Nanobody-Based Enzyme Immunoassay for Aflatoxin in Agro-Products with High Tolerance to Cosolvent Methanol

He, Ting; Wang, Yanru; Li, Peiwu; Zhang, Qi; Lei, Jiawen; Zhang, Zhaowei; Ding, Xiaoxia; Zhou, Haiyan; Zhang, Wen

doi:10.1021/ac502390c

Cited by 130 publications

(111 citation statements)

References 29 publications

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“…Solvent contents in the sample often change the analytical parameters of an immunoassay (He et al, 2014;Mercader & AbadFuentes, 2009). Fermented drinks like wine or cider may contain important concentrations of ethanol, so the influence of alcohol over the inhibition curve of the proposed cELISAs needs to be evaluated.…”

Section: Alcohol Tolerancementioning

confidence: 99%

Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks

Esteve‐Turrillas¹,

Mercader²,

Agulló³

et al. 2015

Food Control

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1Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection 2 and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site 3 heterologies were synthesized and employed in order to produce antibodies with enhanced affinity. 4Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity 5 improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active 6 substance were developed using direct and indirect formats, achieving IC 50 values around 0.15 7 µg/L. Analytical figures of merit and usability of the optimized assays were evaluated with wine and 8 cider as model food processed matrices. The obtained recoveries were from 90% to 120%, and the 9 limit of quantification was in the 1 to 5 µg/L range. Finally, a monitoring study (n=150) was 10

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Section: Alcohol Tolerancementioning

confidence: 99%

Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks

Esteve‐Turrillas¹,

Mercader²,

Agulló³

et al. 2015

Food Control

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“…Compared to the conventional antibody fragments, the evolution of hydrophilic amino acids within framwork 2 of the heavy chain variable domain contributes to the high water solubility of nanobodies [9]. Due to their unique properties, more and more nanobodies for toxic and harmful substances were isolated and used as the detection reagent of immunoassays in food analysis [13, 14]. In addition, Nbs are readily genetically manipulated to construct fusions with green fluorescence protein and alkaline phosphatase [15–17], thus expanding their applications in immunoassay.…”

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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“…Aflatoxin B 1 (AFB 1 ) is the most toxic, and one of the most potent carcinogens in nature. In this study [39], the VHH antibodies were selected using gradually decreasing concentrations of AFB 1 −BSA conjugate and free AFB 1 and more stringent washing. Two unique clones, named Nb26 and Nb28, were selected.…”

Section: Summary Of Vhh Assays To Environmental Chemicalsmentioning

confidence: 99%

“…Similar results were observed with DMSO. The VHHs for aflatoxin described by He et al [39] were evaluated in the presence of MeOH, DMSO, dimethylformamide (DMF), acetone and acetonitrile. The data indicated that the VHHs and mAbs were remarkably resistant to acetone.…”

Section: Advantages Of Vhhsmentioning

confidence: 99%

VHH antibodies: emerging reagents for the analysis of environmental chemicals

et al. 2016

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A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Discovered nearly 25 years ago, they have been investigated for their use in clinical therapeutics and immunodiagnostics, and more recently for environmental monitoring applications. A new and valuable immunoreagent for the analysis of small molecular weight environmental chemicals, VHH will overcome many pitfalls encountered with conventional reagents. In the work so far, VHH antibodies often perform comparably to conventional antibodies for small molecule analysis, are amenable to numerous genetic engineering techniques, and show ease of adaption to other immunodiagnostic platforms for use in environmental monitoring. Recent reviews cover the structure and production of VHH antibodies as well as their use in clinical settings. However, no report focuses on the use of these VHH antibodies to small environmental chemicals (MW <1,500 Da). This review article summarizes the efforts made to produce VHHs to various environmental targets, compares the VHH-based assays with conventional antibody assays, and discusses the advantages and limitations in developing these new antibody reagents particularly to small molecule targets.

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Nanobody-Based Enzyme Immunoassay for Aflatoxin in Agro-Products with High Tolerance to Cosolvent Methanol

Cited by 130 publications

References 29 publications

Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks

Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks

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

VHH antibodies: emerging reagents for the analysis of environmental chemicals

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