Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

Seo, Hyo‐Deok; Lee, Joong-Jae; Kim, Yu Jung; Hantschel, Oliver; Lee, Dae-Hee; Kim, Kwang Ho

doi:10.1016/j.aca.2016.11.013

Cited by 13 publications

(7 citation statements)

References 39 publications

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“…iScience Article modular engineering, various repebodies have been successfully developed with high affinity and selectivity for disease-related targets (Son et al, 2020;Duarte et al, 2020;Seo et al, 2017;Kim et al, 2016;Hwang et al, 2016aHwang et al, , 2016bHwang et al, , 2016cLee et al, 2014Lee et al, , 2015Heu et al, 2014). The human epidermal growth factor receptor (EGFR)-specific repebody exemplifies such a scaffold where the robust targeting moiety has been extensively exploited for targeted therapy and diagnosis with negligible toxicity (Lee et al, 2015(Lee et al, , 2017Ryu et al, 2018Ryu et al, , 2020Yun et al, 2017).…”

Section: Open Accessmentioning

confidence: 99%

Dissecting the impact of target-binding kinetics of protein binders on tumor localization

et al. 2021

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Summary Systematic control of in vivo behavior of protein-based therapeutics is considered highly desirable for improving their clinical outcomes. Modulation of biochemical properties including molecular weight, surface charge, and binding affinity has thus been suggested to enhance their therapeutic effects. However, establishing a relationship between the binding affinity and tumor localization remains a debated issue. Here we investigate the influence of the binding affinity of proteins on tumor localization by using four repebodies having different affinities to EGFR. Biochemical analysis and molecular imaging provided direct evidence that optimal affinity with balanced target binding and dissociation can facilitate deep penetration and accumulation of protein binders in tumors by overcoming the binding-site-barrier effect. Our findings suggest that binding kinetics-based protein design can be implicated in the development of fine-tuned protein therapeutics for cancers.

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Section: Open Accessmentioning

confidence: 99%

Dissecting the impact of target-binding kinetics of protein binders on tumor localization

et al. 2021

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“…VHHs, the smallest specific-binding entities, can be genetically manipulated for a variety of applications [ 72 ]. To simplify immunoassay protocols, fluorescent proteins [ 72 ] or enzymes, such as alkaline phosphatase (AP), can be fused with VHHs for one-step immunoassay [ 73 ]. Besides, avoiding random chemical conjugation and the use of secondary antibodies, VHHs with fused proteins enlarge the paratopes and thus improve the affinities of VHHs due to the homodimeric nature of AP [ 74 ].…”

Section: Vhh-based Recombinant Proteinsmentioning

confidence: 99%

VHH Antibodies: Reagents for Mycotoxin Detection in Food Products

Wang

Mukhtar

et al. 2018

Sensors

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Mycotoxins are the toxic secondary metabolites produced by fungi and they are a worldwide public health concern. A VHH antibody (or nanobody) is the smallest antigen binding entity and is produced by heavy chain only antibodies. Compared with conventional antibodies, VHH antibodies overcome many pitfalls typically encountered in clinical therapeutics and immunodiagnostics. Likewise, VHH antibodies are particularly useful for monitoring mycotoxins in food and feedstuffs, as they are easily genetic engineered and have superior stability. In this review, we summarize the efforts to produce anti-mycotoxins VHH antibodies and associated assays, presenting VHH as a potential tool in mycotoxin analysis.

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“…The decrease in the binding capacities of LAL13 and RANA clones might have resulted from the steric hindrance effect which is probably caused by the fusion between the Lig7 scFv and AP. To avoid such conformational problems, we added the 'ASGAEGGG-SEGGGSG' linker between the scFv and AP [40]. The results show that this linker has no significant effect on the binding capacity of Lig7 to HBsAg.…”

Section: Binding and Biological Activity Of The Bifunctional Recombinmentioning

confidence: 99%

Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

Erdağ

Balcıoğlu

Bahadır

et al. 2017

Biotechnology & Biotechnological Equipment

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Hepatitis B virus (HBV) infection is a worldwide health problem. More than 400 million people are chronic HBV carriers in the world. Infected individuals are at a high risk of developing liver cirrhosis and hepatocellular carcinoma as the main consequences of HBV. The discoveries of fast diagnostic systems and new therapeutic applications are crucial in the fight against viral hepatitis. In this paper we present the generation of a single-chain variable fragment (scFv) from a mouse monoclonal antibody specific to the HBV surface antigen (HBsAg) and demonstrate its expression as a bacterial alkaline phosphatase (AP) fusion protein. In this study, we constructed scFvs from hybridoma cells expressing HBsAg-specific antibody using phage display technology and expressed them in Escherichia coli. The anti-HBsAg scFvs were inserted into pQE-2 vector to produce scFv antibody genetically fused to bacterial AP. Reproducibility of the recombinant HBsAg-scFv fusion protein was tested using Enzyme-linked Immunosorbent Assay (ELISA). Present preliminary findings indicate that the anti-HBsAg-scFv AP conjugate could be further used for the development of one-step ELISA for the detection of HBV.

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Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

Cited by 13 publications

References 39 publications

Dissecting the impact of target-binding kinetics of protein binders on tumor localization

Dissecting the impact of target-binding kinetics of protein binders on tumor localization

VHH Antibodies: Reagents for Mycotoxin Detection in Food Products

Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

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