Dw. Koenig et al., A WESTERN-BLOT ASSAY DETECTS AUTOANTIBODIES TO CRYPTIC ENDOTHELIAL ANTIGENS IN THROMBOTIC MICROANGIOPATHIES, Journal of clinical immunology, 13(3), 1993, pp. 204-211
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.