Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library

Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

doi:10.1007/s00253-018-8797-8

Cited by 23 publications

(9 citation statements)

References 56 publications

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“…As is well known, IPTG concentration, post-induction time, and incubation temperature are the main factors for optimizing protein expression [25]. In the preliminary experiments of this study, the highest scFv expression level was obtained at the following conditions: 37 • C, 16 h, and 1 mM IPTG.…”

Section: Expression Purification Of the Scfv-22mentioning

confidence: 59%

Preparation and Directed Evolution of Anti-Ciprofloxacin ScFv for Immunoassay in Animal-Derived Food

Wang

Zhang

et al. 2021

Foods

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An immunized mouse phage display scFv library with a capacity of 3.34 × 109 CFU/mL was constructed and used for screening of recombinant anti-ciprofloxacin single-chain antibody for the detection of ciprofloxacin (CIP) in animal-derived food. After four rounds of bio-panning, 25 positives were isolated and identified successfully. The highest positive scFv-22 was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residue Val160 was the key residue for the binding of scFv to CIP. Based on the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of contact amino acid residue Val160 to Ser. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant scFv was established for CIP, respectively. The IC50 value of the assay established with the ScFv mutant was 1.58 ng/mL, while the parental scFv was 26.23 ng/mL; this result showed highly increased affinity, with up to 16.6-fold improved sensitivity. The mean recovery for CIP ranged from 73.80% to 123.35%, with 10.46% relative standard deviation between the intra-assay and the inter-assay. The RSD values ranged between 1.49% and 9.81%. The results indicate that we obtained a highly sensitive anti-CIP scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of CIP residue in animal-derived edible tissues.

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Section: Expression Purification Of the Scfv-22mentioning

confidence: 59%

Preparation and Directed Evolution of Anti-Ciprofloxacin ScFv for Immunoassay in Animal-Derived Food

Wang

Zhang

et al. 2021

Foods

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“…As is well known, IPTG concentration, post-induction time, and incubation temperature are the main factors for optimizing protein expression (Dong, Bo, Zhang, Feng, & Liu, 2018).…”

Section: Discussionmentioning

confidence: 99%

Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

Wang¹,

Li²,

Zhang³

et al. 2021

Preprint

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A recombinant anti-enrofloxacin single-chain antibody (scFv) was produced for the detection of enrofloxacin. An immunized mouse phage display scFv library with a capacity of 2.35×109 CFU/mL was constructed and used for anti-enrofloxacin scFv screening. After four rounds of bio-panning, 10 positives were isolated and identified successfully. The highest positive scFv was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residues Leu121 were the key residues for the binding of ScFv to ENR. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Leu121 to Asn. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant ScFv were established for enrofloxacin respectively. The IC50 value of the assay established with the ScFv mutant was 1.63 ng/mL, while the parental ScFv was 21.08 ng/mL, this result showed highly increased affinity with up to 12.9-folds improved sensitivity. The mean recovery for ENR ranged from 71.80% to 117.35% with 10.46% relative standard deviation between the intra-assay and the inter-assay. The results indicate that we have obtained a highly sensitive anti-ENR scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of ENR residue in animal derived edible tissues and milk.

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“…Later, a mouse phage-displayed scFv-3H9 (MG214869) mutant was screened out for broad-spectrum one-step detection of six Cry1 antigens. The LOD of DAS-ELISA reached 3.14–11.07 ng/mL with 84–95% recovery from spiked rice with CV of <8%, respectively . Subsequently, Zhong et al.…”

Section: Elisamentioning

confidence: 97%

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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Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library

Cited by 23 publications

References 56 publications

Preparation and Directed Evolution of Anti-Ciprofloxacin ScFv for Immunoassay in Animal-Derived Food

Preparation and Directed Evolution of Anti-Ciprofloxacin ScFv for Immunoassay in Animal-Derived Food

Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins

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