody will capture the Fg in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to Fg is added to the plate to bind to the captured Fg. After washing the plate to remove unbound conjugated antibody, the peroxidase activity is expressed by incubation with ophenylenediamine (OPD). After a fixed development time the reaction is quenched with the addition of H2SO4 and the color produced is quantified using a microplate reader. The color generated is proportional to the concentration of Fg in the sample.
F4202B: Detection Antibody (HRP), 1x500ulAffinity-purified sheep anti-Fg labeled with HRP suitable for detection of Fg.
Storage and Stability:May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
