Reporter–nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents

Yamagata, Masahito; Sanes, Joshua R.

doi:10.1073/pnas.1722491115

Cited by 53 publications

(43 citation statements)

References 39 publications

(39 reference statements)

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“…Nanobodies have arisen as a substitute to conventional antibodies and show great potential when used as tools in the diagnostic field [45][46][47][48]. One of the main advantages of nanobodies is that several tags can be fused in their tertiary structure by recombinant technology [9][10][11]13]. Based on this advantage, PPV-VP2 specific nanobody-HRP and -EGFP fusions were used for the first time as probes to develop immunoassays for PPV detection in this study.…”

Section: Discussionmentioning

confidence: 99%

“…Because nanobodies contain only one ∼ 130 amino acid variable domain, they can be simply derivatised by coupling to reporters or dyes. For example, one study designed a reporter-nanobody fusion (RANbody) platform, in which RANbody was used in immunohistochemical detection [9]. Other works have reported the application of nanobody-HRP, EGFP, or nano-luciferase fusions derived from nanobodies to develop detection assays, label cells and tissues, and for other purposes [10][11][12][13].…”

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confidence: 99%

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Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

Zhao

et al. 2020

J Nanobiotechnol

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Background: Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV.Results: In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni-NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests. Conclusions:For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article'

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

Zhao

et al. 2020

J Nanobiotechnol

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“…It has been suggested that nAbs preferentially bind to target proteins via a convex paratope (Wurch et al, 2012), which could contribute to the inability of the nAbs against the targets with the exception of Homer1 to detect aldehyde-fixed and/or denatured target proteins. While nAbs have widespread use in binding to native protein, which enhances their utility as chaperones for crystallography , some generated against GFP and other proteins have been used as immunolabels for immunohistochemistry (Fang et al, 2018;Yamagata and Sanes, 2018) and immunoblots (Bruce and McNaughton, 2017) In summary, we have generated a series of validated nAbs against neuronal proteins selectively expressed in specific subcellular compartments in brain neurons. These nAbs represent a valuable toolbox of reagents available to the neuroscience community for diverse applications.…”

Section: Discussionmentioning

confidence: 99%

A toolbox of nanobodies developed and validated for diverse neuroscience research applications

Jiang¹,

Lee²,

Kirmiz³

et al. 2019

Preprint

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Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications.Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.3

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“…Each target has its advantages and disadvantages depending on the model system and experimental question at hand. Nbs of either persuasion can be delivered into cells or animals by genomic engineering; the Nb cDNA sequences are placed under the control of cell-specific, inducible, or ubiquitous reporter sequences (Panza et al, 2015; Rothbauer et al, 2006; Yamagata & Sanes, 2018). In model organisms lacking established transgenic lines like the sea urchin, in vivo Nb expression can be easily encoded in F 0 by using mRNA overexpression (Fig.…”

Section: Introductionmentioning

confidence: 99%

Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos

Schrankel

Gökirmak

Lee

et al. 2019

Echinoderms, Part B

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Single-domain antibodies, also known as nanobodies, are small antigen-binding fragments (~15 kDa) that are derived from heavy chain only antibodies present in camelids (VHH, from camels and llamas), and cartilaginous fishes (VNAR, from sharks). Nanobody V-like domains are useful alternatives to conventional antibodies due to their small size, and high solubility and stability across many applications. In addition, phage display, ribosome display, and mRNA/cDNA display methods can be used for the efficient generation and optimization of binders in vitro. The resulting nanobodies can be genetically encoded, tagged, and expressed in cells for in vivo localization and functional studies of target proteins. Collectively, these properties make nanobodies ideal for use within echinoderm embryos. This chapter describes the optimization and imaging of genetically encoded nanobodies in the sea urchin embryo. Examples of live-cell antigen tagging (LCAT) and the manipulation of green fluorescent protein (GFP) are shown. We discuss the potentially transformative applications of nanobody technology for probing membrane protein trafficking, cytoskeleton re-organization, receptor signaling events, and gene regulation during echinoderm development.

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Reporter–nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents

Cited by 53 publications

References 39 publications

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

A toolbox of nanobodies developed and validated for diverse neuroscience research applications

Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos

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