Tissue-type plasminogen activator (tPA) is a serine protease that activates plasminogen to plasmin in the blood fibrinolytic system. Plasminogen activator inhibitor type 1 (PAI-1) is involved in the regulation of the blood fibrinolytic system and forms a 1:1 covalent complex with tPA and uPA. PAI-1 tPA complex increases during pregnancy, artherosclerosis, sepsis, and are associated with myocardial infarction reoccurrence. High levels of PAI-1 tPA complex in breast cancer cytosols are associated with poor survival. PAI-1 tPA complex levels may also be useful as a prognostic indicator for renal/bladder cancer, multiple organ failure, and stroke. The sensitive quantitative measurement of total human PAI-1 tPA complex antigen in plasma, serum, urine, cell culture media, or tissue extract samples is easily performed with this 96 well strip format ELISA kit. Free tPA and PAI-1 will not be detected by this assay. Concentration of PAI-1 tPA complex in normal human plasma was found to be 2.8 ng/ml. The assay measures PAI-1 tPA complex in the 0.5-200 ng/ml range. Samples with complex levels above 200 ng/ml should be diluted in plasma or similar fluid devoid of PAI-1 or tPA. Human PAI-1 in samples will bind to the monoclonal capture antibody coated on the microtiter plate. Free, latent, and complexed PAI-1 will react with the capture antibody. After appropriate washing steps, polyclonal anti-human tPA primary antibody binds to the captured PAI-1/tPA complex. Excess primary antibody is washed away and bound antibody is then reacted with peroxidase conjugated secondary antibody. Following an additional washing step, 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 PAI-1/tPA complex. Color development is proportional to the concentration of PAI-1/tPA complex in the samples. A standard calibration curve is prepared in PAI-1 depleted plasma using dilutions of purified PAI-1 tPA complex and is measured along with the test samples. All reagents and standards are provided in these ELISA kits.