Human Factor IX will bind to the affinity purified capture antibody coated on the microtiter plate. Factor IX and IXa will react with the antibody on the plate. After appropriate washing steps, peroxidase labeled polyclonal anti-human Factor IX primary antibody binds to the captured protein. Excess antibody is washed away and TMB substrate is used for color development at 450nm. A standard calibration curve is prepared using dilutions of purified Factor IX and is measured along with the test samples. Color development is proportional to the concentration of Factor IX in the samples.