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

Abstract

Experimental design and measurement uncertainty in ligand binding studies by affinity capillary electrophoresisIn 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 Ͼ 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.Abbreviations: , electrophoretic mobility; c KD , theoretical mobility at the ligand concentration equals the K D -value; c , mobility of the complex; f , mobility of the free analyte; AIQ, analytical instrument qualification; aMRR, absolute maximum response range; BGE, background electrolyte; BSA, bovine serum albumin; c, concentration; c 0 , in absence of ligand; c Ý , in presence of a theoretically infinite ligand concentration; CD, sulfobutyl ether betacyclodextrine derivates; CI mean , confidence interval of the mean; const, constant parameter; conti, continuous parameters; DPP, data point position; DPR, data point range; DS, design space; E-systems, example analyte/ligand systems; G, Gradient; HC, hydrocorti-sone; HPAC, high performance affinity chromatography; HSA, human serum albumin; K A , association constant; K D , dissociation constant; LBA, ligand binding assay; LBAs, ligand binding assays; MRR, maximum response range; ms ACE, mobility shift affinity capillary electrophoresis; MUM, measurement uncertainty multiplier; MUP, measurement uncertainty propagation; nDS, normalized design space; nMRR, normalized maximum response range; NoDP, number of data points; PMS, parameter sets; PPS, pentosan polysulfate; R", complexed fraction; RSD, relative standard deviation; SOP, standard operating procedure; SPR, surface plasmon resona...

Investigation of the enantioselective interaction between selected drug enantiomers and human serum albumin by mobility shift‐affinity capillary electrophoresis

“…Due to different degrees of complexation, the enantiomeric analytes will hold different apparent mobilities depending on the CS levels [32,36]. The change of effective mobilities as a function of the ligand concentrations is used to calculate the apparent binding properties in ms‐ACE [37]. A schematic illustration of the ms‐ACE setup is given in Figure 1.…”

“…The interaction between the negatively charged HSA molecules and the positively charged basic chiral molecules was considered to be the main reason for efficient chiral discrimination of the tested basic compounds. The opposite charges of HSA and the analytes support the enantioseparation due to the optimal difference between the effective mobilities of the CS-enantiomer complexes and the free enantiomers [37,46]. The respective electrophoretic mobilities are listed in Table 1.…”

“…This is due to the weak interaction between VER enantiomers and HSA in the applied system, and binding curves are too straight to be evaluated quantitatively. Insufficient description of the binding curve leads to the unavoidable low accuracy of the binding constant prediction [37]. Indeed, the K A values of VER enantiomers at certain CE conditions correspond to the relative measures of the complexes strength instead of apparent association constants.…”

Determining binding constants for 1:1 and 1:2 inclusion complexes of ester betulin derivatives with (2‐hydroxypropyl)‐β‐cyclodextrin by affinity capillary electrophoresis

“…In addition, the ratio of the analyte concentration in samples to the CD concentration in BGE should be less than 10–35 [10], otherwise disturbed triangular peaks with dips are observed for which the parameter a 1 is heavily biased relative to this parameter for peaks at a lower concentration. The analyte concentration should be as low as possible, which allows using a wider range of CD concentration in BGE leading to higher precision in determining the binding constants values [22]. But, unfortunately, not all researchers know about the need to use the HVL function for triangular electrophoretic peaks, and very few studies have been done in this manner [9,10,17,20,23].…”

“…CDs usually form 1:1 complexes with guest compounds according to the following equation [14–30]: where D is the drug (guest compound) under study, is the apparent binding (stability) constant of the complex D/CD, and […] is the equilibrium concentrations of corresponding species. The effective electrophoretic mobility of an analyte (guest compound) for the case of 1:1 complexation is dependent on the ligand (host compound) concentration in BGE as follows: where is the coefficient allowing viscosity change correction for the i th BGE containing CD, is the effective electrophoretic mobility, μ D is the ionic mobility of D (effective electrophoretic mobilities of the analyte when CD concentration in BGE is zero), μ 11 is the ionic mobility of the complex D/CD, [ CD ] i is the CD (ligand) concentration in BGE, μ analyte, i and μ marker, i are the electrophoretic mobilities of the analytes and EOF marker, respectively [14].…”