Affinity maturation of single-chain variable fragment specific for aflatoxin B1 using yeast surface display

Min, Won‐Ki; Kim, Sung-Gun; Seo, Jin‐Ho

doi:10.1016/j.foodchem.2015.04.117

Cited by 23 publications

(15 citation statements)

References 28 publications

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“…32 The mutations that were located outside the canonical antigen-contact loops suggested favorable allosteric effects that could not be predicted by 3D modeling as previously suggested. 26 Therefore, our result also confirmed that case-to-case analysis is necessary to understand the contribution of antibody chains to their binding properties. 33 In conclusion, application of the chain-shuffling technique for improvement of the binding characteristic of human antiaflatoxin (yAFB1-c3) scFv antibody was demonstrated.…”

Section: ■ Materials and Methodssupporting

confidence: 81%

“…25 This result is comparable to another method of affinity maturation using a combination of random mutation and yeast display method, of which the best mouse scFv (obtained from a hybridoma) against AFB1 could be improved 9-fold compared to the parental clone. 26 This improvement was much lower when compared to previous reports on the affinity maturation of chicken scFv antibody against halofuginone, an antiprotozoal drug, where a 185-fold improvement over the original scFv could be obtained by affinity selection from a heavy chain-biased, light-chain-shuffling library. 8 The difference could be due to the nature of the antigen.…”

Section: ■ Materials and Methodsmentioning

confidence: 74%

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Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody–Ligand Interaction Using Chain Shuffling

Rangnoi

Choowongkomon

O’Kennedy

et al. 2018

J. Agric. Food Chem.

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A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.

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Section: ■ Materials and Methodssupporting

confidence: 81%

Section: ■ Materials and Methodsmentioning

confidence: 74%

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody–Ligand Interaction Using Chain Shuffling

Rangnoi

Choowongkomon

O’Kennedy

et al. 2018

J. Agric. Food Chem.

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“…With the advances in molecular cloning and phage display techniques, numerous recombinant antibodies have been produced, which are also known as genetically modified antibodies [3]. Recombinant antibodies such as fragment of antigen-binding (Fab) and single-chain variable fragment (scFv) have been developed and applied to various assays of mycotoxins [4, 5]. However, the low expression yield and poor stability of these recombinant antibodies limit their development [6, 7].…”

Section: Introductionmentioning

confidence: 99%

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference

Liu

Tang

Duan

et al. 2017

Talanta

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A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95 °C for 5 min or to 90 °C for 75 min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC50 of 0.64 ng/mL and a linear range (IC20-IC80) of 0.27–1.47 ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development.

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“…The affinity maturation of scFv‐WT using yeast surface display resulted in the isolation of scFv‐M37 containing six mutations, which exhibited a ninefold higher affinity than scFv‐WT (Min et al . ). Furthermore, when a deleterious mutation (V H ‐A110T) located on the framework region of the scFv‐M37 was reverted in the scFv‐BM3, the affinity to AFB 1 was improved by 31‐fold in comparison with the scFv‐WT (Min et al .…”

Section: Introductionmentioning

confidence: 97%

“…In our previous studies, the single-chain variable fragment (scFv) against AFB 1 (scFv-WT) was constructed from its monoclonal antibody gene, and expressed in Escherichia coli (Min et al 2011). The affinity maturation of scFv-WT using yeast surface display resulted in the isolation of scFv-M37 containing six mutations, which exhibited a ninefold higher affinity than scFv-WT (Min et al 2015). Furthermore, when a deleterious mutation (V H -A110T) located on the framework region of the scFv-M37 was reverted in the scFv-BM3, the affinity to AFB 1 was improved by 31-fold in comparison with the scFv-WT (Min et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

Kim²,

Choi³

et al. 2019

Lett Appl Microbiol

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scFv‐BM3 is a single‐chain variable fragment (scFv) against aflatoxin B1 (AFB1) engineered by affinity maturation and site‐directed mutagenesis, and thus has a 31‐fold higher affinity than its wild‐type. To apply scFv‐BM3 to immunological detection of AFB1, periplasmic expression in Escherichia coli was attempted to produce a functional form of scFv‐BM3. scFv‐BM3 accumulated as inactive aggregates in the cells. However, it was found that scFv‐BM3 secreted into the culture medium had binding activity to AFB1. Expression conditions for scFv‐BM3 were further manipulated to enhance secretion into the culture medium. This extracellular secretion of functional scFv‐BM3 was significantly improved by supplementation with Triton X‐100 and optimization of expression conditions. The scFv‐BM3 purified from the culture medium exhibited a typical antiparallel β‐sheet structure and adopted a proper conformation to bind AFB1 with high affinity and specificity in various biophysical and biochemical analyses. Significance and Impact of the Study Single‐chain variable fragments (scFvs) are recombinant antibodies that are difficult to produce as a functional form in Escherichia coli. This study demonstrates the production of functional scFvs against aflatoxin B1 (AFB1) (scFv‐BM3) using Escherichia coli by extracellular secretion. While periplasmic expression of scFv‐BM3 resulted in formation of inactive aggregates in E. coli, the scFv‐BM3 secreted into the culture medium adopted a properly folded structure for specific binding to AFB1. This study promotes the application of functional scFv‐BM3 to the immunological detection of AFB1 in biotechnology fields.

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Affinity maturation of single-chain variable fragment specific for aflatoxin B1 using yeast surface display

Cited by 23 publications

References 28 publications

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody–Ligand Interaction Using Chain Shuffling

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody–Ligand Interaction Using Chain Shuffling

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

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Affinity maturation of single-chain variable fragment specific for aflatoxin B1 using yeast surface display

Cited by 23 publications

References 28 publications

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody–Ligand Interaction Using Chain Shuffling

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody–Ligand Interaction Using Chain Shuffling

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference

Extracellular production of functional single-chain variable fragment against aflatoxin B1usingEscherichia coli

Contact Info

Product

Resources

About

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli