Rapid and sensitive SARS-CoV-2 detection using a homogeneous fluorescent immunosensor Quenchbody with crowding agents

Zhu, Bo; Nosaka, Nobuyuki; Kanamaru, Shuji; Dong, Jinhua; Dai, Yancen; Inoue, Akihito; Yang, Yinghui; Kobayashi, Kaori; Kitaguchi, Tetsuya; Iwasaki, Hiroshi; Koike, Ryuji; Wakabayashi, Kenji; Ueda, Hiroshi

doi:10.1039/d2an01051h

Cited by 10 publications

(9 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The purified nucleocapsid protein reported in this study can be used for several applications. It can be used as an immunogen in animals such as mice or rabbits to generate monoclonal or polyclonal antibodies for the development of antigen detection-based immunochemical tests in different formats such as lateral flow assays or fluorescent immunosensors [12][13][14][15]28]. The purified nucleocapsid protein can also be used as a bait or capture molecule for the development of antibody detection-based serodiagnostic tests [11,16,17].…”

Section: Discussionmentioning

confidence: 99%

Production of SARS-CoV-2 nucleocapsid protein in Escherichia coli and its characterization

Verma¹,

Abhishek²

2022

J App Biol Biotech

View full text Add to dashboard Cite

SARS-CoV-2 encodes a nucleocapsid protein that binds to the single-stranded RNA genome present inside the viral particles. It is one of the predominant proteins expressed during infection and tends to be highly immunogenic in infected individuals. Consequently, it has been recognized as an important diagnostic marker for the development of antigen or antibody detection-based diagnostic platforms for COVID-19. In this work, we have described cloning, autoinduction-based expression, and purification of full-length SARS-CoV-2 nucleocapsid protein using Escherichia coli as the heterologous host. We have characterized the purified protein using commercial rapid antigen test kits and indirect ELISA to demonstrate its suitability as bait for diagnostic assays. The purified recombinant protein reported in this study can be useful for numerous applications including the development of monoclonal or polyclonal antibodies, studying protein structure and function, and mapping B-and T-cell epitopes for designing suitable vaccines and therapeutics.

show abstract

Section: Discussionmentioning

confidence: 99%

Production of SARS-CoV-2 nucleocapsid protein in Escherichia coli and its characterization

Verma¹,

Abhishek²

2022

J App Biol Biotech

View full text Add to dashboard Cite

show abstract

“…Next, maleimide dyes were conjugated with the cysteine residue at the N-terminal of the variable region of the antibody fragments, which has made it affordable and easy; hence this labeling method is well accepted for creating Q-bodies against many analytes including antigens from SARS-Cov-2. 33,34 To take a shortcut for removing free dyes during construction, another method using a stable and high-affinity coiled-coil peptide pair, E4 and K4, was employed, and the prepared Q-bodies were named coiled Q-body. It was formed by mixing a Fab fragment fused with an E4 peptide at the N-terminal with fluorescently labeled K4.…”

Section: Single Fluorophore Immunosensorsmentioning

confidence: 99%

Recent progress in homogeneous immunosensors based on fluorescence or bioluminescence using antibody engineering

Rani¹,

Zhu

Ueda

et al. 2023

Analyst

Self Cite

View full text Add to dashboard Cite

show abstract

“…Measurements can be carried out by simply mixing the Q-body with an antigen and detecting the fluorescence, thereby reducing time consumption. Additionally, the target compound can be a wide array of molecules, from small molecules, including narcotics such as morphine [ 4 , 9 , 10 , 11 ] and small peptides such as BGP-C7 [ 12 ], to large proteins such as influenza hemagglutinin and SARS-CoV-2 proteins [ 13 , 14 , 15 , 16 ]. Both cell-free and Escherichia coli -based methods have been previously applied to produce Q-bodies.…”

Section: Introductionmentioning

confidence: 99%

VH-Based Mini Q-Body: A Novel Quench-Based Immunosensor

Dong

Banwait

Ueda

et al. 2023

Sensors

Self Cite

View full text Add to dashboard Cite

Quenchbodies (Q-bodies), a type of biosensor, are antibodies labeled with a fluorescent dye near the antigen recognition site. In the absence of an antigen, the dye is quenched by tryptophans in the antibody sequence; however, in its presence, the dye is displaced and therefore de-quenched. Although scFv and Fab are mainly used to create Q-bodies, this is the first report where a single-domain heavy chain VH from a semi-synthetic human antibody library formed the basis. To create a proof of concept “mini Q-body”, a human anti-lysozyme single-domain VH antibody C3 was used. Mini Q-bodies were successfully developed using seven dyes. Different responses were observed depending on the dye and linker length; it was concluded that the optimal linker length for the TAMRA dye was C5, and rhodamine 6G was identified as the dye with the largest de-quenching response. Three single-domain antibodies with sequences similar to that of the C3 antibody were chosen, and the results confirmed the applicability of this method in developing mini Q-bodies. In summary, mini Q-bodies are an easy-to-use and time-saving method for detecting proteins.

show abstract

Rapid and sensitive SARS-CoV-2 detection using a homogeneous fluorescent immunosensor Quenchbody with crowding agents

Abstract: Antigen tests for SARS-CoV-2 are widely used by the public during the ongoing COVID-19 pandemic, which demonstrates the societal impact of homogeneous immunosensor-related technologies. In this study, we used the...

Cited by 10 publications

References 44 publications

Production of SARS-CoV-2 nucleocapsid protein in Escherichia coli and its characterization

Production of SARS-CoV-2 nucleocapsid protein in Escherichia coli and its characterization

Recent progress in homogeneous immunosensors based on fluorescence or bioluminescence using antibody engineering

VH-Based Mini Q-Body: A Novel Quench-Based Immunosensor

Contact Info

Product

Resources

About