A WESTERN-BLOT ASSAY DETECTS AUTOANTIBODIES TO CRYPTIC ENDOTHELIAL ANTIGENS IN THROMBOTIC MICROANGIOPATHIES

Autoantibodies detected by immunofluorescence, ELISA, and complement-f ixation techniques have provided discriminatory markers for many human diseases. However, these commonly applied assays may fail to detect a ntibodies against antigenic sites which are either inaccessible or not displayed in recognizable cellular structures. Moreover, molecular id entities of recognized antigen(s) are not determined with such methods . We have used Western blot analysis of cellular proteins derived from human renal microvascular endothelial cells (HRMEC) to identify autoa ntibodies in patients with pathological endothelial injury. Exploring the possibility that endothelial injury may expose cryptic endothelial antigens to immune recognition, we detected antibodies binding a numb er of distinct HRMEC proteins. Among these, antibodies recognizing spe cific HRMEC proteins of 43 kDa were commonly detected in plasmas from patients with thrombotic thrombocytopenic purpura (TTP) (13 of 14) and hemolytic uremic syndrome (HUS) (4 of 5) but were absent in 9 of 10 h ealthy subjects and 11 patients with a range of diseases not associate d with endothelial injury or insult. Antibodies binding 43-kDa HRMEC a ntigens were detected in individual patients with systemic lupus eryth ematosus, anti-glomerular basement membrane nephropathy, and heparin-a ssociated thrombocytopenia, as well as in one of three patients with i mmune thrombocytopenic purpura. Similar antibodies were detected in on e hypercholesterolemic subject. Antibodies from four TTP patients were affinity purified and shown by two-dimensional analysis to recognize 43-kDa proteins having identical pl's (5.9, 6.0, and 6.1). Subcellular fractionation localized these antigens to cytosolic and nuclear compa rtments, sites presumably protected from immune recognition in the abs ence of endothelial injury. Western blot recognition of antiendothelia l antibodies offers opportunities to define molecular characteristics and cellular distribution of antigens while generating reagents useful in their purification.