Feng Wang,Zhen-Feng Li,De-Bin Wan,Natalia Vasylieva,Yu-Dong Shen,Zhen-Lin Xu,Jin-Yi Yang,Jan Gettemans,Hong Wang,Bruce D. Hammock,Yuan-Ming Sun
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.
Alternaria spp. infect and accumulate various toxic secondary metabolites in apples, posing a threat to human and animal health. This study established instrumental analytical methods for six Alternaria toxins, systematically investigated the contamination levels of Alternaria toxins in apples and their reduction and transfer patterns during processing. Apples exhibited heightened disease susceptibility at 25 °C and produced 6 Alternaria toxins in different degrees, while Alternaria toxins were not observed within 4 cm of the symptomatic site. The transfer rate to pomace after juice extraction was higher than that in juice, with transfer rates ranging from 51.9 to 62.75 % for pomace and from 21.44 to 35.79 % for juice. Canning reduced Alternaria toxins in pulp, with the highest reduction rate of 93.3 % for tenuazonic acid. A total of four Alternaria toxins were detected in the testing of market samples of apple products, and none of the assessed total daily exposure doses of toxins ingested by humans through apple products exceeded the daily intake level recommended by the EFSA. The evaluation results indicate that the risk of dietary exposure of humans is relatively low through apple products. These findings provide valuable data and theoretical support for ensuring food safety in apples.
危险品化学品经营许可证（带存储）