College of Food Science, Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, P. R. China.
Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.
J Agric Food Chem. 2021 Apr 28;69(16):4911-4917. doi: 10.1021/acs.jafc.1c01128. Epub 2021 Apr 18.
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 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 (IC) 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.
由于使用抗独特型抗体替代毒素具有更高的安全性和成本节约优势，针对真菌毒素的无毒免疫分析受到了更多关注。在这项研究中，以具有高毒性的棒曲霉素（TeA）为目标，基于展示在铁蛋白上的抗独特型纳米抗体-纳米荧光素酶多聚体，开发了一种增强型无毒免疫分析方法。首先，从免疫噬菌体展示文库中筛选出三个具有 TeA 真菌毒素内影像的特异性β型抗独特型纳米抗体（AId-Nb）。然后，利用表现最佳的 AId-Nb 2D 生成纳米荧光素酶（Nluc）功能化融合单体，建立了一步式无毒免疫检测 TeA 的方法，证明其有效。为了进一步提高单体的亲和力，采用铁蛋白展示策略制备 2D-Nluc 融合多聚体。最终，建立了增强型生物发光酶免疫分析（BLEIA），TeA 的半抑制浓度（IC）为 6.5 ng/mL，比基于 2D 的酶联免疫吸附测定（ELISA）提高了 10.5 倍。该检测方法具有较高的选择性和良好的回收率（80.0-95.2%）。所生成的 AId-Nb 和铁蛋白展示的 AId-Nb-Nluc 多聚体成功扩展到食品样品中 TeA 的应用。该研究为生产多价抗独特型纳米抗体和痕量有毒污染物的无毒免疫分析带来了新策略。
