Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

Erdağ, Berrin; Balcıoğlu, Bertan Koray; Bahadır, Aylin Özdemir; Hinc, Duygu; Ibrahimoglu, O. Ozlem; Bahar, Aydin; Başalp, Aynur; Yücel, Fatıma

doi:10.1080/13102818.2017.1348256

Cited by 4 publications

(3 citation statements)

References 42 publications

(37 reference statements)

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“…More broadly, this study represents one of the first instances in which a full-length antibody is genetically fused to an enzyme for the purposes of detection. The vast majority of previous work has generated scFv–enzyme fusion proteins − or fragment antigen-binding (Fab)–enzyme fusion proteins. , scFvs and Fabs contain only one antigen-binding site per molecule and thus do not benefit from the avidity advantage afforded by the two binding sites in a full-length antibody. To our knowledge, only one platform has been developed to produce enzymes that are genetically fused to full-length antibodies, specifically a fusion protein scaffold linking an enzyme to IgM, yielding a highly multimeric antibody–enzyme pentamer .…”

Section: Discussionmentioning

confidence: 99%

Antibody–Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers

et al. 2022

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Rapid diagnostics that can accurately inform patients of disease risk and protection are critical to mitigating the spread of the current COVID-19 pandemic and future infectious disease outbreaks. To be effective, such diagnostics must rely on simple, cost-effective, and widely available equipment and should be compatible with existing telehealth infrastructure to facilitate data access and remote care. Commercial glucometers are an established detection technology that can overcome the cost, time, and trained personnel requirements of current benchtop-based antibody serology assays when paired with reporter molecules that catalyze glucose conversion. To this end, we developed an enzymatic reporter that, when bound to disease-specific patient antibodies, produces glucose in proportion to the level of antibodies present in the patient sample. Although a straightforward concept, the coupling of enzymatic reporters to secondary antibodies or antigens often results in low yields, indeterminant stoichiometry, reduced target binding, and poor catalytic efficiency. Our enzymatic reporter is a novel fusion protein that comprises an antihuman immunoglobulin G (IgG) antibody genetically fused to two invertase molecules. The resulting fusion protein retains the binding affinity and catalytic activity of the constituent proteins and serves as an accurate reporter for immunoassays. Using this fusion, we demonstrate quantitative glucometer-based measurement of anti-SARS-CoV-2 spike protein antibodies in blinded clinical sample training sets. Our results demonstrate the ability to detect SARS-CoV-2-specific IgGs in patient serum with precise agreement to benchmark commercial immunoassays. Because our fusion protein binds all human IgG isotypes, it represents a versatile tool for detection of disease-specific antibodies in a broad range of biomedical applications.

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Section: Discussionmentioning

confidence: 99%

Antibody–Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers

et al. 2022

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“…The cells were then centrifuged at 10,000 × g for 10 min at 4 °C and the pellet was resuspended in 5 mM Mg 2 SO 4 and incubated for 10 min on ice with shaking. The cells were centrifuged at 10,000 × g for 10 min at 4 °C and the pellet was recovered 27 .…”

Section: Methodsmentioning

confidence: 99%

Development of a single-chain variable antibody fragment against a conserved region of the SARS-CoV-2 spike protein

Gao,

Irie,

Kouwaki

et al. 2024

Sci Rep

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prolonged the duration of the pandemic because of the continuous emergence of new variant strains. The emergence of these mutant strains makes it difficult to detect the virus with the existing antibodies; thus, the development of novel antibodies that can target both the variants as well as the original strain is necessary. In this study, we generated a high-affinity monoclonal antibody (5G2) against the highly conserved region of the SARS-CoV-2 spike protein to detect the protein variants. Moreover, we generated its single-chain variable antibody fragment (sc5G2). The sc5G2 expressed in mammalian and bacterial cells detected the spike protein of the original SARS-CoV-2 and variant strains. The resulting sc5G2 will be a useful tool to detect the original SARS-CoV-2 and variant strains.

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“…Here, we have hypothesized that if the framework regions of a suitable intrinsically soluble scFV are substituted by the framework regions of an inclusion body forming scFv, the resulting scFv would be a soluble molecule. Fortunately for us, a periplasmic expression scFv molecule, namely Lig7 25 developed in our group against a different antigen, shares a moderate sequence similarity to anti‐KDR 1.3, bears the same FR lengths, and most importantly demonstrates a dramatically higher yield of soluble protein expression. This engineering approach is a promising strategy that can resolve the inclusion body problem of a scFv antibody, eliminating the need for time‐consuming optimization procedures.…”

Section: Introductionmentioning

confidence: 99%

Structure‐based engineering of an antiangiogenic scFv antibody for soluble production in E. coli without loss of activity

Öncü

Balcıoğlu

Özgür

et al. 2021

Biotech and App Biochem

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Development of monoclonal antibody therapeutics against vascular endothelial growth factor receptor 2 (VEGFR‐2) protein, which is the main regulator in angiogenesis, has been a major challenge for years. In the current study, we engineer an inclusion body forming single‐chain variable fragment (scFv) against VEGFR‐2 by using complementarity determining regions (CDR) grafting technique to improve its solubility and investigate the activity of the engineered molecule. CDR sequences of the target scFv were grafted into the framework of another intrinsically soluble scFv molecule. Based on the computational results, CDR grafting has increased the solubility of the grafted scFv molecule. Results confirmed that the grafting approach increased in vivo folding properties of the target scFv molecule compared with the original scFv molecule. Similar binding affinities to the VEGFR‐2 were observed for the original and the grafted scFv by surface plasmon resonance assays. Biological activity assays, including human umbilical vein endothelial cells proliferation and wound healing assays, showed that grafted scFv molecule has an antiangiogenic property. This study suggests that an antiangiogenic scFv fully expressed as an inclusion body can be rescued by grafting its CDR regions to a scFv expressed in a soluble form without any loss in its binding property and its activity.

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Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

Cited by 4 publications

References 42 publications

Antibody–Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers

Antibody–Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers

Development of a single-chain variable antibody fragment against a conserved region of the SARS-CoV-2 spike protein

Structure‐based engineering of an antiangiogenic scFv antibody for soluble production in E. coli without loss of activity

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