Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yanna; Zhang, Weifen; Tang, Jin‐Bao

doi:10.1016/j.aca.2016.10.039

Cited by 14 publications

(2 citation statements)

References 36 publications

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“…In recent years, several groups have reported the use of photo-cross-linkable Fc-binding protein domains as a generic approach to synthesize antibody conjugates with a well-defined stoichiometry. − Here, we chose to use a protein G domain variant developed by Tsourkas and co-workers for LASIC, light-activated site-specific conjugation of native IgG’s . This small protein domain, which we here refer to as Gx, contains the photoreactive non-natural amino acid para -benzoylphenylalanine (pBPA) at position 24.…”

Section: Resultsmentioning

confidence: 99%

Bioluminescent Antibodies through Photoconjugation of Protein G–Luciferase Fusion Proteins

et al. 2020

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Bioluminescent antibodies represent attractive detection agents in both bioanalytical assays and imaging. Currently, their preparation relies on genetic fusion of luciferases to antibodies or nonspecific chemical conjugation strategies. Here, we report a generic method to generate well-defined covalent antibody− luciferase conjugates starting from commercially available monoclonal antibodies. Our approach uses fusion proteins consisting of the bright blue light-emitting luciferase NanoLuc (NL) and an Fcbinding protein domain (Gx) that can be photo-cross-linked to the antibody using UV light illumination. Green and red color variants were constructed by tight fusion of the NanoLuc with a green fluorescent acceptor domain and introduction of Cy3, respectively. To increase the already bright NanoLuc emission, tandem fusions were successfully developed in which the Gx domain is fused to two or three copies of the NanoLuc domain. The Gx-NL fusion proteins can be efficiently photo-cross-linked to all human immunoglobulin G (IgG) isotypes and most mammalian IgG's using 365 nm light, yielding antibodies with either one or two luciferase domains. The bioluminescent antibodies were successfully used in cell immunostaining and bioanalytical assays such as enzyme-linked immunosorbent assay (ELISA) and Western blotting.

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Section: Resultsmentioning

confidence: 99%

Bioluminescent Antibodies through Photoconjugation of Protein G–Luciferase Fusion Proteins

et al. 2020

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“…It has been successfully used for example for coupling of the biotinylated antibody to the SPR platform made of a diblock of poly[oligo(ethylene glycol) methyl ether methacrylate] and click chemistry biotinylated poly(glycidyl methacrylate). 32,131,132 The method is also frequently used for the attachment of aptamers, nucleic acid probes, 104,133,134 or for enhancing the detection response using functionalized Au nanoparticles. 89 Even though the biotin-streptavidin immobilization technique is popular, biotin is naturally abundant in bodily fluids or foods, where more biotin-binding molecules may occur.…”

Section: Functionalization Techniquesmentioning

confidence: 99%

Biorecognition antifouling coatings in complex biological fluids: a review of functionalization aspects

Víšová

Houska

Vaisocherová‐Lísalová

2022

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Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken

et al. 2018

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A sensitive competitive immunoassay with simple operation was developed for the detection of the anti-virus drug amantadine (AMD). The single-chain variable fragment (scFv) antibody against AMD was site-specific biotinylated and overexpressed as a secreted body in Escherichia coli AVB101. Horseradish peroxidase-labeled streptavidin-biotinylated scFv antibody (HRP-SA-BIO-scFv) could specifically bind to AMD-functionalized magnetic beads (MBs) and then the immune complexes were separated from the matrix solution by magnet. The concentration of the AMD could be known by the measurement of the signal produced by the horseradish peroxidase. The newly established assay provides a significant improvement in comparison to the conventional ELISA without SA-BIO signal amplification and MBs separation. The limit of detection and assay time was 0.64 vs. 8.4 ng/mL and 50 vs. 150 min, respectively. The recoveries ranged from 77.8 to 112% with the coefficient of variation less than 13%. The immunoassay exhibited an obvious cross-reactivity to rimantadine (84%), 1-(1-adamantyl)ethylamine (72%), and somantadine (63%). These results demonstrated that the developed immunoassay provided a sensitive, rapid, and accurate approach for the detection of AMD in chicken by employing MBs as solid phase and SA-BIO as signal amplification. When applied in natural chicken samples, the newly established method provided results consistent with those from UPLC-MS/MS, suggesting that the proposed method could be used for rapid screening of the target of interest; the new immunoassay could also be extended to other small molecular contaminants and thus represents a universal strategy for food safety analysis. Graphical abstract ᅟ.

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Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

Cited by 14 publications

References 36 publications

Bioluminescent Antibodies through Photoconjugation of Protein G–Luciferase Fusion Proteins

Bioluminescent Antibodies through Photoconjugation of Protein G–Luciferase Fusion Proteins

Biorecognition antifouling coatings in complex biological fluids: a review of functionalization aspects

Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken

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