QUANTITATIVE-EVALUATION OF PORCINE ENDOTHELIAL-CELL ANTIGENS RECOGNIZED BY HUMAN NATURAL ANTIBODIES - AN ANALYSIS BY WESTERN BLOTTING

Xenoreactive natural antibodies recognize a series of glycoproteins in porcine endothelial cell membranes, the epitopes being Gal alpha 1-3G al substitutions on those proteins. We recently identified the glycopr otein antigens as members of the integrin family, von Willebrand facto r, and DM-GRASP. The antibodies that react with these structures are a dsorbed during perfusion of a porcine organ, indicating that these ant igens may be the biologically relevant targets. To evaluate the relati ve importance of the glycoproteins as targets of xenoreactive natural antibodies, immunoreactive electrophoretic bands containing these glyc oproteins were analyzed by photodensitometry. The relevance of these a ntigens in the recognition of endothelial cell antigens by xenoreactiv e natural antibodies was assessed by an analysis of the differences be tween antigens recognized by normal human serum and those recognized b y human serum that had been depleted of xenoreactive natural antibodie s. Depletion of xenoreactive natural antibodies was performed by adsor ption of serum on cultured porcine aortic endothelial cell monolayers and by perfusion through a porcine kidney, The analysis revealed that greater than 91% of the antibodies binding to porcine cell surfaces ar e specific for antigens identified in porcine endothelial cell membran e extracts. Quantitative analysis of antigens recognized by affinity p urified antibodies confirmed that integrins are indeed the primary xen ogeneic targets recognized on Western blots of porcine aortic endothel ial cell membranes.