Dose-dependent inhibition of BrdU detection in the cell proliferation ELISA by culture medium proteins.
Determination of the proliferation rate of cultured mammalian cells is widely done using incorporation of 5-bromo-2-deoxyuridine into replicating DNA followed by quantitative detection in ELISA using a specific monoclonal antibody. However, we noted that the BrdU ELISA results did not correlate with viable cell counts when increasing concentrations of proteins were added to test their effects on proliferating cells. This observation suggested that proteins could interfere with BrdU incorporation or detection in the commercial BrdU ELISA used. We show here that the presence of exogenous proteins during cell fixation and DNA denaturation significantly inhibited BrdU detection presumably by coating the extracted DNA by a concentration-dependent protein film. A simple modification to the manufacturer's protocol (cell washing) permitted to avoid this interference and resulted in a significant increase of the assay sensitivity.
Padet
Lauriane
L
Department of Research and Development, Héma-Quebec, Québec, QC, Canada.
St-Amour
Isabelle
I
Aubin
Eric
E
Proulx
Dominic Paquin
DP
Bazin
Renée
R
Lemieux
Réal
R
D001973:Bromodeoxyuridine / Q000378:metabolism*; D002452:Cell Count / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism; D011506:Proteins / Q000008:administration & dosage / Q000378:metabolism*; D012680:Sensitivity and Specificity
D003470:Culture Media; D011506:Proteins; D001973:Bromodeoxyuridine