Figure 2
Figure 2. Characterization of heat-inactivated FVIII. (A) FVIII can be inactivated by heating to 56°C for 30 minutes, and the activity level can be measured using a chromogenic FXa substrate that can be read at A405. This graph demonstrates that preparations of FVIII can be heat-inactivated at concentrations as high as 100 μg/mL. (B) FVIII samples were heated in a fluorometer monitoring the ratio of fluorescence intensity at 370 nm to that at 330 nm with 280 nm excitation. Both the native and heated FVIII samples exhibit a sigmoidal transition between 46°C and 66°C, reflecting the distribution of the folded and unfolded protein in this temperature range. From this plot, it can be inferred that approximately one-third of the B-cell epitopes in the heat-inactivated FVIII are in their native conformation because the magnitude of the shift of the heat-inactivated FVIII is approximately one-third of the magnitude of the shift of the native, untreated FVIII. (C) ELISA plates were coated with either native or heat-inactivated FVIII for analysis of B-cell epitopes. The primary antibodies in this assay were monoclonals that bind to known domains of the native FVIII protein. If the antibodies were also able to bind heat-inactivated FVIII, then they have been described as binding stable domains. If the antibody cannot bind heat-inactivated FVIII, then its epitope has been lost during heating. This figure depicts a drawing of FVIII and its domains. Each monoclonal antibody used has been listed under the domain it binds. If the antibody binds a stable epitope, then it is labeled in black; if the epitope was lost during heating, then the name is labeled in gray. These data correlate with the fluorescence experiment because approximately one-third of the antibodies bind stable B-cell epitopes. (D) Five FVIII−/−/C57BL/6 mice were immunized with native FVIII, and proliferation assays were performed to compare the profile when cells are stimulated, in vitro, with native or heat inactivated FVIII. There is no significant difference between T-cell recall responses to native or heat-inactivated FVIII, indicating that heating does not alter uptake and presentation.

Characterization of heat-inactivated FVIII. (A) FVIII can be inactivated by heating to 56°C for 30 minutes, and the activity level can be measured using a chromogenic FXa substrate that can be read at A405. This graph demonstrates that preparations of FVIII can be heat-inactivated at concentrations as high as 100 μg/mL. (B) FVIII samples were heated in a fluorometer monitoring the ratio of fluorescence intensity at 370 nm to that at 330 nm with 280 nm excitation. Both the native and heated FVIII samples exhibit a sigmoidal transition between 46°C and 66°C, reflecting the distribution of the folded and unfolded protein in this temperature range. From this plot, it can be inferred that approximately one-third of the B-cell epitopes in the heat-inactivated FVIII are in their native conformation because the magnitude of the shift of the heat-inactivated FVIII is approximately one-third of the magnitude of the shift of the native, untreated FVIII. (C) ELISA plates were coated with either native or heat-inactivated FVIII for analysis of B-cell epitopes. The primary antibodies in this assay were monoclonals that bind to known domains of the native FVIII protein. If the antibodies were also able to bind heat-inactivated FVIII, then they have been described as binding stable domains. If the antibody cannot bind heat-inactivated FVIII, then its epitope has been lost during heating. This figure depicts a drawing of FVIII and its domains. Each monoclonal antibody used has been listed under the domain it binds. If the antibody binds a stable epitope, then it is labeled in black; if the epitope was lost during heating, then the name is labeled in gray. These data correlate with the fluorescence experiment because approximately one-third of the antibodies bind stable B-cell epitopes. (D) Five FVIII−/−/C57BL/6 mice were immunized with native FVIII, and proliferation assays were performed to compare the profile when cells are stimulated, in vitro, with native or heat inactivated FVIII. There is no significant difference between T-cell recall responses to native or heat-inactivated FVIII, indicating that heating does not alter uptake and presentation.

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