State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang, 330047, China.
College of Food Science, Nanchang University, Nanchang, 330031, China.
Anal Bioanal Chem. 2024 Nov;416(28):6199-6208. doi: 10.1007/s00216-024-05498-0. Epub 2024 Sep 12.
A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B (AFB) was developed using an AFB-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (T) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB as well as phase transition when the temperature rose above T. To establish the optimal conditions for cleaning AFB, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods.
一种无柱免疫亲和净化（CFIP）技术，用于黄曲霉毒素 B（AFB）的样品制备，该技术使用了一种 AFB 特异性纳米抗体（命名为 G8）和弹性蛋白样多肽（ELP）。ELP 来源于人弹性蛋白，具有对温度变化的液-固可逆相转变特性。G8 通过递归定向连接（RDL）在 C 端与不同长度（20、40、60 和 80 个重复单元）的 ELP 连接。然后，将编码序列亚克隆到 pET30a 的多克隆位点。通过在大肠杆菌 BL21（DE3）中表达包涵体来生产生物活性的重组蛋白，然后溶解和重折叠。间接竞争酶联免疫吸附测定（icELISA）和转变温度（T）测量分析证实，重折叠的 G8-ELP 保持了识别 AFB 的能力，并且在温度升高超过 T 时发生相转变。为了确定净化 AFB 的最佳条件，研究了各种参数对回收率的影响。在优化的 CFIP 工作流程下，ELISA 测试中的回收率为 95±3.67%。此外，CFIP 制备的样品用于高效液相色谱（HPLC）检测。在 CFIP-HPLC 测试中，玉米、大米、酱油和植物油样品的回收率范围为 54±1.86%至 98±3.58%。据我们所知，这是首次将纳米抗体和 ELP 的功能结合起来，开发用于复杂基质中小分子的净化技术。用于样品预处理的 CFIP 易于使用且价格低廉。与传统的免疫敏感性材料相比，CFIP 中使用的试剂完全是生物合成的，没有任何化学偶联反应。这表明，纳米抗体-ELP 可能成为开发样品净化或纯化方法的有用的双功能试剂。
