A novel in‐capillary assay for dynamically monitoring fast binding between antibody and peptides using CE

Abstract

Nowadays, despite numerous techniques available for the detection of antibody–peptide binding, sensitivity and detection limit inflow still remains a major challenge. Herein, we report a strategy for the detection of binding inflow of monoclonal anti‐FLAG M2 antibody and 5,6‐carboxyfluorescein‐labeled FLAG tag in capillary. Antibody and peptides were sequentially injected into the capillary where they mixed and bound together. Capillary electrophoresis with fluorescence detection was employed to monitor the binding process, and the results showed that the efficacy of the antibody–peptide binding in capillary (in‐capillary assay) is affected by the stoichiometry. Compared to the out‐capillary assay, this novel assay showed different KD values and required a very short detection time, thus showing great potential for rapid detection as well as other applications. Additionally, our novel assay can be used to investigate the stability of the antibody–peptide complex. The addition of excess DYKDDDDK or TAMRA‐DYKDDDDK, with similar binding priorities with M2, can leads to dissociation of the complex with a two‐step mechanism including dissociation and association. We believed that our developed method can be extended to monitor wide range of other biomolecule interactions.

Atypical applications of transverse diffusion of laminar flow profiles methodology for in‐capillary reactions in capillary electrophoresis

“…To date, the direct in-capillary affinity studies performed so far have been purely based on the plug-plug format of EMMA methodology. Its combination with fluorescence detection and one of the interaction partners being fluorescently labeled, resulting in the author designation of "in-capillary detec-tion", were employed for an investigation of interactions between mAbs and peptides [85], the nanoconjugates of polycysteine ligand and quantum dots [86], or between peptides conjugated to these quantum dots and other peptides or proteins [87,88]. In one study dealing with mAbs and peptide interactions, the authors even used capillary bending to increase the mixing efficiency [89].…”

Multienzyme detection and in‐situ monitoring of enzyme activity by bending CE using quantum dots‐based polypeptide substrate

“…Although many research groups have tried using CE to investigate the interaction between biomolecules and QDs, it is still difficult to make biomolecules close enough to interact through capillary migration. Further research is needed to detect multienzyme using CE combined with FRET technology by approach of adding bends in capillary [27,28].…”