False positivity in a cyto-ELISA for anti-endothelial cell antibodies caused by heterophile antibodies to bovine serum proteins

Background: ELISAs with fixed endothelial cells or cell lines are widely us ed screening tests for anti-endothelial-cell antibodies (AECAs), but spurio us increases occur. We examined interferences by heteroantibodies and means to eliminate them. Methods: AECAs were measured by ELISA on fixed layers of the human endothel ial cell line, EA.hy 926, in a panel of 60 patient serum samples diluted in bovine serum albumin. Heteroantibodies against fetal calf serum(FCS) prote ins were demonstrated and characterized in an ELISA-the interference assay- that used FCS-coated plates and Tween 20-containing buffer as blocking agen t:and sample diluent, as well as by immunoblotting. Results:In 12 of 60 patient serum samples, spurious increases of AECA titer s were produced by endogenous antibodies reacting with FCS proteins from cu lture medium that were coated onto the solid-phase at the time of cell plat ing. This mechanism of interference was supported experimentally by exposin g extracellular matrix, varying cell density, and incubating wells with FCS alone. The heterophile antibodies were mainly IgG and IgA, and in inhibiti on 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. (C) 2000 Amer ican Association for Clinical Chemistry.