Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-Ro, Seodaemun-gu, Seoul 03722, Republic of Korea.
OPTOLANE Technologies Inc., 20 Pangyoyeok-ro 241beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do 13494, Republic of Korea.
Anal Chem. 2022 Jul 12;94(27):9627-9635. doi: 10.1021/acs.analchem.2c00716. Epub 2022 Jun 28.
In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants () have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.
在这项研究中，提出了一种基于切换肽的均相一步免疫测定法，用于检测甲型和乙型流感病毒（分别为 Inf-A 和 Inf-B）。一步免疫测定法代表了一种不涉及任何洗涤步骤的免疫测定方法，仅需处理样品。在这种方法中，荧光标记的切换肽从免疫球蛋白 G（IgG）的抗原结合位点定量解离。特别是，基于可溶性检测抗体与切换肽的一步免疫测定法称为均相一步免疫测定法。开发的免疫测定法使用用两种类型荧光染料（FAM 和 TAMRA）标记的切换肽和用两种类型荧光猝灭剂（FAM 的 TQ2 和 TAMRA 的 TQ3）标记的检测抗体。已选择用于检测 Inf-A 和 Inf-B 的最佳切换肽为 L1-肽和 H2-肽。使用计算对接模拟和 SPR 生物传感器分析了四种切换肽与 IgG 之间的相互作用。根据切换肽的荧光染料与荧光猝灭剂之间的距离，基于荧光猝灭效率，使用福斯特方程确定荧光猝灭剂的标记位置。为了证明一步免疫测定法的可行性，使用等温模型计算了针对目标抗原（Inf-A 和 Inf-B）和切换肽（L1-和 H2-肽）的 Inf-A 和 Inf-B 检测抗体的结合常数（）。使用抗原以及 Inf-A 和 Inf-B 的真实样本证明了免疫测定法是可行的，临界循环数（Ct）。还将免疫测定法与其他市售的用于 Inf-A 和 Inf-B 的快速检测试剂盒进行了比较，发现它在整个检测范围内对 Inf-A 和 Inf-B 的检测灵敏度要高得多。
