False Positivity in a Cyto-ELISA for Anti-Endothelial Cell Antibodies Caused by Heterophile Antibodies to Bovine Serum Proteins Ronan Revelen, Anne Bordron, Maryvonne Dueymes, Pierre Youinou, and Josiane Arvieux * Background: ELISAs with fixed endothelial cells or cell lines are widely used screening tests for anti-endothe- lial cell antibodies (AECAs), but spurious increases occur. We examined interferences by heteroantibodies and means to eliminate them. Methods: AECAs were measured by ELISA on fixed layers of the human endothelial cell line, EA.hy 926, in a panel of 60 patient serum samples diluted in bovine serum albumin. Heteroantibodies against fetal calf se- rum (FCS) proteins were demonstrated and character- ized in an ELISA—the interference assay—that used FCS-coated plates and Tween 20-containing buffer as blocking agent and sample diluent, as well as by immu- noblotting. Results: In 12 of 60 patient serum samples, spurious increases of AECA titers were produced by endogenous antibodies reacting with FCS proteins from culture medium that were coated onto the solid-phase at the time of cell plating. This mechanism of interference was supported experimentally by exposing extracellular ma- trix, varying cell density, and incubating wells with FCS alone. The heterophile antibodies were mainly IgG and IgA, and in inhibition experiments, they recognized serum proteins from goat, sheep, and horse. Washing cells free of FCS before plating, or adding FCS (100 mL/L) to the patient sample diluent eliminated spurious signals from all 30 tested sera, but the latter method had practical advantages. Conclusions: Antibodies against animal serum proteins are a frequent cause of erroneous results in cyto-ELISAs. The interference can be eliminated by simple antibody absorption in FCS-containing dilution buffer. © 2000 American Association for Clinical Chemistry Anti-endothelial cell antibodies (AECAs) 1 represent a heterogeneous group of antibodies against poorly charac- terized targets that have been reported in a variety of inflammatory disorders, and they may be valuable as markers of disease activity (1). Often considered as an epiphenomenon of vascular injury, AECAs may also play a role in the pathophysiology of associated diseases, especially by inducing endothelial cell activation and/or apoptosis (1, 2). Numerous methods have been devel- oped to assay AECAs, including immunofluorescence, immunoblotting, functional assays, and more commonly, solid-phase immunoassays such as ELISAs and RIAs, but parallel studies of the same samples in different tests have been infrequent (3–5 ). One of the main problems faced in this field is the lack of agreement on a standardized method to detect AECAs, which renders the interlabora- tory comparison of the results somewhat hazardous (6, 7). The large discrepancies observed in clinical correlations of AECA concentrations may be ascribed to several variables that affect their measurement. For example, the choice of cells (with possible interdonor variability) or the use of cell membrane extracts, cell culture and fixation condi- tions, heating of serum samples (8), and the expression of results may affect the outcome. While attempting to optimize cell fixation in such an ELISA, we identified another possible confounding factor: a substantial proportion of patient samples containing IgG antibodies against bovine serum proteins gave false- positive results in the AECA test. Human antibodies reactive with animal proteins, including immunoglobu- Laboratory of Immunology, Institut de Synergie des Sciences et de la Sante ´, Brest University Medical School, F29609 Brest Cedex, France. *Address correspondence to this author at: Laboratory of Immunology, Brest University Medical School Hospital, BP 824, F 29609, Brest Cedex, France. Fax 33-298-80-10-76; e-mail youinou@univ-brest.fr. Received October 20, 1999; accepted November 10, 1999. 1 Nonstandard abbreviations: AECA, anti-endothelial cell antibody; BSA, bovine serum albumin;  2 GPI,  2 -glycoprotein I; FCS, fetal calf serum; PBS, phosphate-buffered saline; MAb, monoclonal antibody; and ECM, extracellu- lar matrix. Clinical Chemistry 46:2 273–278 (2000) Clinical Immunology 273