Abstract：
To measure toxins using immunoassays, hazardous toxin standards need to be added for quantification. To solve this problem, we propose to use aptamers as competitors to replace toxin standards. In this work, aptamers specific for ochratoxin A (OTA) nanobodies were selected using a DNA library containing a 36 nucleotide random region. The obtained sequences were highly aligned and the best competitor was identified to be a sequence named apt2-OT based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). The Kd of apt2-OT was measured to be 2.86 μM using local surface plasmon resonance spectroscopy. The optimal apt2-OT was identified to substitute the OTA standard with a concentration needed for 50% inhibition of binding (IC50) of 3.26 μM based on a nontoxic direct competitive ELISA. The equivalence relationship between the aptamer and OTA was established in a flour sample, and a recovery experiment was performed. The detection limit for this method was 0.23 ng/mL, with a linear range from 0.25 to 10.50 ng/mL. The recovery rate was 97.5%-115.5%. This study provides a low-cost, rapid and environmentally friendly alternative to the development of immunoassays for toxins.
摘要：
用免疫测定法测定毒素,需要添加危险毒素标准以进行量化。为了解决这个问题,我们建议使用适体作为竞争对手来取代毒素标准。在这项工作中,使用含有36个核苷酸随机区域的DNA文库选择对曲霉毒素A(OTA)纳米抗体特异性的适体。将获得的序列进行高度比对，并根据间接竞争性酶联免疫吸附测定（ELISA）将最佳竞争者鉴定为名为apt2-OT的序列。使用局部表面等离子体共振光谱法测量apt2-OT的Kd为2.86μM。基于无毒的直接竞争ELISA,鉴定出最佳的apt2-OT以3.26μM的50%结合抑制(IC50)所需的浓度代替OTA标准品。在面粉样品中建立了适体与OTA之间的等效关系，并进行了恢复实验。该方法的检出限为0.23ng/mL，线性范围为0.25至10.50ng/mL。回收率为97.5%～115.5%。这项研究提供了一种低成本的，快速和环境友好的替代毒素免疫测定的发展。
