Evaluation of a water-soluble adjuvant for the development of monoclonal antibodies against small-molecule compounds

Zhang

et al. 2017

J. Agric. Food Chem.

Carabrone, a botanical bicyclic sesquiterpenic lactone, has broad-spectrum antifungal activity and is particularly efficient against the devastating phytopathogen Gaeumannomyces graminis var. tritici (Ggt). The antifungal mechanism of carabrone against Ggt, however, remains unclear. The main objective of this study was to investigate the subcellular localization of carabrone in Ggt to gain a better understanding of its mechanism of action. When Ggt was exposed to carabrone (EC value of 28.45 μg/mL) for 7 days, a decline in mitochondrial concentration together with some obvious alternations in mitochondrial structure, including hazy outlines, medullary transitions, excess accumulation of unclear settlings, and vacuolar degeneration, were observed, indicating that carbrone may act on the mitochondria directly. A fluorescent conjugate (TTY) was thus designed and synthesized as a surrogate of carabrone that possessed comparable antifungal activity against Ggt (EC of 33.68 μg/mL). Additionally, a polyclonal antibody specific to carabrone and with a high titer (256 000) was also prepared by immunizing mice. Subsequently, two imaging techniques, the use of the fluorescent conjugate (FC) and immunofluorescence (IF), were applied to determine the subcellular localization of carabrone. Both FC and IF fluorescent signals demonstrated its mitochondrial localization with a Pearson's coefficient of 0.83 for FC and 0.86 for IF. These results imply that carabrone exerts its antifungal activity against Ggt by interfering with mitochondrial function.

Section: ■ Materials and Methodsmentioning

confidence: 99%

Mitochondrial Signs and Subcellular Imaging Provide Insight into the Antifungal Mechanism of Carabrone against Gaeumannomyces graminis var. tritici

Zhang

et al. 2017

J. Agric. Food Chem.

“…Thiacloprid-C4C4 mAb was prepared according to the protocol described in our previous work [ 47 ]. The thiacloprid–bull serum albumin (BSA) conjugate reported by Zhenjiang Liu et al [ 32 ] was used as the artificial immunogen to induce the immune response.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody

Liu

Guo

Jiao

et al. 2020

IJMS

Self Cite

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 μg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH–π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

J. Zhejiang Univ. Sci. B

“…Enzyme-linked immunosorbent assay (ELISA) was used to confirm the conjugates by the standard procedure (Liu R et al, 2016;Song et al, 2017). OTA-BSA (OTA-OVA) or ZEN-BSA (ZEN-OVA) was immobilized on polystyrene 96-well plates and then the coated antigens were probed with their specific monoclonal antibody, followed by the addition of goat anti-rabbit IgG-horseradish peroxidase (HRP) to each well.…”

Section: Synthesis Of Ota and Zen Conjugatesmentioning

confidence: 99%

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples

Zhang

Fang³

et al. 2018

A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53-12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06-39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.