Background/Purpose: Sera from patients with systemic vasculitis or inflammatory conditions have been reported to contain antibodies that bind to endothelial cells (EC), i.e., AECA (anti-endothelial cell antibodies). AECA are known to play immunogenic effects by triggering EC activation and vascular damage, but the immunopathological role of AECA is not clear. SDS-PAGE and Western blotting have previously been used for detecting target antigens of AECA. However, we assumed that these methods are not appropriate for searching genuine target antigens on cell surface, and developed a novel solubilized cell surface protein-capture ELISA (CSP-ELISA).

Methods: Antigens were obtained as cell surface proteins from the plasma membrane of human umbilical vein endothelial cells (HUVEC); these cell surface proteins were biotinylated, solubilized with detergent, and captured on ELISA wells coated with NeutrAvidin biotin binding protein. AECA titers in serum from 126 autoimmune disease patients and 122 healthy controls (HC) were tested. Additionally, sera from 52 patients with biopsy-proven lupus nephritis (LN), 25 with systemic lupus erythematosus (SLE) without renal involvement (non-LN SLE), 10 disease controls (DC) and 81 healthy controls were tested for IgG- and IgA-AECA to human glomerular EC (HGEC) by CSP-ELISA.

Results: IgG-AECA were detected in 28 of 36 (78%) of SLE patients; in 13 of 16 (81%) of mixed connective tissue disease (MCTD) patients; in 5 of 9 (56%) of systemic sclerosis (SSc); and in 4 of 122 (3%) of healthy controls. Relatively weak denaturation of antigens on ELISA wells caused loss of binding of these autoantibodies. Titers of IgG- and IgA- AECA to HGEC were significantly higher in LN and non-LN SLE patients than in the combined DC and HC (P < 0.001) groups. The level of IgG-AECA did not correlate with active lesions, but the level of IgA-AECA to HGEC did correlate with histological evidence of active lesions in LN patients (P < 0.001). Immunocytochemical analysis showed AECA recognized membrane proteins on HGEC. The significant correlation of titer of AECA to both HGEC and HUVEC (R = 0.95 for IgG-, 0.93 for IgA-AECA, respectively) indicated AECA in LN patients recognize membrane proteins expressed on HGEC and HUVEC. To identify specific antigens against AECA, biotinylated CSPs were incubated with sera from LN patients with high titers of IgG-AECA, immunoprecipitated with immobilized protein G followed by immobilized avidin, and blotted with NeutrAvidin. A 150-kDa protein band that shifted to a 55-kDa protein band under reducing conditions was detected in patients with LN, but not in HC.

Conclusion: This newly developed CSP-ELISA method enables the detection of antibodies to the labile epitopes of autoantigens such as membrane proteins, and this method is generally applicable to various kinds of membrane proteins and the antibodies against them. IgA-AECA was observed to be associated with pathological activity in LN. These EC membrane components recognized by AECA may be linked with the pathogenesis of LN. We propose CSP-ELISA for measuring AECA in serum samples for routine laboratory testing.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/autoantibodies-directed-against-cell-surface-components-in-autoimmune-disease-patients-proposal-of-a-novel-elisa-for-the-detection-of-anti-endothelial-cell-antibodies/