Application of capillary electrophoresis to the development and evaluation of aptamer affinity probes

Sooter, Letha J.; McMasters, Sun; Stratis‐Cullum, Dimitra N.

doi:10.1117/12.731962

Cited by 1 publication

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“…To date, much of the work with CE and optical biosensors has focused on aptamers or nucleic acid molecular beacons for target detection, typically with a mobility shift for the aptamer [3][4][5][6]. In this study, we show that affinity capillary electrophoresis (ACE) can be used to measure peptide specificity and binding affinity of a synthetic peptide selected from a bacterial display library to bind exclusively to PA of B. anthracis.…”

Section: Introductionmentioning

confidence: 99%

Binding specificity and affinity analysis of an anti-protective antigen peptide reagent using capillary electrophoresis

Kogot¹,

Sarkes²,

Pennington³

et al. 2014

ABB

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Peptide biosensor reagents are emerging as an alternative to typical antibody-based detection methods. Peptides can be rapidly isolated using bacterial display methods for new and emerging biothreats and can be chemically synthesized for rapid, large-scale production. With the emergence of peptide biosensor reagents, there is a growing need to develop methods for characterizing binding interactions. Capillary electrophoresis (CE) is a free-solution separation method that is able to determine target and analyte binding association (K b ) and dissociation constants (K d ). In this study, the K b , K d , and peptide specificity of an isolated peptide binding reagent to protective antigen (PA) of Bacillus anthracis were evaluated using capillary electrophoresis at 10 and 20 kV. The relative binding specificity was rapidly assessed by measuring the peptide relative mobility shift at 20 kV at nonequilibrium using bovine serum albumin (BSA), horseradish peroxidase (HRP), and an anti-PA monoclonal antibody (mAb). The αPA peptide was shown to be highly specific for PA, with a K d = 177 nM measured at 20 kV and K d = 312 nM measured at 10 kV. These results show that peptides from bacterial display libraries can be rapidly tested for specificity and binding affinity, in solution, for use as a potential biosensor reagent against new and emerging biothreats.

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Section: Introductionmentioning

confidence: 99%