Contents of Kit
1. Capture antibody (yellow):0.5 ml of affinity-purified polyclonal anti-fibrinogen antibody for coating plates.
2. Detecting antibody (red):0.5 ml of affinity-purified peroxidase conjugated polyclonal anti-fibrinogen antibody for detection of captured fibrinogen.
Note: Reagents are sufficient for at least 5×96 well plates using recommended protocols. Antibodies are supplied in a 50% (v/v) glycerol solution for storage at –10 to -20°C. Keep vials tightly capped. Do not store in frost-free freezers.
Fibrinogen is an abundant plasma protein (5-10 uM) produced in the liver. The intact protein has a molecular weight of 340 kDa and is composed of 3 pairs of disulphide-bound polypeptide chains named Aα, Bβ and γ. 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, Aα1-16) followed by Fibrinopeptide B (FPB, Bβ1-14) 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 α, β and γ, due to the removal of FPA and FPB. The polymerised fibrin is subsequently stabilized by the transglutaminase activated Factor XIII that forms amide linkages between γ chains and, to a lesser extent, α chains of the fibrin molecules. Proteolysis of fibrinogen by plasmin initially liberates C-terminal residues from the Aα 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 γ 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: 184 kDa for fragment DD, 92 kDa for D, 50 kDa for E, 1.54 kDa for FPA and 1.57 kDa for FPB.