Anti vwf ab

ELISA measured the binding of a panel of anti-D’D3 mAbs to substrates immobilized with 10μg/mL human VWF constructs: hV, hNV or hCV [8 (link)]. mAb (DD3.3) binding hNV at higher levels compared to hV and hCV, were further tested under physiological shear using a cone-plate viscometer. Here, 10μg/mL hV (recombinant wild-type VWF) was captured onto 3μm polystyrene beads bearing anti-VWF Ab (Dako) over 30min at room temperature, blocked with PBS containing 1%BSA and washed to remove unbound VWF. The beads were then subjected to fluid shear at 0-9600 s⁻¹ for 5min, in the presence of 10μg/mL anti-VWF FITC (Dako) to measure total bound VWF and 20 μg/mL DD3.3-Alexa 647 to detect protein confirmation change. The fluorescence intensity of anti-D’D3 mAb binding was normalized to anti-VWF FITC signal. In independent runs, the anti-D’D3 mAb DD3.5 replaced DD3.3 Alexa 647. Here, bead-bound DD3.5 was quantified after shear stoppage, using a secondary anti-mouse Alexa 647 Ab.

VWF concentration was determined using a flow cytometer-bead sandwich assay, using multimeric VWF purified from humate-P (CSL Behring) as a standard [4 (link)]. Human-murine VWF chimera levels in mouse plasma was determined using calibration curves generated by spiking VWF^−/− mouse plasma with human plasma pooled from 5 normal, adult volunteers.
VWF multimer distribution was assayed using 0.6-1.2% agarose gel electrophoresis [21 (link)]. To assay VWF susceptibility to proteolysis by ADAMTS13, 10μg/mL VWF was incubated with 10U/mL ADAMTS13 (WHO standard) and 1.6M urea at 37°C for 24h. Western blotting of cleaved VWF products was assayed under standard reducing conditions using 4%-8% SDS-PAGE followed by detection using anti-VWF Ab (Dako).