Generation of bioluminescent enzyme immunoassay for ferritin by single-chain variable fragment and its NanoLuc luciferase fusion

He, Qiyi; Li, Yang; Lin, Mingxia; Yang, Huiyi; Cui, Xiping; McCoy, Mark R.; Hammock, Bruce D.; Fang, Yanxiong; Zhao, Suqing

doi:10.1007/s00216-022-04261-7

Cited by 3 publications

(3 citation statements)

References 27 publications

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“…When the same amount of the nanobody–NanoBiT fusion protein was used, a weaker signal was observed with increasing volume. This outcome may be due to the increasing volume of the buffer, which can cause a signal decline in the system . To obtain the strongest signal, 10 μL of immune reagents, 10 μL of the substrate solution, and 10 μL of samples were selected for further analysis.…”

Section: Resultsmentioning

confidence: 99%

“…The nanoluciferase reporter gene has been widely used in developing immunoassays in a conventional sandwich ELISA for protein determination. − The nanoluciferase can split into a nonfunctional large binary subunit (LgBiT) and a small binary subunit (SmBiT) and reassemble and recover the enzymatic activity when approaching. Two nanobodies that had been previously used to successfully establish a double nanobody sandwich ELISA were chosen as the model to generate the homogeneous split-luciferase assay.…”

Section: Resultsmentioning

confidence: 99%

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Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase

McCoy

Yang

et al. 2023

Anal. Chem.

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The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT–nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase

McCoy

Yang

et al. 2023

Anal. Chem.

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“…ScFv, including IgY-scFv, has the advantages of easy penetration into the target tissue, low side effects, high specificity, and high purity; therefore, it has a great biomedical potential of being widely used in clinic [3,8]. Particularly, avian IgY-scFv can fuse to alkaline phosphatase for the detection of aspergillus pathogens [9] and fuse to NanoLuc luciferase for the detection of ferritin [10]. Therefore, IgY-scFv can bind to the corresponding antigen in the target tissue of the host, thereby leading to enhanced diagnostic and therapeutic effects.…”

Section: Introductionmentioning

confidence: 99%

Chimerism of avian IgY‐scFv and truncated IgG‐Fc: A novel strategy in cross‐species antibody generation and enhancement

Ge,

Dias,

Zhang

2024

Immunology

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Chicken single‐chain fragment variable (IgY‐scFv) is a functional fragment and an emerging development in genetically engineered antibodies with a wide range of biomedical applications. However, scFvs have considerably shorter serum half‐life due to the absence of antibody Fc region compared with the full‐length antibody, and usually requires continuous intravenous administration for efficacy. A promising approach to overcome this limitation is to fuse scFv with immunoglobulin G (IgG) Fc region, for better recognition and mediation by the neonatal Fc receptor (FcRn) in the host. In this study, engineered mammalian ΔFc domains (CH2, CH3, and intact Fc region) were fused with anti‐canine parvovirus‐like particles avian IgY‐scFv to produce chimeric antibodies and expressed in the HEK293 cell expression system. The obtained scFv‐CH2, scFv‐CH3, and scFv‐Fc can bind with antigen specifically and dose‐dependently. Surface plasmon resonance investigation confirmed that scFv‐CH2, scFv‐CH3, and scFv‐Fc had different degrees of binding to FcRn, with scFv‐Fc showing the highest affinity. scFv‐Fc had a significantly longer half‐life in mice compared with the unfused scFv. The identified ΔFcs are promising for the development of engineered Fc‐based therapeutic antibodies and proteins with longer half‐lives. The avian IgY‐scFv‐mammalian IgG Fc region opens up new avenues for antibody engineering, and it is a novel strategy to enhance the rapid development and screening of functional antibodies in veterinary and human medicine.

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Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals

Wen,

Huang,

Sun

et al. 2024

Anal Bioanal Chem

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Generation of bioluminescent enzyme immunoassay for ferritin by single-chain variable fragment and its NanoLuc luciferase fusion

Cited by 3 publications

References 27 publications

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase

Chimerism of avian IgY‐scFv and truncated IgG‐Fc: A novel strategy in cross‐species antibody generation and enhancement

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals

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