Prothrombin is a single-chain vitamin K dependent 579 amino acid glycoprotein zymogen. Prothrombin levels are decreased by anticoagulant therapy, vitamin K deficiency and severe liver disease. Elevated plasma prothrombin is associated with a single nucleotide change at position 20210. The sensitive quantitative measurement of total human prothrombin antigen in plasma samples is easily performed with this 96 well strip format ELISA kit. Thrombin and thrombin-antithrombin complex will be detected by the assay. Prothrombin in normal human plasma ranges from 110-212 ug/ml with an average concentration of 150 ug/ml. The assay measures total human prothrombin in the 0.25-100 ng/ml range. Samples giving human prothrombin levels above 100 ng/ml should be diluted in blocking buffer before use. A 1:5,000 to 1:40,000 dilution for plasma is suggested for best results. Human prothrombin will bind to the capture antibody coated on the microtiter plate. After appropriate washing steps, anti-human prothrombin primary antibody binds to the captured protein. Excess primary antibody is washed away and bound antibody is reacted with the secondary antibody conjugated to HRP. Following an additional washing step, TMB substrate is used for color development at 450nm. Color development is proportional to the concentration of prothrombin in the samples. A standard calibration curve is prepared using dilutions of purified prothrombin and is measured along with the test samples. All reagents and standards are provided in these ELISA kits.

The human prothrombin standard provided is calibrated against a reference plasma standardized to the SSC/ISTH Secondary Coagulation Standard distributed by NIBSC, South Mimms, Potters Bar, Hertfordshire, UK.