| 主权项 |
1. An in vitro immunoassay for quantifying thrombin in a sample in the presence of anti-thrombin III (AT-III), the immunoassay comprising the steps of: contacting the sample with a small molecule that recognizes a substrate binding site of thrombin and is selected from the group consisting of naturally occurring hirudin, synthetic hirudin variants, hirudin derivatives, recombinant hirudin variants, hirudin mimics, and a combination thereof; adding to the sample mixture a thrombin specific antibody, wherein the thrombin specific antibody consists of a polyclonal antibody or a fragment thereof and was raised against a thrombin having a blocked active site with a molecule selected from the group consisting of Phe-Pro-Arg-chloromethylketone (PPACK), 4-aminophenylpyruvic acid (APPA), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and a combination thereof; measuring the binding of the polyclonal antibody or fragment thereof to thrombin in the sample mixture; and quantifying the amount of thrombin by comparison and extrapolation from a predetermined thrombin standard curve using known amounts of purified thrombin. |
