A double antibody radioimmunoassay for the ristocetin aggregation factor

Patients with von Willebrand's disease lack a plasma factor (Ristocetin Aggregation Factor, RAF) required for ristocetin induced platelet aggregation. RAF free of contaminating factor VIII activity was prepared by the agarose chromatography of hemophilic plasma and used to immunize rabbits. The rabbit antisera inhibited ristocetin induced aggregation of normal platelets and prevented platelet retention by glass bead columns. While the antisera reduced the factor VIII activity present in whole plasma, there was no inactivation of factor VIII procoagulation activity dissociated from RAF by chromatography using 1 M NaCl as eluant. High potency RAF by radiolabelling was prepared free of factor VIII by the 1 M NaCl agarose chromatogrphy of a commercial AMF concentrate (Profilate, Abbott Lab.). This material was labelled with ¹²⁵I using the peroxide lactoperoxidase method. Radioiodination of RAF did not affect biological activity, whereas similar treatment of factor VIII resulted in a reduction of activity proportional to the intensity of labelling. Chromatography of the labelling material showed an identical peak of elution of the radioactivity and biological activity. Radioimmunoelectrophoresis of the labelled peak demonstrated a single precipitin arc with the rabbit anti RAF serum. Dilutions of an RAF standard were incubated first with rabbit antiserum, then with the radiolabelled RAF, and finally with goat anti rabbit serum. Precipitated radioactivity from serial dilutions of RAF standard ranged from 19% (undiluted) to 61% (1:31 dilution) while the serum from a patient with severe von Willebrand's disease showed 65% precipitation. These values compared favorably (r = 0.93) with those obtained using the washed platelet assay system of Weiss et al.