A novel monitoring approach of antibody-peptide binding using “bending” capillary electrophoresis

Wang, Jianhao; Zhu, Zhilan; Wang, Xiang; Yang, Li; Liu, Li; Igbinigie, Eseosaserea; Liu, Xiaoqian; Li, Jinping; Qiu, Lin; Li, Yongqiang; Jiang, Pengju

doi:10.1016/j.ijbiomac.2018.03.032

Cited by 8 publications

(5 citation statements)

References 34 publications

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“…FAM‐DYKD was manually synthesized based on standard 9‐fluorenylmethoxycarbonyl (Fmoc) solid‐phase peptide synthesis protocol using 2‐(1 H ‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU)/ N ‐hydroxybenzotriazole (HOBt) activation strategy . The identity of FAM‐DYKD was characterized and confirmed by LC–MS: m/z calculated for [M+4H] 4+ 773.01, found 773.22 (Supporting Information Figure S1).…”

Section: Methodsmentioning

confidence: 99%

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

Wang

Qiu

You

et al. 2018

J of Separation Science

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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.

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Section: Methodsmentioning

confidence: 99%

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

Wang

Qiu

You

et al. 2018

J of Separation Science

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“…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]. Besides peptides and proteins, it has also been used for studying the interaction between G-quadruplex aptamer and thrombin [90], and for the study of DNA hybridization [91].…”

Section: Affinity Interactionmentioning

confidence: 99%

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

Bržezická,

Kohútová,

Glatz

2024

J of Separation Science

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Capillary electrophoresis (CE) is a powerful separation technique offering quick and efficient analyses in various fields of bioanalytical chemistry. It is characterized by many well‐known advantages, but one, which is perhaps the most important for this application field, is somewhat overlooked. It is the possibility to perform chemical and biochemical reactions at the nL scale inside the separation capillary. There are two basic formats applicable for this purpose, heterogeneous and homogeneous. In the former, one reactant is immobilized onto a particle or monolithic support or directly on the capillary wall, and the other is injected. In the latter, the reactant mixing inside a capillary is based on electromigration or diffusion. One of the diffusion‐based methodologies, termed Transverse Diffusion of Laminar Flow Profiles, is the subject of this review. Since most studies utilizing in‐capillary reactions in CE focus on enzymes, which are being continuously and exhaustively reviewed, this review covers the atypical applications of this methodology, but still in the bioanalytical field. As can be seen from the demonstrated applications, they are not limited to reactions, but can also be utilized for other biochemical systems.

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“…A home-built CE system was presented using a high voltage supply (0-30 kV) (Shanghai Nuclear Research Institute, Shanghai, China), an inverted IX71 fluorescence microscope (Olympus, Tokyo, Japan) and a fiber optic spectrometer QE65000 (Ocean Optics, Dunedin, FL, USA) attached to the side port [13][14][15]. The capillary with a total length of 75 cm (50 cm effective length) was used.…”

Section: Instrumentation and Spectrometrymentioning

confidence: 99%

“…His‐tagged polypeptides have been successfully applied as a means of functionalizing QDs by metal affinity. His‐tag has a high affinity, and related reports have confirmed that it has spontaneously bound to the surface of QDs by oligohistidine, allowing various ligands to functionalize QDs [13–15]. Preliminary studies have revealed the probing enzyme's activity by the QD‐peptide substrate [16, 17].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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Multienzyme detection and monitoring enzyme activity in situ are significant for the disease to diagnose. This study aims to develop a quantum dots (QDs)‐based nanoprobe Cyanine5‐DDDLEVLFQFPGLVPRGSGGHHHHHH‐QDs (Cy5‐LEVLVP‐QD), which is able to detect two enzymes inside a bent capillary using CE. Cy5‐LEVLVP and QDs were allowed to bind with each other through metal affinity interaction and then injected the Cy5‐LEVLVP‐QD complex into a capillary with different bends, followed by related enzyme that can cleave the Cy5‐LEVLVP peptide. The fluorescence of Cy5 was excited by QDs due to Förster resonance energy transfer. By monitoring the peaks produced by the original Cy5‐LEVLVP‐QD complex and a significant fluorescence change, sensitive analysis of two different enzymes was conducted. Therefore, the novel approach of using capillaries with semicircular bends could prove particularly useful for enzyme investigating in disease.

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A novel monitoring approach of antibody-peptide binding using “bending” capillary electrophoresis

Cited by 8 publications

References 34 publications

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

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

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

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

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