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 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.
Revelen
R
R
Laboratory of Immunology, Institut de Synergie des Sciences et de la Santé, Brest University Medical School, F29609 Brest Cedex, France.
Bordron
A
A
Dueymes
M
M
Youinou
P
P
Arvieux
J
J
D000818:Animals; D000906:Antibodies / Q000097:blood*; D000910:Antibodies, Heterophile / Q000097:blood*; D002460:Cell Line; D003470:Culture Media; D004727:Endothelium / Q000166:cytology / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005189:False Positive Reactions; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D012710:Serum Albumin, Bovine / Q000276:immunology*
D000906:Antibodies; D000910:Antibodies, Heterophile; D003470:Culture Media; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine