A simple enzyme-immunoassay test for von Willebrand factor binding in human arterial subendothelium

In order to determine the binding of vWF, subendothelium from everted human umbilical arteries was perfused with dialysed serum containing different concentrations of purified vWF using an annular perfusion chamber at a wall shear rate of 1100 sec-1 for 30 min. After perfusion, control (not perfused) and perfused vessel segments were washed and incubated with a diluted rabbit antibody against human vWF. Then the nonbound anti-vWF from both samples were used to determine indirectly vWF by EIA. Although in our experiments normal vWF serum concentrations were not enough to exert vWF binding, a substantial binding could be attained with vWF levels around 2.5 U/ml. To estimate the pre-existing subendothelial vWF amount, three different experiments were developed: a) diluted IgG from a nonimmunized rabbit, b) a diluted rabbit antibody to human vWF, c) PBS-BSA. After washing, vessel segments were incubated with rabbit antibody to human vWF. After incubation, the nonbound anti-vWF was used to determine indirectly vWF by EIA. The results obtained showed that the amount of pre-existing vWF was approximately 1.1x10(-3) U vWF/cm2 subendothelium.