Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Speci...

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Autores principales:	Zhuolin Song, Lin Feng, Yuankui Leng, Mingzhu Huang, Hao Fang, Weipeng Tong, Xuelan Chen, Yonghua Xiong
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	cascade-amplifying enzyme loading M13 bacteriophage polymeric horseradish peroxidases ochratoxin A Medicine R
Acceso en línea:	https://doaj.org/article/03d61e8a01ce4e0cb699073d137fa607
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spelling	oai:doaj.org-article:03d61e8a01ce4e0cb699073d137fa6072021-11-25T19:08:47ZDramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading10.3390/toxins131107812072-6651https://doaj.org/article/03d61e8a01ce4e0cb699073d137fa6072021-11-01T00:00:00Zhttps://www.mdpi.com/2072-6651/13/11/781https://doaj.org/toc/2072-6651Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.Zhuolin SongLin FengYuankui LengMingzhu HuangHao FangWeipeng TongXuelan ChenYonghua XiongMDPI AGarticlecascade-amplifying enzyme loadingM13 bacteriophagepolymeric horseradish peroxidasesochratoxin AMedicineRENToxins, Vol 13, Iss 781, p 781 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cascade-amplifying enzyme loading M13 bacteriophage polymeric horseradish peroxidases ochratoxin A Medicine R
spellingShingle	cascade-amplifying enzyme loading M13 bacteriophage polymeric horseradish peroxidases ochratoxin A Medicine R Zhuolin Song Lin Feng Yuankui Leng Mingzhu Huang Hao Fang Weipeng Tong Xuelan Chen Yonghua Xiong Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading
description	Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.
format	article
author	Zhuolin Song Lin Feng Yuankui Leng Mingzhu Huang Hao Fang Weipeng Tong Xuelan Chen Yonghua Xiong
author_facet	Zhuolin Song Lin Feng Yuankui Leng Mingzhu Huang Hao Fang Weipeng Tong Xuelan Chen Yonghua Xiong
author_sort	Zhuolin Song
title	Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading
title_short	Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading
title_full	Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading
title_fullStr	Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading
title_full_unstemmed	Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading
title_sort	dramatically enhancing the sensitivity of immunoassay for ochratoxin a detection by cascade-amplifying enzyme loading
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/03d61e8a01ce4e0cb699073d137fa607
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Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

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