Development of a highly thermostable immunoassay based on a nanobody-alkaline phosphatase fusion protein for carcinoembryonic antigen detection

Lin, Jingtao; Yu, Jianliang; Wang, Huan; Xu, Yanru; Li, Fēi; Chen, Xiaoheng; Liang, Yunlong; Tang, Jinsong; Li-li, WU; Zhou, Zhengwei; Chen, Cailing; Liu, Minjuan; Chun, Xuan; Nian, Rui; Song, Haipeng

doi:10.1007/s00216-020-02456-4

Cited by 15 publications

(13 citation statements)

References 14 publications

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“…Meanwhile, there was a trend in enhanced expression level with an increasing number of the SFP hexapeptide fusion tags. Therefore, 10-, 30-, 40-, 50-SFP hexapeptide units were fused to another model nanobody 11C12, which was isolated from immune libraries to target carcinoembryonic antigen (CEA) (Lin et al 2020).…”

Section: Discussionmentioning

confidence: 99%

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

Yang

et al. 2022

Appl Microbiol Biotechnol

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Nanobodies show a great potential in biomedical and biotechnology applications. Bacterial expression is the most widely used expression system for nanobody production. However, the yield of nanobodies is relatively low compared to that of eukaryotic systems. In this study, the repetitive amino acid sequence motifs (GAGAGS) found in silk fibroin protein (SFP) were developed as a novel fusion tag (SF-tag) to enhance the expression of nanobodies in Escherichia coli. SF-tags of 1 to 5 hexapeptide units were fused to the C-terminus of 4G8, a nanobody against human epididymis protein 4 (HE4). The protein yield of 4G8 variants was increased by the extension of hexapeptide units and achieved a 2.5 ~ 7.1-fold increase compared with that of untagged 4G8 (protein yield of 4G8-5C = 0.307 mg/g vs that of untagged 4G8 = 0.043 mg/g). Moreover, the fusion of SF-tags not only had no significant effect on the affinity of 4G8, but also showed a slight increase in the thermal stability of SF-tag-fused 4G8 variants. The fusion of SF-tags increased the transcription of 4G8 by 2.3 ~ 7.0-fold, indicating SF-tags enhanced the protein expression at the transcriptional level. To verify the applicability of the SF-tags for other nanobody expression, we further investigated the protein expression of two other anti-HE4 nanobodies 1G8 and 3A3 upon fusion with the SF-tags. Results indicated that the SF-tags enhanced the protein expression up to 5.2-fold and 5.7-fold for 1G8 and 3A3, respectively. For the first time, this study reported a novel and versatile fusion tag system based on the SFP for improving nanobody expression in Escherichia coli, which may enhance its potential for wider applications. Key points • A silk fibroin protein-based fusion tag (SF-tag) was developed to enhance the expression of nanobodies in Escherichia coli.• The SF-tag enhanced the nanobody expression at the transcriptional level.• The fusion of SF-tag had no significant effect on the affinity of nanobodies and could slightly increase the thermal stability of nanobodies.

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Section: Discussionmentioning

confidence: 99%

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

Yang

et al. 2022

Appl Microbiol Biotechnol

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“…Lin et al. [ 27 ] used phage‐display technology to screen two nanobodies, termed Nb11C12 and Nb2D5 that could recognize different epitopes of CEACAM‐5 with high affinity. Nb2D5 was further fused with three different types of ALP and expressed in human embryonic kidney cells.…”

Section: Nanobody‐implemented Immunoassaysmentioning

confidence: 99%

Nanobody and Nanozyme‐Enabled Immunoassays with Enhanced Specificity and Sensitivity

