A multispecies enzyme-linked immunosorbent assay for von Willebrand's factor

A modified double-sandwich enzyme-linked immunosorbent assay (ELISA), which was developed for human and canine von Willebrand's factor (vWF), was adapted for quantitation of vWF in other species. In addition to human and dog plasmas, 12 other mammalian plasmas that were surveyed exhibited significant cross-species reactivity with antibodies specific for canine vWF or monoclonal antibodies against porcine or bovine vWF. Mixed combinations of monoclonal antibodies and various polyclonal antibodies were also used as sandwich or capture antibodies. The ability of this multispecies ELISA to detect less than 0.002 U of vWF per milliliter of plasma in a large number of species enhances its utility for both research and clinical diagnostic applications. A quantitative assay for rabbit vWF, which exhibits poor cross-species reactivity with most antibodies, was constructed with anti-porcine monoclonal antibody W1-5 and goat-anti-canine vWF as capture and sandwich antibodies, respectively. The same conjugate antibody configuration was used to visualize rabbit vWF multimers by immunoblotting. Specificity of the assay for vWF in human, dog, pig, and horse plasmas was confirmed by use of species-specific vWF-deficient plasmas. In other species, for which vWD plasmas were not available, ELISA specificity for vWF was demonstrated by recovery of greater than 75% of the ELISA-reactive antigen coincident with vWF multimers in the high-molecular-weight (greater than 500 kd) fractions of purified plasmas. This multispecies ELISA permits, for the first time, the measurement of vWF in a variety of mammals for which species-specific immunologic reagents do not currently exist. The results also suggest that certain vWF epitopes have been highly conserved among phylogenetically diverse species.