Aflatoxin B (AFB) is a well-known carcinogen for human health, and requires sensitive and rapid methods for monitoring. Here, we developed an Avi-tagged nanobody (AviTag-Nb) and incorporated it into the biotin-streptavidin-amplified ELISA (BA-ELISA) and magnetic bead-based ELISA (MB-ELISA) to detect AFB. After optimization, BA-ELISA and MB-ELISA achieved 50% inhibitory concentrations (IC) of 0.28 and 0.53 ng/mL, respectively, and provided results within 50 and 26 min, making them more sensitive and time-saving compared to the classic ELISA. The detection limits (LODs) of BA-ELISA and MB-ELISA were down to 0.07 and 0.12 ng/mL, respectively, ensuring their practical application in AFB analysis. Additionally, both assays provided satisfactory recoveries (90.0%–97.2%) and favorable relative standard deviation (4.00%–10.5%) when tested with spiked cereal samples, aligning well with the results obtained from HPLC. Therefore, the AviTag-Nb shows promise as a valuable reagent in immunoassays, and the proposed BA-ELISA and MB-ELISA methods can serve as practical analytical tools for determining AFB in real samples.

中文翻译：

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基于 AviTag 纳米抗体的酶免疫分析法灵敏测定谷物中的黄曲霉毒素 B1

黄曲霉毒素 B (AFB) 是一种众所周知的人类健康致癌物，需要灵敏、快速的监测方法。在这里，我们开发了一种 Avi 标记的纳米抗体 (AviTag-Nb)，并将其纳入生物素-链霉亲和素放大 ELISA (BA-ELISA) 和基于磁珠的 ELISA (MB-ELISA) 中以检测 AFB。优化后，BA-ELISA和MB-ELISA的50%抑制浓度（IC）分别为0.28和0.53 ng/mL，并在50和26分钟内提供结果，与经典ELISA相比更加灵敏且节省时间。 BA-ELISA和MB-ELISA的检测限（LOD）分别低至0.07和0.12 ng/mL，确保了它们在AFB分析中的实际应用。此外，当使用加标谷物样品进行测试时，两种测定方法均提供了令人满意的回收率 (90.0%–97.2%) 和良好的相对标准偏差 (4.00%–10.5%)，与 HPLC 获得的结果非常吻合。因此，AviTag-Nb有望成为免疫测定中有价值的试剂，并且所提出的BA-ELISA和MB-ELISA方法可以作为测定实际样品中AFB的实用分析工具。