Preparation And Application Of Monoclonal Antibody Against Human Coagulation Factor VII

Objective: (1) To isolate and purify human coagulation factor VII from human plasma Cohn fraction III. (2) To prepare hybridoma cell lines which can secrete monoclonal antibody against human coagulation factor VII; (3) To purify and preliminarily apply monoclonal antibody against human coagulation factor VII. Methods: (1) The purification procedure of human factor VII from Cohn fraction III involves resolving fraction III, absorbing factor VII onto barium citrate and eluting it, ammonium sulfate fractionation and DEAE-Sepharose Fast Flow ion exchange chromatography, Sephadex G-100 gel filtration chromatography. (2) BALB/c mice were immunized with the purified FVII. Hybridoma technique and ELISA were used to prepare and screen hybridoma cell lines which secreted monoclonal antibody against human coagulation factor VII. Hybridoma cells were injected to abdominal cavity of BALB/c mice for ascites. Monoclonal antibodies were purified from ascites with ammonium sulfate fractionation and DEAE-affi-gel-blue chromatography. The purity and subclasses of monoclonal antibody were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Mouse Monoclonal Antibody Isotyping Kit. The characteristics of these antibodies were analyzed, including the cross reaction between monoclonal antibody and factor II (FII), factor IX (FIX), factor X (FX), respectively, and the effect of divalent mental ion on the combination between monoclonal antibody and FVII. (3) The purified monoclonal antibodies were coupled to GoldMag-@ magnetic particulate. The efficiency of coupled reaction was detected. Antibodies coupled to magnetic particulate were reacted with plasma and concentrated FVII. The coupled FVII was eluted from the surface of magnetic...