Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody

Dong, Sa; Gao, Meijing; Bo, Zongyi; Guan, Lingjun; Hu, Xiaodan; Zhang, Hanxiaoya; Liu, Beibei; Li, Pan; He, Kangli; Liu, Xianjin; Zhang, Cunzheng

doi:10.1016/j.ijbiomac.2020.01.152

Cited by 14 publications

(19 citation statements)

References 44 publications

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“…Recently, Dong et al developed a scFv by site-directed mutagenesis combined with hybridoma cell culture, homology modeling, and molecular docking to detect multiple Cry1A endotoxins. The LOD reported by the scFv-2G12 mutant was 4.6–9.2 ng/mL as determined by DAS-ELISA, and the equilibrium dissociation constant was surprisingly lower compared to that of scFv-5B5, which was obtained by biopanning . Recently, the development of genetically engineered antibodies is in its infancy.…”

Section: Elisamentioning

confidence: 99%

“…The LOD reported by the scFv-2G12 mutant was 4.6−9.2 ng/mL as determined by DAS-ELISA, and the equilibrium dissociation constant was surprisingly lower compared to that of scFv-5B5, which was obtained by biopanning. 47 Recently, the development of genetically engineered antibodies is in its infancy. Although the sensitivity was lower than some of the reported assays and commercial kits as a result of several reasons, including limited mutations, library capacity, and screening procedure, the advances in molecular approaches would likely transform detection methods and scope of immunoassays.…”

Section: Elisamentioning

confidence: 99%

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Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins

Faheem

Qin

Nan

et al. 2021

J. Agric. Food Chem.

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Insect-resistant genetically modified organisms have been globally commercialized for the last 2 decades. Among them, transgenic crops based on Bacillus thuringiensis crystalline (Cry) toxins are extensively used for commercial agricultural applications. However, less emphasis is laid on quantifying Cry toxins because there might be unforeseen health and environmental concerns. Immunoassays, being the preferred method for detection of Cry toxins, are reviewed in this study. Owing to limitations of traditional colorimetric enzyme-linked immunosorbent assay, the trend of detection strategies shifts to modified immunoassays based on nanomaterials, which provide ultrasensitive detection capacity. This review assessed and compared the properties of the recent advances in immunoassays, including colorimetric, fluorescence, chemiluminescence, surface-enhanced Raman scattering, surface plasmon resonance, and electrochemical approaches. Thus, the ultimate aim of this study is to identify research gaps and infer future prospects of current approaches for the development of novel immunosensors to monitor Cry toxins in food and the environment.

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Section: Elisamentioning

confidence: 99%

Section: Elisamentioning

confidence: 99%

Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins

Faheem

Qin

Nan

et al. 2021

J. Agric. Food Chem.

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“…Among them, the most frequently used format is double-antibody sandwich ELISAs (DAS-ELISAs), which generally depend on the traditional polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) [ 17 , 18 ]. Besides, genetically engineered antibodies (e.g., ScFvs) and phage-displayed peptides have been applied in DAS-ELISA for Cry toxin analysis [ 19 , 20 ]. However, ScFvs and peptides usually exhibited relatively low affinity and poor stability [ 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

Quantum-Dot-Bead-Based Fluorescence-Linked Immunosorbent Assay for Sensitive Detection of Cry2A Toxin in Cereals Using Nanobodies

Qiu

You

et al. 2022

Foods

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In this study, a quantum-dot-bead (QB)-based fluorescence-linked immunosorbent assay (FLISA) using nanobodies was established for sensitive determination of the Cry2A toxin in cereal. QBs were used as the fluorescent probe and conjugated with a Cry2A polyclonal antibody. An anti-Cry2A nanobody P2 was expressed and used as the capture antibody. The results revealed that the low detection limit of the developed QB-FLISA was 0.41 ng/mL, which had a 19-times higher sensitivity than the traditional colorimetric ELISA. The proposed assay exhibited a high specificity for the Cry2A toxin, and it had no evident cross-reactions with other Cry toxins. The recoveries of Cry2A from the spiked cereal sample ranged from 86.6–117.3%, with a coefficient of variation lower than 9%. Moreover, sample analysis results of the QB-FLISA and commercial ELISA kit correlated well with each other. These results indicated that the developed QB-FLISA provides a potential approach for the sensitive determination of the Cry2A toxin in cereals.

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“…Considering the effect of a substitution in multiple genetic contexts-in other words, considering combinations of substitutions-can better illuminate the contributions of interface residues to specificity (Dutta et al, 2010;Jenson et al, 2018;Salinas & Ranganathan, 2018). Many studies have leveraged large, combinatorial libraries to identify protein variants that bind a desired target such as bacterial Cry toxins (Dong et al, 2020;Jiao et al, 2017), HIV antigens (Barbas et al, 1994;Burton et al, 1991), or cytokine receptors (Fairlie et al, 2004). However, these studies did not systematically examine the effects of substitutions at a given protein interface.…”

Section: Introductionmentioning

confidence: 99%

Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library

Lite

Grant

Nocedal

et al. 2020

eLife

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Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.

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Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody

Cited by 14 publications

References 44 publications

Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins

Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins

Quantum-Dot-Bead-Based Fluorescence-Linked Immunosorbent Assay for Sensitive Detection of Cry2A Toxin in Cereals Using Nanobodies

Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library

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