Clinical Chemistry
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Clinical Chemistry 30: 1452-1456, 1984;
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Clinical Chemistry, Vol 30, 1452-1456, Copyright © 1984 by American Association for Clinical Chemistry

Zymogen activation: a new system for homogeneous ligand-binding assay

DA Blake, MT Skarstedt, JL Shultz and DP Wilson

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.




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Copyright © 1984 by the American Association for Clinical Chemistry.