Enhanced Non-Toxic Immunodetection of Alternaria Mycotoxin Tenuazonic Acid Based on Ferritin-Displayed Anti-Idiotypic Nanobody-Nanoluciferase Multimers

• Published in: Journal of agricultural and food chemistry 2021-04, Vol.69 (16), p.4911-4917
• Main Authors: Wang, Feng, Li, Zhen-Feng, Wan, De-Bin, Vasylieva, Natalia, Shen, Yu-Dong, Xu, Zhen-Lin, Yang, Jin-Yi, Gettemans, Jan, Wang, Hong, Hammock, Bruce D, Sun, Yuan-Ming
• Format: Article
• Language: English
• Subjects: Alternaria; Enzyme-Linked Immunosorbent Assay; Ferritins; Mycotoxins; New Analytical Methods; Single-Domain Antibodies - genetics; Tenuazonic Acid
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.1c01128

The non-toxic immunoassay for mycotoxins is being paid more attention due to its advantages of higher safety and cost savings by using anti-idiotype antibodies to substitute toxins. In this study, with tenuazonic acid (TeA), a kind of highly toxic Alternaria mycotoxin as the target, an enhanced non-toxic immunoassay was developed based on the ferritin-displayed anti-idiotypic nanobody-nanoluciferase multimers. First, three specific β-type anti-idiotype nanobodies (AId-Nbs) bearing the internal image of TeA mycotoxin were selected from an immune phage display library. Then, the AId-Nb 2D with the best performance was exploited to generate a nanoluciferase (Nluc)-functionalized fusion monomer, by which a one-step non-toxic immunodetection format for TeA was established and proven to be effective. To further improve the affinity of the monomer, a ferritin display strategy was used to prepare 2D-Nluc fusion multimers. Finally, an enhanced bioluminescent enzyme immunoassay (BLEIA) was established in which the half maximal inhibitory concentration (IC50) for TeA was 6.5 ng/mL with a 10.5-fold improvement of the 2D-based enzyme-linked immunosorbent assay (ELISA). The proposed assay exhibited high selectivities and good recoveries of 80.0–95.2%. The generated AId-Nb and ferritin-displayed AId-Nb-Nluc multimers were successfully extended to the application of TeA in food samples. This study brings a new strategy for production of multivalent AId-Nbs and non-toxic immunoassays for trace toxic contaminants.