College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China.
Guangdong Provincial Key Laboratory of Emergency Test for Dangerous Chemicals, Guangdong Institute of Analysis, Guangzhou, Guangdong 510070, People's Republic of China.
J Agric Food Chem. 2020 May 27;68(21):5959-5968. doi: 10.1021/acs.jafc.0c00422. Epub 2020 May 18.
As one of the leading causes of food poisoning, staphylococcal enterotoxins (SEs) secreted by pose a serious threat to human health. The immunoassay has become the dominant tool used for the rapid detection of harmful bacteria and toxins as a result of its excellent specificity. However, with regard to SEs, staphylococcal protein A (SpA) is likely to bind with the fragment crystallizable (Fc) terminal of the traditional antibody and result in a false positive, limiting the practical application of this method. Therefore, to eliminate the bottleneck problem, the sandwich immunoassay was development by replacing the traditional antibody with a nanobody (Nb) that lacked a Fc terminal. Using 0.5 × 10 colony-forming units, the Nb library was constructed using Bactrian camels immunized with staphylococcal enterotoxin B (SEB) to obtain a paired Nb against SEB with good affinity. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using one Nb as the capture antibody and a phage-displayed Nb with signal-amplifying properties as the detection antibody. In optimal conditions, the current immunoassay displayed a broad quantitative range from 1 to 512 ng/mL and a 0.3 ng/mL limit of detection. The recovery of spiked milk, milk powder, cheese, and beef ranged from 87.66 to 114.2%. The Nbs-ELISA was not influenced by SpA during the detection of SEB in food poisoning. Therefore, the Nb developed here presented the perfect candidates for immunoassay application during SE determination as a result of the complete absence of SpA interference.
作为食物中毒的主要原因之一，金黄色葡萄球菌肠毒素（SEs）的分泌对人类健康构成了严重威胁。由于其出色的特异性，免疫测定已成为快速检测有害细菌和毒素的主要工具。然而，对于 SEs，金黄色葡萄球菌蛋白 A（SpA）很可能与传统抗体的片段结晶（Fc）末端结合，导致假阳性，限制了该方法的实际应用。因此，为了消除瓶颈问题，通过用缺乏 Fc 末端的纳米抗体（Nb）代替传统抗体，开发了夹心免疫测定法。使用 0.5×10 个菌落形成单位，用免疫金黄色葡萄球菌肠毒素 B（SEB）的双峰驼构建了 Nb 文库，获得了对 SEB 具有良好亲和力的配对 Nb。使用一种 Nb 作为捕获抗体，并用具有信号放大特性的噬菌体展示 Nb 作为检测抗体，开发了夹心酶联免疫吸附测定法（ELISA）。在最佳条件下，该免疫测定法的定量范围从 1 到 512 ng/mL 很宽，检测限为 0.3 ng/mL。牛奶、奶粉、奶酪和牛肉中添加的回收率在 87.66%至 114.2%之间。在检测食物中毒中的 SEB 时，SpA 不会影响 Nbs-ELISA。因此，由于完全没有 SpA 干扰，这里开发的 Nb 作为 SE 测定的免疫测定应用的理想候选物。
