title: Comparison of potency assays using different read-out systems and their suitability for quality control.
abstract: In this study, three of the most commonly used non-radioactive read-out systems for bioassays, the tetrazolium salt MTS/PMS, the fluorescent dye Alamar Blue and the ATP bioluminescence assay were compared regarding their suitability for quality control purposes. In this regard, three different potency assays using murine CTLL-2, as well as human DiFi and Kit 225 cells were performed. No major differences regarding accuracy and precision were detected between the different read-out systems. Both workload and hands-on time were similar for all three assays used. All read-out systems were suitable for use in quality control. However, luminescence and fluorescence techniques were much more sensitive than the colorimetric system. The first two could determine approximately ten times lower drug concentrations, and the assay could be performed by using considerably lower cell numbers. Moreover, in two of the three potency assays, the luminescence and fluorescence read-out systems provided higher signal to noise ratios leading to a higher precision of the assays. Regarding the comparison of the luminescence and fluorescence system, the ATP assay has the advantage to be much faster than the Alamar Blue assay. Consequently, in this study, the luminescence technique turned out to be the most advantageous of the three read-out systems.
authors.affiliation: Analytical Development Biotech Products, Merck KGaA, Frankfurter Str. 250, Darmstadt, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Zumpe
authors.forename: C L
authors.initials: CL
authors.lastname: Bachmann
authors.forename: A U
authors.initials: AU
authors.lastname: Metzger
authors.forename: N
authors.initials: N
authors.lastname: Wiedemann
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003124:Colorimetry; D066246:ErbB Receptors / Q000276:immunology; D005240:Feasibility Studies; D005453:Fluorescence; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D051379:Mice; D010078:Oxazines / Q000378:metabolism*; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013778:Tetrazolium Salts / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007376:Interleukin-2; D015851:Interleukin-7; D010078:Oxazines; D011993:Recombinant Fusion Proteins; D013778:Tetrazolium Salts; D014966:Xanthenes; C005843:resazurin; D000255:Adenosine Triphosphate; C512478:EGFR protein, human; D066246:ErbB Receptors