Wang

Xianyu

2022

Small Methods

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treatment of cancers, the second leading cause of death in the world that are hard to be treated in the terminal stages. [2] For instance, liver cancer is one of the most lethal cancers and has a poor prognosis with a 5-year survival rate of less than 20%. [3] The early detection and timely treatment of cancers are very important for the patients to save their lives. Microorganism contamination in food is the main cause of foodborne diseases and 70% of patients with foodborne illnesses get sick after taking foods or water contaminated with pathogenic microorganisms. [4] Viruses are one of the major causes of infectious diseases and various public health events like the acquired immunodeficiency syndrome (AIDS), the severe acute respiratory syndrome (SARS), and the coronavirus disease 2019 (COVID-19). [5] Therefore, the timely and rapid detection of microorganisms is of great importance to ensure human health and public safety. [6] In addition, the detections of small molecules including mycotoxins and exogenous pollutions play an important role in food safety. Mycotoxins such as aflatoxin B1, ochratoxin A, and tenuazonic acid are classified as naturally occurring carcinogens by the World Health Organization that are extremely harmful to both humans and animals. [7] They not only directly threaten human life by contaminating food, but also indirectly affect human health through the food chain by contaminating feeds of animals. [8,9] Commonly used immunoassays include enzyme-linked immunosorbent assay (ELISA), quantum dot fluorescence immunoassay, fluorescence polarization immunoassay, colloidal gold immunoassay, chemiluminescent immunoassay (CLIA), electrochemical immunoassay, and microarray-based immunoassay. In spite of the different formats of immunoassays, they share the same principle that relies on the specific binding between antigenic determinants and antibody recognition sites. Therefore, it is of great significance to prepare specific antibodies with high affinity towards the target molecules to improve the specificity of immunoassays. Conventional immunoassays generally use polyclonal and monoclonal antibodies, but both types of antibodies have limitations such as poor specificity, low stability, and complicated preparation. To meet these challenges, a new type of antibody called "nanobody" has emerged. Nanobody is the heavy-chain-only antibody with the advantages of small size, high specificity, mass expression and high stability, which can act as a substitute for conventional antibodies in immunoassays and a promising low-cost immuno-affinity detection reagent.Immunoassay as a rapid and convenient method for detecting a variety of targets has attracted tremendous interest with its high specificity and sensitivity. Among the commonly used immunoassays, enzyme-linked immunosorbent assay has been widely used as a gold standard method in various fields that consists of two main components including a recognition element and an enzyme label. With the rapid advances in nanotechnology, nanobodies and nanozym...

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“…We have also developed a diagnosis kit for CEA based on these nanobodies, which can still accurately detect the content of CEA in clinical serum after storage at 37 1C and 50% relative humidity for 64 days. 26 More interestingly, the epitope prediction analysis showed that the binding site of 11C12 was in the near membrane region of CEA. Therefore, 11C12 has the potential to be used as a targeting antibody to modify nanoparticles and achieve drug targeted delivery.…”

Section: Introductionmentioning

confidence: 99%

“…25 In our previous study, we screened and produced a pair of nanobodies, 11C12 and 2D5, that specifically bind CEA (dissociation constants of 12 nM and 774 pM, respectively). 26 They demonstrated superior specificity, with no binding to highly similar homologous proteins such as CEACAM1, 3, 6, 8 and fetoprotein, HE4 and CA125. They also exhibited exceptional stability.…”

Section: Introductionmentioning

confidence: 99%

Dual CEA/CD44 targeting to colorectal cancer cells using nanobody-conjugated hyaluronic acid-modified mesoporous silica nanoparticles with pH- and redox-sensitivity

et al. 2022

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Development of a highly thermostable immunoassay based on a nanobody-alkaline phosphatase fusion protein for carcinoembryonic antigen detection

Cited by 15 publications

References 14 publications

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

A novel silk fibroin protein–based fusion system for enhancing the expression of nanobodies in Escherichia coli

Nanobody and Nanozyme‐Enabled Immunoassays with Enhanced Specificity and Sensitivity

Dual CEA/CD44 targeting to colorectal cancer cells using nanobody-conjugated hyaluronic acid-modified mesoporous silica nanoparticles with pH- and redox-sensitivity

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