Journal of Clinical Immunology

, Volume 13, Issue 3, pp 204–211

A western blot assay detects autoantibodies to cryptic endothelial antigens in thrombotic microangiopathies


  • Daniel W. Koenig
    • Division of Nephrology, Departments of Medicine and Cell BiologyVanderbilt University
  • Lise Barley-Maloney
    • Division of Nephrology, Departments of Medicine and Cell BiologyVanderbilt University
  • Thomas O. Daniel
    • Division of Nephrology, Departments of Medicine and Cell BiologyVanderbilt University
Original Articles

DOI: 10.1007/BF00919973

Cite this article as:
Koenig, D.W., Barley-Maloney, L. & Daniel, T.O. J Clin Immunol (1993) 13: 204. doi:10.1007/BF00919973


Autoantibodies detected by immunofluorescence, ELISA, and complement-fixation techniques have provided discriminatory markers for many human diseases. However, these commonly applied assays may fail to detect antibodies against antigenic sites which are either inaccessible or not displayed in recognizable cellular structures. Moreover, molecular identities 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 autoantibodies 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 number of distinct HRMEC proteins. Among these, antibodies recognizing specific 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 healthy subjects and 11 patients with a range of diseases not associated with endothelial injury of insult. Antibodies binding 43-kDa HRMEC antigens were detected in individual patients with systemic lupus erythematosus, anti-glomerular basement membrane nephropathy, and heparin-associated thrombocytopenia, as well as in one of three patients with immune thrombocytopenic purpura. Similar antibodies were detected in one 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 compartments, sites presumably protected from immune recognition in the absence of endothelial injury. Western blot recognition of antiendothelial antibodies offers opportunities to define molecular characteristics and cellular distribution of antigens while generating reagents useful in their purification.

Key words

Western blotautoantibodiescryptic endothelial antigensthrombotic microangiopathies

Copyright information

© Plenum Publishing Corporation 1993