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Keywords = fluobodies

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10 pages, 1246 KiB  
Article
A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies
by Urša Štrancar, Claudia D’Ercole, Lucia Cikatricisová, Mirna Nakić, Matteo De March and Ario de Marco
Biomolecules 2024, 14(5), 587; https://doi.org/10.3390/biom14050587 - 15 May 2024
Viewed by 1823
Abstract
Background: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. Methods: We analyzed the quality of such biomolecules by a combination of gel filtration [...] Read more.
Background: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. Methods: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. Results: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. Conclusions: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments. Full article
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20 pages, 1074 KiB  
Review
Fluobodies against Bioactive Natural Products and their Application in Fluorescence-Linked Immunosorbent Assay
by Seiichi Sakamoto, Benyakan Pongkitwitoon, Hiromichi Nakahara, Osamu Shibata, Yukihiro Shoyama, Hiroyuki Tanaka and Satoshi Morimoto
Antibodies 2012, 1(2), 239-258; https://doi.org/10.3390/antib1020239 - 11 Sep 2012
Cited by 10 | Viewed by 9354
Abstract
An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a [...] Read more.
An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA). In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli) and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article. Full article
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