Figure 5.
Detection of intact VWF in congenital VWD. (A) VWF-deficient plasma was spiked with different amounts of purified rVWF and degraded VWF, and incubated in microtiter plates coated with KB-VWF-D3.1. Bound VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent mean ± SD of 3 to 4 independent measurements. The solid line illustrates the best linear fit, with 95% confidence intervals indicated with the dotted lines. The vertical line indicates 90% intact rVWF supplemented with 10% degradedVWF. (B, C) Patient plasma samples were analyzed for total antigen using polyclonal antibodies, and for intact VWF using KB-VWF-D3.1. Normal pooled plasma (NPP) was used as a calibrator. Presented is the ratio of intact VWF to total VWF antigen. Each individual sample is represented by a closed symbol. Statistical analysis was performed via a one-way analysis of variance with Dunnett’s correction for multiple comparisons (B) or Mann-Whitney (C). (D) Multimers were analyzed via SDS-agarose (Ag) electrophoresis. The relative amount of multimers exceeding 10 bands was determined via comparison to NPP. (E) Plotted is the ratio of intact VWF to total VWF antigen vs the relative amount of large multimers. Correlation was determined using GraphPad Prism Software (GraphPad, La Jolla, CA).

Detection of intact VWF in congenital VWD. (A) VWF-deficient plasma was spiked with different amounts of purified rVWF and degraded VWF, and incubated in microtiter plates coated with KB-VWF-D3.1. Bound VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent mean ± SD of 3 to 4 independent measurements. The solid line illustrates the best linear fit, with 95% confidence intervals indicated with the dotted lines. The vertical line indicates 90% intact rVWF supplemented with 10% degradedVWF. (B, C) Patient plasma samples were analyzed for total antigen using polyclonal antibodies, and for intact VWF using KB-VWF-D3.1. Normal pooled plasma (NPP) was used as a calibrator. Presented is the ratio of intact VWF to total VWF antigen. Each individual sample is represented by a closed symbol. Statistical analysis was performed via a one-way analysis of variance with Dunnett’s correction for multiple comparisons (B) or Mann-Whitney (C). (D) Multimers were analyzed via SDS-agarose (Ag) electrophoresis. The relative amount of multimers exceeding 10 bands was determined via comparison to NPP. (E) Plotted is the ratio of intact VWF to total VWF antigen vs the relative amount of large multimers. Correlation was determined using GraphPad Prism Software (GraphPad, La Jolla, CA).

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