Alsulami Tawfiq, Nath Nidhi, Flemming Rod, Wang Hui, Zhou Wenhui, Yu Jae-Hyuk
Department of Food Science, University of Wisconsin-Madison, Madison, WI, 53706, USA; Department of Food Science and Nutrition, King Saud University, Saudi Arabia.
Promega Corporation, 2800 Woods Hollow Road, Madison, WI, 53711, USA.
Biosens Bioelectron. 2021 Apr 1;177:112939. doi: 10.1016/j.bios.2020.112939. Epub 2020 Dec 30.
Compared to the traditional heterogeneous assays, a homogeneous immunoassay is a preferred format for its simplicity. By cloning and isolating luminescent proteins from bioluminescent organisms, bioluminescence has been widely used for various biological applications. In this study, we present the development of a homogeneous luminescence immunoassay (FNanoBiT assay) for detection of fumonisin B1 (FB1), based on the binding of two subunits of an engineered luminescent protein (NanoLuc). For the detection of the mycotoxin FB1 in foods, the anti-fumonisin antibody was conjugated to the large subunit of NanoLuc (FLgBiT), and the FB1 was conjugated to the small subunit (FSmBiT). The conjugates were used for the detection of FB1 in a competitive immunoassay format without the need of a secondary antibody, or washing steps. The developed FNanoBiT assay revealed high specificity toward FB1 with no cross-reactivity with other mycotoxins, and it demonstrated acceptable recovery (higher than 94%) and relative standard deviation from spiked maize samples. Further, the assay was successfully applied for the detection of FB1 in naturally contaminated maize, with a dynamic range of 0.533-6.81 ng mL-1 and a detection limit of 0.079 ng mL-1. The results derived with FNanoBiT assay of all spiked samples showed a strong correlation to those obtained by the High-performance liquid chromatography method. Thus, the FNanoBiT based homogeneous immunoassay could be used as a rapid, and simple tool for the analysis of mycotoxin-contaminated foods.
与传统的非均相分析方法相比，均相免疫分析因其简单性而成为一种更优的分析形式。通过从生物发光生物体中克隆和分离发光蛋白，生物发光已被广泛应用于各种生物学领域。在本研究中，我们基于一种工程化发光蛋白（NanoLuc）的两个亚基的结合，开发了一种用于检测伏马菌素B1（FB1）的均相发光免疫分析方法（FNanoBiT分析）。为了检测食品中的霉菌毒素FB1，将抗伏马菌素抗体与NanoLuc的大亚基（FLgBiT）偶联，将FB1与小亚基（FSmBiT）偶联。这些偶联物用于竞争性免疫分析形式中检测FB1，无需二抗或洗涤步骤。所开发的FNanoBiT分析对FB1显示出高特异性，与其他霉菌毒素无交叉反应，并且在加标玉米样品中表现出可接受的回收率（高于94%）和相对标准偏差。此外，该分析方法成功应用于天然污染玉米中FB1的检测，动态范围为0.533 - 6.81 ng mL-1，检测限为0.079 ng mL-1。所有加标样品的FNanoBiT分析结果与高效液相色谱法获得的结果显示出强烈的相关性。因此，基于FNanoBiT的均相免疫分析可作为一种快速、简单的工具用于分析受霉菌毒素污染的食品。
