Flow-through partial-filling affinity capillary electrophoresis for the estimation of binding constants of ligands to receptors

Mito, Erica; Gomez, Frank A.

doi:10.1007/bf02497305

Cited by 25 publications

(20 citation statements)

References 34 publications

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“…This method does not require the knowledge of the mobility of the solute-complex (i.e., DM max ). By using mobility ratios rather than mobilities, the data analysis method is not sensitive to voltage fluctuations [16,23,24]. Values calculated using this method were found to be in agreement with those given by other techniques [23,24].…”

Section: Partial-filling Techniquesupporting

confidence: 71%

“…The partial-filling ACE technique was recently introduced [22][23][24]. The technique is illustrated in Figs.…”

Section: Partial-filling Techniquementioning

confidence: 99%

“…Similar studies were performed using vancomycin and carbonic anhydrase B systems [23,24). In these studies, the sample and noninteracting standards were injected together into the capillary partial filled with ligand solution.…”

Section: Partial-filling Techniquementioning

confidence: 99%

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Methods for the determination of binding constants by capillary electrophoresis

2001

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Apparent equilibrium constants for molecular association (e.g., association constants, binding constants, dissociation constants, partition coefficients) can be determined with a variety of different capillary electrophoresis (CE) approaches. In many cases, the investigated association behavior is between a smaller molecule or ion (i.e., the solute, drug, or analyte of interest) and a larger entity (e.g., proteins, micelles, polymers, chiral selectors such as cyclodextrins, etc.). Each experimental approach has advantages and disadvantages. Frequently, it is the nature of the system being evaluated that determines the optimal experimental approach. Six different CE-based techniques for evaluating binding constants are reviewed. Examples of each method, and recent references on its use are given.

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Section: Partial-filling Techniquesupporting

confidence: 71%

“…The partial-filling ACE technique was recently introduced [22][23][24]. The technique is illustrated in Figs.…”

Section: Partial-filling Techniquementioning

confidence: 99%

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Methods for the determination of binding constants by capillary electrophoresis

2001

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“…for the interaction of L 2 , and CAB on a relative time scale with noninteracting markers [10]. Equation (2) represents the general equation used to obtain a linear plot and hence the K b of the competitive binding system we are investigating:…”

Section: Form Of Analysismentioning

confidence: 99%

Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresis

2008

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Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresisThis work expands the knowledge of the use of chemometric response surface methodology (RSM) in optimizing conditions for competitive binding partial filling ACE (PFACE). Specifically, RSM in the form of a Box-Behnken design was implemented in flow-through PFACE (FTPFACE) ACE has been shown to be a versatile microanalytical technique to estimate affinity constants, and has emerged as a useful and sensitive method for studying bimolecular noncovalent interactions and for determining binding and dissociation constants of formed complexes. The first reports detailing the use of ACE to measure affinity parameters between biological species were published in the early 1990s [3][4][5][6][7]. Since these informative studies, a multitude of other interactions including protein-ligand, peptide-peptide, proteinpeptide, protein-antibody, polymer-peptide, and antibodyantigen have been examined successfully using ACE .ACE differentiates between bound and unbound receptor (R) as a function of free ligand (L) concentration only when the R-L complexation yields a sizable difference in mass or charge-to-mass ratio. In a typical ACE experiment, a sample of receptor and noninteracting markers are reacted with an increasing concentration of ligand in a running buffer, thereby, causing a shift in the migration of the receptor peak. Subsequent analysis of these changes in migration time yields a value for K b [20].To minimize the amount of sample needed in an ACE assay, partial filling techniques in ACE were developed. In PFACE, the capillary is partially filled with ligand (or receptor) Correspondence: Dr. Frank A. Gomez, Department of Chemistry and Biochemistry, California State University, Los Angeles, CA, USA E-mail: fgomez2@calstatela.edu Fax: 11-323-343-6490 Abbreviations: CAB, carbonic anhydrase B; FTPFACE, flowthrough partial filling ACE; HHM, horse heart myoglobin; MO, mesityl oxide; RMTR, relative migration time ratio; RSM, response surface methodology

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“…The complex formed (or the amount of free binding partner left after incubation) provides information about the affinity and binding kinetics and stoichiometry [7][8][9][10][11][12][13]. In clinical analysis, a large variety of immunoassays is used for the identification of hormones, drugs and other antigens in blood [14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Use of bioaffinity interactions in electrokinetically controlled assays on microfabricated devices

2002

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In this contribution, the role of bioaffinity interactions on electrokinetically controlled microfabricated devices is reviewed. Interesting applications reported in the literature include enzymatic assays, where enzyme and enzyme inhibition kinetics were studied, often in combination with electrophoretic separation. Attention is paid towards developments that could lead to implementation of electrokinetically controlled microdevices in high-throughput screening. Furthermore, enzyme-facilitated detection in combination with electrophoretic separation on microdevices is discussed. Various types of immunoassays have been implemented on the microchip format. The selectivity of antibody-antigen interaction has been exploited for the detection of analytes in complex sample matrices as required, for example, in clinical chemistry. Binding kinetics as well as stoichiometry were studied in chip-based assays. Automated mixing protocols as well as the demonstration of a parallel immunoassay allow implementation of microdevices in high-throughput screening. Furthermore, demonstration of immunoassays on cheap polymeric microdevices opens the way towards the fabrication of disposable devices, a requirement for commercialization and therefore for application in routine analyses.

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Flow-through partial-filling affinity capillary electrophoresis for the estimation of binding constants of ligands to receptors

Cited by 25 publications

References 34 publications

Methods for the determination of binding constants by capillary electrophoresis

Methods for the determination of binding constants by capillary electrophoresis

Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresis

Use of bioaffinity interactions in electrokinetically controlled assays on microfabricated devices

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