Fig 1.
Scheme for measurement of active VWF.
(A) The protein domain structure of mature VWF (adapted from Crawley et al.[27]). (B) The set-up of the immunosorbent assay described here, based on a variable heavy chain antibody fragment (VHH) directed against a cryptic epitope in the A1 domain of VWF as capture antibody, and a polyclonal rabbit anti-human VWF antibody conjugated with HRP as detecting antibody.
Fig 2.
Analytical characteristics of the active VWF assay.
(A) Binding of the VHH to R1306W VWF (●), R1306Q VWF (○), HVWF (σ), VWF in NPP under static conditions (π) and NPP after vortexing to simulate shear stress (▪, only 10x diluted). Dilution factors are indicated at the x-axis. (B) Mean active VWF levels in plasma pools (with low (91–110%), medium (150–200%) and high (>220%) active VWF levels) with corresponding SDs for 20 replicates on a single plate on the same day (intra-assay, white bars) or on 20 separate days (inter-assay, grey bars). (C) Recovery (%) of increasing levels of R1306W VWF spiked to plasma pool. The acceptance range of 90–110% of expected values is indicated by dotted lines. (D) Assay response (OD at 490 nm) for a 12-step, 1.2-fold dilution series of plasma spiked with R1306W. Linear regression parameters (±SD) are: slope 0,21 ± 0.006, intercept 0.03 ± 0.005, SD of residuals 0.007. (E) Low (L) and high (H) active VWF plasma pools were mixed in varying proportions to assess dilutional linearity. (F) Stability of active VWF in a plasma pool at baseline and after multiple freeze-thaw steps (Thaw 1–4). The acceptance criterium of <10% analyte loss is indicated by the dotted line. Data represent the mean±SD (n = 3) unless otherwise stated.
Table 1.
Analytical performance characteristics of the active VWF ELISA assay.
Table 2.
Demographic and laboratory results of healthy donors in reference interval study.
Table 3.
Reference intervals for active VWF and other VWF parameters in 120 healthy individuals.
Fig 3.
Correlation of active VWF with other VWF parameters.
Scatterplots and corresponding Spearman rank correlations (r) between active VWF assay (VWF:Act) and (A) VWF:Ag, (B) VWF:RCo, (C) VWF:GP1bM, (D) VWFpp and (E) Plt:VWF binding. Dotted lines delineate reference intervals determined in this study, with the centre square containing 95% of values. Spearman rank correlation coefficients (r) are indicated in the upper left corner of each panel.