title: Dose-dependent inhibition of BrdU detection in the cell proliferation ELISA by culture medium proteins.
abstract: 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.
authors: Department of Research and Development, Héma-Quebec, Québec, QC, Canada.
authors: Lauriane
authors: L
authors: Padet
authors: Isabelle
authors: I
authors: St-Amour
authors: Eric
authors: E
authors: Aubin
authors: Dominic Paquin
authors: DP
authors: Proulx
authors: Renée
authors: R
authors: Bazin
authors: Réal
authors: R
authors: Lemieux
mesh_terms: 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
chemical_list: D003470:Culture Media; D011506:Proteins; D001973:Bromodeoxyuridine