College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi, 712100, People's Republic of China.
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, People's Republic of China.
Talanta. 2024 Jul 1;274:126021. doi: 10.1016/j.talanta.2024.126021. Epub 2024 Apr 1.
α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.
α-溶血素（Hla）是金黄色葡萄球菌（S. aureus）分泌的一种毒素，已被证明与食物中毒的发生和加重有关。因此，建立其快速检测方法以保证食品安全是非常必要的。基于纳米抗体的夹心 ELISA 已被证明可用于检测毒素，但目前还没有针对 Hla 的纳米抗体，更不用说针对它的配对纳米抗体了。因此，在本文中，我们通过生物淘选筛选出了特异性纳米抗体，并首次获得了用于夹心 ELISA 的最佳纳米抗体对。然后，通过分子重组技术，在单个步骤中以低成本产生了一种新型的纳米抗体 RANbody，它同时具有识别和催化能力。随后，基于纳米抗体和 RANbody 开发了用于检测 Hla 的夹心 ELISA，与传统的夹心 ELISA 相比，它不仅消除了对二抗和动物源性抗体的使用，还缩短了检测时间和降低了检测成本。最后，对该方法的性能进行了评估，特别是特异性，其对其他溶血素没有反应，检测限低至 10ng/mL。此外，该夹心 ELISA 具有良好的可行性，并成功用于检测牛奶和猪肉样品中的 Hla。
