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Cited by 25Monoclonal and Polyclonal Antibodies ResearchAdvanced Biosensing Techniques and Applicationsinterferon and immune responses

Screening of a ScFv Antibody With High Affinity for Application in Human IFN-γ Immunoassay

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Yang, Hang, Zhong, Yanfang, Wang, Juncheng, Zhang, Qinghong, Li, Xiulan, Ling, Sumei, Wang, Shihua, Wang, Rongzhi·2018·Frontiers in Microbiology
DOI 10.3389/fmicb.2018.00261PMC PMC5850876
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Abstract

Interferon gama (IFN-γ), a signal proinflammatory cytokine secreted by immune cell, and plays a critical role in the pathogenesis and progression of many diseases. It has been regarded as an important marker for determination of disease-specific immune responses. Therefore, it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real blood samples. Until now, the immunoassay based on scFv antibody for human IFN-γ is still not reported. In the present study, an scFv antibody named scFv-A8 with high specificity was obtained by phage display and biopanning, with the affinity 2.6×109 L/mol. Maltose binding protein(MBP) was used to improve the solubility of scFv by inserting an linker DNA between scFv and MBP tag, and the resulted fusion protein (MBP-LK-scFv) has high solubility and antigen biding activity. The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive ELISA (ic-ELISA) for detection of human IFN-γ, and the result indicated that the linear range to detect IFN-γ was 6-60 pg/mL with IC50 of 25 pg/mL. The limit of detection was 2 pg/mL (1.3 fm), and the average recovery was 85.05%, further demonstrating that the detection method based on scFv has higher recovery and accuracy. Hence, the developed ic-ELISA can be used to detect IFN-γ in real samples, and it may be further provided a scientific basis for disease diagnosis.

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  • Use of fusion partners such as GST, MBP, SUMO and Trx to target proteins, especially scFv fragments, results in solubility-enhancing properties and increased yields of soluble and active products.

    “Several reports have also suggested that the use of fusion partners such as GST [118], maltose-binding protein (MBP) [119,120], small ubiquitin-related modifier (SUMO) [87,121] and thioredoxin (Trx) [122] to the target proteins, especially to scFv fragments, results in solubility-enhancing properties and in increased yields of soluble and active products.”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [120]

    The Results (Soluble Expression of ScFv) show that MBP fusion improved scFv solubility, supporting the MBP portion to which this citation is attached. The Discussion says MBP fusion alone did not improve binding activity; a linker was needed for the highest activity. The paper does not establish increased yields of active products from MBP fusion alone. (LLM, conf=0.88)

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Cited by

Sorted by citations

Authors
Yang, H., Zhong, Y., Wang, J., Zhang, Q., Li, X., Ling, S., Wang, S., & Wang, R.
Affiliations
Fujian Agriculture and Forestry University (CN)
Corresponding author
  • Wang, Shihua
  • Wang, Rongzhi
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2018-03-07
Volume
9
Pages
261-261
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2018.00261
PMC
PMC5850876
OpenAlex
W2791522427
Cited by
25
References
0
Citations by year
2026: 2 · 2025: 4 · 2024: 4 · 2023: 4 · 2022: 3 · 2021: 1 · 2020: 3 · 2019: 3 · 2018: 1
FWCI
1.38
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Microbiology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Fujian Agriculture and Forestry University
Topics
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
Keywords
interferon-gamma, scFv antibodies, single-chain antibody, phage display, indirect competitive ELISA, maltose-binding protein fusion, cytokine detection, immunoassay, high-affinity antibodies

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