Anti-endothelial cell antibodies in sera of patients with autoimmune diseases: comparison between ELISA and FACS analysis

Abstract

SUMMARYIn some patients suffering from rheumatoid arthritis (RA), vasculitis is a clear clinical manifestation, mentioned as rheumatoid vasculitis (RV). Autoantibodies directed againsi endothelial cells (AEA) have been implicated in the pathogenesis of this disorder, and it has been suggested in a number of studies that testing for AEA should be included in diagnosing RV. To test this hypothesis, we have evaluated the presence of AEA in sera of patients suffering from various autoimmune diseases, employing an ELISA with fixed cultured endothelial cells (EC). In ail the groups of patients ELlSA-positivc sera were present. A significant diflerence in percentage of positivity was found between the RA and RV group {P < 005). In addition, our results indicated that nol only antibodies directed against antigens on the EC membrane were detected, but atso antibodies directed against intracellular components like DNA. histones and cytoskeletal components. Therefore, we also tested all these patient sera on unfixed intact EC using indirect immunofluorescence followed by FACS analysis. Whereas in the total patient population 34 out of 65 patients were AEA-positivc as determined in the ELISA, only seven patienis were weakly positive when examined by flow cytomelry. We conclude that: (I) an ELISA on fixed EC does not specificaHy detect AEA, A positive test result is. however, to some extent correlated with the occurrence of vasculitis. and tnay therefore be helpful in diagnosing this disease: (ii) EACS analysis is a more suitable method than ELISA to measure the presence of membrane-specific AEA in patient sera; (iii) speciftc IgG-AEA are less common in patients suffering from autoimmune disorders than was assumed previously.

Anti-endothelial cell antibodies in systemic sclerosis

“…During the fixing process, permeabilisation of the substrate EC membranes occurs and intracellular antigens become accessible to autoantibodies which would not interact with ECs in vivo. This problem can be overcome by using unfixed cells 17 18…”

Markers of endothelial dysfunction in patients with non‐alcoholic fatty liver disease and coronary artery disease

“…Fluorescence signals were counted and the positive cells were recorded as a channel shifts value after subtraction of the negative control. This method was modified from Westphal et al …”

Anti‐Endothelial Cell Antibodies in Dogs with Immune‐Mediated Hemolytic Anemia and Other Diseases Associated with High Risk of Thromboembolism

“…Nonetheless, AECA have been widely detected in humans, by the same flow cytometry and IHC techniques we utilized in this study 11,21–23 . For example, AECA have been detected in humans with connective tissue diseases, systemic vasculitis or arteritis, systemic immune‐mediated diseases, and with accelerated coronary artery disease after cardiac transplant 9–11,21,24,25 .…”

“…Other approaches for screening for the presence of AECA in humans include use of cell‐based ELISA, cell cytotoxicity, and Western blotting 10 . However, flow cytometry appears to have the greatest sensitivity and specificity for the detection of AECA in humans 22 …”