Experimental design and measurement uncertainty in ligand binding studies by affinity capillary electrophoresis

Matthias Stein, Rob Haselberg, Mona Mozafari-Torshizi, Hermann Wätzig*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


In all life sciences ligand binding assays (LBAs) play a crucial role. Unfortunately these are very error prone. One part of this uncertainty results from the unavoidable random measurement uncertainty, another part can be attributed to the experimental design. To investigate the latter, uncertainty propagation was evaluated as a function of the given experimental design. A design space including the normalized maximum response range (nMRR), the data point position (DPP), the data point range (DPR) and the number of data points (NoDP) was defined. Based on ten measured ms ACE source data sets 20 specific parameter sets were selected by Design of Experiments. Monte Carlo simulations using 100 000 repeats for every parameter set were employed. The resulting measurement uncertainty propagation factors (measurement uncertainty multiplier: MUM) were used to describe the whole design space by polynomial regression. The resulting 5-dimensional response surface was investigated to evaluate the design parameter's influence and to find the minimal uncertainty propagation. It could be shown, that the nMRR is of highest importance, followed by DPP and DPR. Interestingly, the NoDP is less relevant. However, the interactions of the four parameters need to be carefully considered during design optimization. Using at least five data points which cover over 40% of the upper part of the binding hyperbola (DPP > 0.57) the MUM will be minimized (MUM approximately 1.5) when the nMRR is appropriate. It is possible to reduce the measurement uncertainty propagation more than one order of magnitude.

Original languageEnglish
Pages (from-to)1-14
Number of pages14
Issue number7
Publication statusPublished - 14 Jan 2019


The authors thank Polymicro Technologies for providing the capillaries, CASSS for ideational and financial (travel grant) support and Rüdiger Kessel from Metrodata for technical and scientific assistance. The authors also thank Julia Kahle, Lutz Preu and Rebecca Wiesner for proofreading and helping to edit the manuscript.

FundersFunder number
Polymicro Technologies


    • Affinity capillary electrophoresis
    • Drug binding studies
    • Experimental design
    • Ligand binding assay
    • Measurement uncertainty


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