Clinical Chemistry
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Clinical Chemistry 46: 273-278, 2000;
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(Clinical Chemistry. 2000;46:273-278.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

False Positivity in a Cyto-ELISA for Anti-Endothelial Cell Antibodies Caused by Heterophile Antibodies to Bovine Serum Proteins

Ronan Revelen1, Anne Bordron1, Maryvonne Dueymes1, Pierre Youinou1 and Josiane Arvieuxa,1

1 Laboratory of Immunology, Institut de Synergie des Sciences et de la Santé, Brest University Medical School, F29609 Brest Cedex, France.
a 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{at}univ-brest.fr

Background: ELISAs with fixed endothelial cells or cell lines are widely used screening tests for anti-endothelial 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 serum (FCS) proteins were demonstrated and characterized 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 immunoblotting.

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 matrix, 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.




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