Figure 10.
Identification of components in immune complexes in baboon serum after porcine pulmonary xenotransplantation. Sera obtained from baboon blood taken before and 15 minutes after pulmonary xenotransplantation were diluted 5:1 with 12% PEG 8000 and 60 mmol/L EDTA in VBS for 16 hours at 4°C and then centrifuged at 2,000 × g for 20 minutes at 4°C to pellet immune complexes. Precipitates were resuspended in PBS and incubated with Galα1-3Galβ (Bdi) -PAA immobilized on Sepharose CL6B. Pig serum was incubated with immobilized Galα1-3Galβ as a control. The beads were washed with PBS and then boiled in reducing sample buffer to release bound material. The released material was separated by SDS-PAGE (7.5% PA), transferred to PVDF, and stained as follows. Lane 1, released material from pretransplant serum reacted with goat anti-human IgM. Lane 2, released material from posttransplant serum reacted with goat anti-human IgM. Lane 3, released material from pretransplant serum reacted with mouse anti-porcine vWF and then with goat anti-mouse IgG. Lane 4, released material from posttransplant serum reacted with mouse anti-porcine vWF and then with goat anti-mouse IgG. Lane 5, released material from pig serum reacted with mouse anti-porcine vWF factor and then with goat anti-mouse IgG. Porcine vWF is associated with baboon IgM.