Figure 5
Figure 5. Quantitation by ELISA and measurement of the specific activity of wild-type and Δ6/7 recombinant FXIs. pcDNA3/FXI, or pcDNA3/FXI-Δ6/7, or equimolar amounts of both plasmids (heterozygous condition) were transiently transfected in COS-1 cells. Equal numbers of cells and equal amounts of plasmids were used in transfection experiments, as described in “Expression and quantitation of recombinant Δ6/7 FXI.” (A) Antigen levels of recombinant FXI were measured in both conditioned media and the corresponding cell lysates using an ELISA assay. Bars represent relative concentrations of protein in media and cell lysates compared with the mean antigen level measured in the wild-type, set as 100%. Results are given as mean ± SEM. (B) The specific activities of recombinant proteins were determined by calculating the ratio between FXI activity (measured using a one-stage method based on a modified partial thromboplastin time) and FXI antigen levels. Bars represent mean ± SEM of 3 independent experiments, each performed in duplicate on different days in different cell batches. The mean value of wild-type FXI was set as 100%. The results were analyzed by unpaired t test. *P < .05; **P < .01; ***P < .001; n.d. indicates not detected. The detection limits of the FXI functional and immunologic assays were 1% and 0.1%, respectively.

Quantitation by ELISA and measurement of the specific activity of wild-type and Δ6/7 recombinant FXIs. pcDNA3/FXI, or pcDNA3/FXI-Δ6/7, or equimolar amounts of both plasmids (heterozygous condition) were transiently transfected in COS-1 cells. Equal numbers of cells and equal amounts of plasmids were used in transfection experiments, as described in “Expression and quantitation of recombinant Δ6/7 FXI.” (A) Antigen levels of recombinant FXI were measured in both conditioned media and the corresponding cell lysates using an ELISA assay. Bars represent relative concentrations of protein in media and cell lysates compared with the mean antigen level measured in the wild-type, set as 100%. Results are given as mean ± SEM. (B) The specific activities of recombinant proteins were determined by calculating the ratio between FXI activity (measured using a one-stage method based on a modified partial thromboplastin time) and FXI antigen levels. Bars represent mean ± SEM of 3 independent experiments, each performed in duplicate on different days in different cell batches. The mean value of wild-type FXI was set as 100%. The results were analyzed by unpaired t test. *P < .05; **P < .01; ***P < .001; n.d. indicates not detected. The detection limits of the FXI functional and immunologic assays were 1% and 0.1%, respectively.

Close Modal

or Create an Account

Close Modal
Close Modal