Tissue factor pathway inhibitor (TFPI) is a circulating plasma component which binds to coagulation Factor Xa (F Xa) and inhibits F Xa activity. This bimolecular complex can then bind to the Factor VIIa-tissue factor complex (VIIa-TF) and, in so doing, inhibit the Factor X (F X) and Factor IX (F IX) -activating ability of the VIIa-TF. Because TFPI exhibits many functions, a monoclonal antibody which blocked the activity of any specific function would be a useful tool for characterizing this molecule. For this reason mice were immunized with purified TFPI, splenic cells isolated and fused with myeloma cells and the resulting hybrid populations were screened for anti-TFPI activity. In order to identify monoclonal antibodies against the various specific regions of TFPI, a number of assays were developed and used in the screening of the isolated monoclonal hybridomas. One assay was developed to detect antibody which neutralized the inhibitory ability of TFPI for F Xa cleavage of a synthetic substrate. A second activity assay was developed to detect the ability of antibody to block the VIIa-TF inhibitory activity of the F Xa-TFPI complex. Because an antibody which neutralized the F Xa inhibitory activity would be expected to function in the second type of neutralization as well, both assays were necessary to characterize antibody activity. A third antibody screening assay was also used. This technique incorporated the standard ELISA methodology for detection of antibodies bound to immobilized TFPI. To date no anti- TFPI monoclonal antibodies have been detected with any of these three assays in spite of reasonable sensitivity demonstrated using rabbit antisera as positive controls.
