Zymogen activation: a new system for homogeneous ligand-binding assay.
In this ligand-binding assay procedure, sensitivity is enhanced by successive generation of enzyme active sites via two zymogens from the blood-coagulation cascade: Factor X and prothrombin. A protease fraction from Russell's viper venom (RVV) acts upon Factor X, initiating a two-step cascade that culminates in generation of thrombin, the activity of which is monitored with a chromogenic substrate. In the model presented here, the analyte of interest, biotin, is covalently coupled to Factor X. In the presence of avidin, a biotin-binding protein, RVV cannot initiate cascade activity; however, the inhibition can be competitively overcome by addition of free biotin to the reaction mixture. In a complete system, the dose-response curve is linear from 20 to 100 nmol of biotin per liter. Such an assay offers improved sensitivity over many radioisotope-independent immunoassay methods, and may be applicable to a wide variety of analytes.
D001360:Avidin; D001710:Biotin / Q000032:analysis*; D010450:Endopeptidases; D004792:Enzyme Precursors; D005170:Factor X; D006801:Humans; D008024:Ligands; D008666:Metalloendopeptidases; D010047:Ovalbumin / Q000031:analogs & derivatives; D011485:Protein Binding; D011516:Prothrombin; D013056:Spectrophotometry, Ultraviolet
D004792:Enzyme Precursors; D008024:Ligands; D001360:Avidin; D001710:Biotin; D011516:Prothrombin; D005170:Factor X; D010047:Ovalbumin; D010450:Endopeptidases; D008666:Metalloendopeptidases; C033858:russellysin