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

• Published in: Toxins Vol. 13; no. 11; p. 781
• Main Authors: Song, Zhuolin, Feng, Lin, Leng, Yuankui, Huang, Mingzhu, Fang, Hao, Tong, Weipeng, Chen, Xuelan, Xiong, Yonghua
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 05.11.2021; MDPI
• Subjects: Antibodies; Antigens; Bacteriophages - chemistry; Binding sites; Biotin; Biotinylation; cascade-amplifying enzyme loading; Chromatography; Competition; Cross-reaction; Enzyme-linked immunosorbent assay; Enzymes; Food contamination; Immunoassay; Immunoassay - methods; International organizations; M13 bacteriophage; Monoclonal antibodies; Mycotoxins; Ochratoxin A; Ochratoxins - analysis; Peptides; Phages; Poisons - analysis; polymeric horseradish peroxidases; Proteins; Sensitivity enhancement; Spectrum analysis; Streptavidin; ochratoxin A; polymeric horseradish peroxidases; cascade-amplifying enzyme loading; M13 bacteriophage
• ISSN: 2072-6651; 2072-6651
• DOI: 10.3390/toxins13110781

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.