PREPARATION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODY AGAINST RECOMBINANT HUMAN TISSUE FACTOR PATHWAY INHIBITOR

Objective To prepare and identify monoclonal antibody (McAb) against r ecombinant human tissue factor pathway inhibitor (rhTPPl) and to use i t for measurement of TFPI by ELISA, and to evaluate the effects of the McAb on dilute prothrombin time (PT) and activated partial thrombopla stin time (APTT). Methods After intrasplenic immunization of Balb/c mo use with TFPI, hybridoma technique was used to raise monoclonal antibo dy against rhTFPl. The McAb was well-characterized and labelled with h orseradish peroxidase (HRP) by using assay of TFPI in ELISA. Furthermo re, the McAb was added to normal and factor IX deficient plasma for ob servation of dilute PT and APTT. Results Two hybridomas (4F4, 4F8) sec reting McAb against TFPI were established. The Ig class and subclass o f the McAb purified from 4F8 was IgG(1). Immunoblotting results indica ted that the McAb4F8 only recognized a single band of TFPI with molecu lar weight of 34. 8 KD. The results of Sandwich enzyme-linked immunoso rbent assay (ELISA) by using the HRP labelled McAb4F8 showed that the mean of TFPI in normal human plasma is 103.2+/-11.5 mu g/L. The McAb 4 F8 was also proved to shorten markedly dilute prothrombin time of fact or IX deficient plasma and normal plasma. Conclusions We established t wo hybridomas cell lines (4F4, 4F8) and obtained the McAb4F8 against T FPI and reported the levels of TFPI in healthy adult human plasma by S andwich ELISA with HRP labelled McAb4F8 in Chinese.