Methods for the detection of anti-endothelial antibodies by enzyme-linked immunosorbent assay

doi:10.1016/0022-1759(91)90207-V

Journal of Immunological Methods

Volume 141, Issue 1, 26 July 1991, Pages 33-39

https://doi.org/10.1016/0022-1759(91)90207-V Get rights and content

Abstract

In order to obtain a routine simple screening test for the detection of anti-endothelial antibodies (AEA) we developed a highly reproducible and sensitive enzyme-linked immunosorbent assay (ELISA) on fixed endothelial hybridoma cells. Detection of AEA with this type of monolayer appeared to be superior to ELISAs with monolayers of human umbilical vein endothelial cells, unfixed endothelial hybridoma cells or assays with membranes of endothelial cells. Glutaraldehyde treated endothelial hybridoma cells are most appropriate for use in ELISA procedures to detect AEA because the endothelial hybridoma cells are easy to culture, form a constant antigenic source and when plated and fixed to microtitre plates they can be stored without losing their ability to bind AEA.

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Cited by (26)

Antiendothelial cell antibodies in systemic lupus erythematosus
2002, Autoimmunity Reviews
Sera from patients with systemic lupus erythematosus have been reported to contain IgM and/or IgG binding to endothelial cells (EC), i.e. anti-EC antibodies (AECA). Similar autoantibodies have been claimed to occur in a number of conditions associated with vasculitis. The original cyto-enzyme-linked immunosorbent assay (ELISA) remains the most widely used method for the detection of AECAs, although numerous pitfalls have been identified since then. These difficulties may explain why a consensus on the prevalence of AECAs has not been reached thus far. It is therefore desirable to confirm a positive result in the cyto-ELISA using other methods, such as flow cytometry, immunoprecipitation or Western blot. Yet, these methods appear to be difficult to use on a routine basis. With regard to the AECA effects, their binding induces activation of ECs, as substantiated by up-regulation of adhesion molecules, and synthesis of cytokines and chemokines, followed by their secretion. Some of these autoantibodies encourage the local production of tissue factor, and thereby favour coagulation. Other AECAs trigger apoptosis of ECs, although the Fas receptor does not seem to be involved in this process. In fact, since the target antigens are not well defined, the current challenge is to identify EC target molecules, and thus to gain further insights into the pathogenesis of diseases with vasculitis.
Peptide-retargeted adenovirus encoding a tissue inhibitor of metalloproteinase-1 decreases restenosis after intravascular gene transfer
2002, Molecular Therapy
Citation Excerpt :
Several virus complexes were made for the experiments: the first virus complex had one lysine residue on the surface (AdlacZ(lys)), the second complex had poly-lysine (AdlacZ(p-lys)), the third complex had HWGF-targeted peptide poly-lysine (AdlacZ(HWGF)), and the fourth complex had the mismatch-peptide poly-lysine on the surface (AdlacZ(MM)). β-Galactosidase activity in RAASMC [31], EaHy [32], and HEP-G2 (HB-8065) cell lines were measured after transduction at MOIs of 1 and 0.1 for four hours. O-Nitrophenyl β-D-galactopyranoside (ONPG) was used as a colorimetric substrate as described [31].
In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P<0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.
Variability in E-selectin expression, mRNA levels and sE-selectin release between endothelial cell lines and primary endothelial cells
2000, Cell Biology International
Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β), median (range) percentage of E-selectin-positive HUVECs increased from 1.6(0.9–6.2)% to 91.4(83.0–96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-α/IL-1β. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of adhesion.
An association between antibodies specific for endothelial cells and renal transplant failure
1998, Transplant Immunology
Human leucocyte antigen (HLA)-specific antibodies, present at the time of transplant, cause renal transplant rejection but cases of rejection of HLA-identical renal transplants indicate that antibodies to non-HLA antigens may also be detrimental. There is increasing evidence that antibodies to antigens present on endothelial cells and monocytes, and on endothelial cells alone, are associated with transplant rejection.
We investigated 105 patients with failed renal transplants for the presence of endothelial cell reactive antibodies and compared them with 94 successful transplant patients to determine the role of non-HLA antibodies in transplant failure. Patient sera were tested by enzyme-linked immunoabsorbent assay (ELISA) using as a target fixed cells either from the endothelial/epithelial cell line EAHy.926 or primary cultures of human umbilical vein endothelial cells. Antibody binding was detected using an alkaline phosphatase-conjugated anti-human immunoglobulin G (IgG) antibody.
Fourteen of the 105 failed transplant patients had endothelial cell-reactive antibodies as compared with only three of the 94 patients with successful transplants (Fisher's exact test, p = 0.02). Antibody-positive sera were absorbed with the epithelial cell line A549 to remove antibodies directed against the epithelial component of EAH.926 and with a pool of lymphoblastoid cell line cells to remove HLA-specific antibodies. Absorption did not reduce antibody activity showing the antibodies to be directed against endothelial cell determinants. Antibody-positive sera were also tested by flow cytometry against the monocyte cell line THP-1 and 13 of the 14 patients were negative.
In conclusion, we have demonstrated the presence of IgG antibodies directed against endothelial cell determinants in renal transplant recipients in association with renal transplant failure.
Anti-endothelial and epithelial antibodies in renal transplantation
1998, Transplantation Proceedings
Cytokines, adhesion molecules, antiendothelial cell autoantibodies and vascular disease
1997, Cardiovascular Pathology
Vascular endothelial cells present a surface to circulating blood elements that is continuously changing its phenotype under the influence of cytokines and other soluble mediators, and as a result of cell-cell interactions. Phenotypic changes enable endothelial cells to respond to local environmental conditions, for example, promoting pro-inflammatory or pro-coagulant properties. Antibodies may also develop to structures on this changing endothelial surface. The characteristics of antiendothelial cell antibodies identified to date and their perceived role in vascular disease is discussed.

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Methods for the detection of anti-endothelial antibodies by enzyme-linked immunosorbent assay

Abstract

Am. J. Med.

Clin. Immunol. Immunopathol.

Blood

Lancet

J. Immunol. Methods

A possible role for anti-endothelial antibodies in vasculitis

Arthritis Rheum.

Detection and quantitation of human Ia-type antigens with iodinated protein A and specific purification of antibodies against Ia-type alloantigens

Scand. J. Immunol.

Identification of the antibody to vascular endothelial cells in patients undergoing cardiac transplantation

Transplantation

Presence of complement-fixing anti-endothelial cell antibodies in systemic lupus erythematosus

J. Clin. Invest.

Permanent cell line expressing human factor VIII-related antigen established by hybridization

Anti-endothelial cell antibodies: detection and characterization in sera from patients with autoimmune hypoparathyroidism

Anti-endothelial cell antibodies: Detection and characterization using a cellular enzyme-linked immunosorbent assay

J. Lab. Clin. Med.