Antithrombin III is a glycosylated plasma serine protease inhibitor that forms a stoichiometric complex with coagulation cascade enzymes. Thrombin is a two-chain vitamin K-dependent glycosylated serine protease that is activated from prothrombin in the coagulation cascade. Antithrombin III inhibits thrombin with heparin enhanced kinetics and forms a 1:1 covalent complex. The sensitive quantitative measurement of total human Thrombin Antithrombin (TAT) Complex antigen in plasma and serum samples is easily performed with this 96 well strip format ELISA kit. Free thrombin, prothrombin and antithrombin III will not be detected by this assay. In-house testing determined human TAT complex concentrations of 10 ng/ml in plasma and 52 ug/ml in serum. The assay measures total human TAT complex in the 1-1,000 ng/ml range. Samples giving human TAT complex levels above 1,000 ng/ml should be diluted in blocking buffer before use. Normal human plasma samples should be applied directly to the plate without dilution. A 1:500 to 1:1,000 dilution for normal human serum is suggested for best results. Human TAT complex in samples will bind to the anti-human thrombin capture antibody coated on the microtiter plate. After appropriate washing steps, monoclonal anti-human antithrombin primary antibody binds to TAT complex captured on the plate. Excess antibody is washed away and bound monoclonal antibody is then reacted with the secondary antibody conjugated to horseradish peroxidase. TMB substrate is used for color development at 450nm. A standard calibration curve is prepared along with the samples to be measured using dilutions of TAT complex. Color development is proportional to the concentration of TAT complex in the samples. All reagents and standards are provided in these ELISA kits.