Preparation and characterization of monoclonal antibody against recombinant human tissue factor pathway inhibitor

OBJECTIVE: To prepare and identify monoclonal antibody (McAb) against recombinant human tissue factor pathway inhibitor (rhTFPI) and to use it for measurement of TFPI by ELISA, and to evaluate the effects of the McAb on dilute prothrombin time (PT) and activated partial thromboplastin time (APTT). METHODS: After intrasplenic immunization of Balb/c mouse with TFPI, hybridoma technique was used to raise monoclonal antibody against rhTFPI. The McAb was well-characterized and labelled with horseradish peroxidase (HRP) by using assay of TFPI in ELISA. Furthermore, the McAb was added to normal and factor IX deficient plasma for observation of dilute PT and APTT. RESULTS: Two hybridomas (4F4, 4F8) secreting McAb against TFPI were established. The Ig class and subclass of the McAb purified from 4F8 was IgG1. Immunoblotting results indicated that the McAb4F8 only recognized a single band of TFPI with molecular weight of 34.8 KD. The results of Sandwich enzyme-linked immunosorbent assay (ELISA) by using the HRP labelled McAb4F8 showed that the mean of TFPI in normal human plasma is 103.2 +/- 11.5 micrograms/L. The McAb 4F8 was also proved to shorten markedly dilute prothrombin time of factor IX deficient plasma and normal plasma. CONCLUSIONS: We established two hybridomas cell lines (4F4, 4F8) and obtained the McAb4F8 against TFPI and reported the levels of TFPI in healthy adult human plasma by Sandwich ELISA with HRP labelled McAb4F8 in Chinese.