| Objectives:To investigate how to purify human coagulation factorⅦ(FⅦ) from Cohn fraction III precipitate combining DEAE ion exchange chromatography with Heparin affinity chromatography, and to identify purification product. To produce monoclonal antibodies of FⅦ(FⅦMcAb) by using hybridoma cell lines recovered and selected. To produce coagulation factorⅦdeficient plasma (FⅦDP) and evaluate the function of the plasma.Methods:Initially, FⅦwas isolated and purified from Cohn fraction III precipitate via dissolving fractionⅢ, absorbing FⅦonto barium citrate and eluting, and ammonium sulfate fractionation. Secondly, by the methods of DEAE-Sepharose Fast Flow ion exchange chromatography and Heparin Sepharose CL-4B affinity chromatography, FⅦwas purified further. And then purity of FⅦwas identified by SDS-PAGE. FⅦcoefficient (FVE:C) and concentration were measured by the methods of one-stage coagulation assays and ultraviolet spectrometry separately.The hybridoma cell lines were recovered and injected to abdominal cavity of BALB/c mice for ascites. The FⅦMcAb were obtained from ascitic fluid of BALB/c mice in which the antibody-producing cells had been grown as ascitis tumor. Then, FⅦMcAb was purified from ascitic fluid using HiTrap rProtein A HP. The purity and isotype of McAb 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 factorⅡ(FⅡ), factorⅨ(FⅨ), factorⅩ(FⅩ), respectively, and the effect of divalent mental ion on the combination between monoclonal antibody and FⅦ.The McAb was coupled with CNBr-activated Sepharose 4B to form an affinity chromatographic column. The FⅦDP was obtained when normal fresh plasma passed through the column. Coagulation factors concentrations were measured by the methods of one-stage coagulation assays. To evaluate the precision of the immunodepleted plasma substrate, the linear ranges of the immunodepleted plasma substrate were investigated, comparing with imported commercial products.Results:With above approaches, the purification factor was (4954.22±164.43) fold, the specific clotting activity of purified FⅦwas (147.19±4.89) U/mg, and the overall yield recovery rate of FⅦwas (21.09±1.93)% (n=5). There was only one clear protein band at about 50 kD site of Protein Molecular Weight Marker on the gel.Ten hybridoma cell lines were recovered. Eight monoclonal antibodies were purified from ascites which were induced by eight selected hybridoma cell lines and renamed with 1 to 8 respectively. The isotype of the antibodies were IgGl. All antibodies hadn't cross reaction with coagulation FⅡ, FⅨ, FⅩrespectively. Four antibodies could inhibited the clotting activity of FⅦin plasma. No mental ion could remarkably restrain the combination of FⅦwith these antibodies against FⅦ.The FⅦDP were produced by using affinity chromatographic column coupled with the artificial FⅦMcAb. The FW in this biodepleted FWDP gained was removed completely (FⅦ:C<1%) and the amount or activity of other coagulation factors remained unchanged (FⅡ:C,FⅤ:C,FⅧ:C,FⅨ:C and FⅩ:C>70%).The precisions and coefficients of variation of three batches of production within batches were Sr=3.30, Sr=3.36, Sr=3.30 and CV=4.12%, CV=4.11%, CV=3.98%, respectively, while the between-run precision and coefficient of variation are Srr=4.20 and CV=4.99%, respectively. The linear range of three batches of production and the measured results of a series of samples were evaluated by making a contrast with congeneric commercially available FⅦDP. The preliminary evaluation indicates that. This biodepleted substrate is of high precision and with broad linear range. Moreover, it gave an excellent standard curve (related coefficients are r=-0.998, r=-0.996, r=-0.996, respectively) over a wide range of normal plasma dilutions (1.625%-100%) and a good correlation of FⅦ:C concentrations with those obtained using a commercial substrate (r=-0.998) for a series of samples (r=0.97,r=0.96,r=0.97).Conclusions:DEAE-Sepharose Fast Flow ion exchange chromatography and Heparin affinity chromatography are effective in concentrating FⅦfrom fractionⅢ, which will optimize FⅦpurification technology. The hybridoma cell lines which secreted monoclonal antibodies against factorⅦwere successfully recovered. Purified antidodies were obtained.The plasma could be used as a substrate in a one stage coagulation assay and performed as well as commercially available FⅦDP.
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