Rabbit anti human vwf pab

The concentration of recombinant VWF (rVWF) in each of the harvest fractions was determined by enzyme-linked immunosorbent assay, using two anti-VWF mAbs for capture: AVW-1 and 105.4 (BloodCenter of Wisconsin). Bound VWF was detected using a rabbit-anti-human VWF pAb (Dako, Carpinteria, CA), and a horseradish peroxidase-conjugated goat-anti-rabbit antibody. Sample concentrations were calculated by comparing sample signals to a known range of pooled normal plasma (PNP) dilutions on the same 96-well plate, where undiluted PNP VWF concentration was assumed to be 1 U/mL. Reported values of percent retention were calculated by dividing the total amount of rVWF in the lysate plus pellet fractions by the total amount of rVWF detected for each transfection (supernatant, plus lysate, plus pellet). Reported values of percent secretion were calculated by dividing the total amount of rVWF in the conditioned media by the total amount of VWF detected for each transfection (supernatant, plus lysate, plus pellet).

ELISA plates (Nunc-Immuno Maxisorp; Nunc A/S, Roskilde, Denmark) were coated with 1 μg/mL human type III collagen (Southern Biotech, Birmingham, AL) diluted in a calcium carbonate buffer and incubated at 4°C overnight. The plate was then washed and blocked with a 1% BSA solution for an hour before the samples were added at various dilutions and left to incubate for an hour. The plate was then washed, and samples were incubated with a rabbit-anti-human VWF pAb (Dako, Carpinteria, CA) before a wash and the addition of a HRP-conjugated goat-anti-rabbit antibody (Bio-Rad Laboratories, Hercules, CA). Absorbance was read at 450 nm in a plate reader (Synergy 2, BioTek). Collagen-binding ratio was calculated by evaluating the ratio between collagen-binding and VWF Ag levels. This ratio represents the biological capacity of the available VWF to bind to collagen. As in prior studies,^{59 (link)} we calculated ratios of VWF:collagen binding/VWF:Ag with normal defines as ≥0.7.