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.
Biosens Bioelectron. 2021 Apr 15;178:112996. doi: 10.1016/j.bios.2021.112996. Epub 2021 Jan 19.
Herein, we present switching-peptides for a one-step immunoassay, without the need for additional antibody treatment or washing steps to detect antigen-antibody interactions. Fluorescently labeled switching-peptides were dissociated from the immobilized antibody soon after the antigens were bound to the binding pockets. In this study, four different parts of the antibody (IgG) frame regions were chemically synthesized, and these peptides were bound to immobilized antibodies as switching-peptides. We presented the design principle of switching-peptides and used Pymol software, based on the changes in thermodynamic parameters, to study the interaction between antibodies and switching-peptides. The binding properties of switching-peptides were analyzed based on Förster resonance energy transfer between switching-peptides as well as between switching-peptides and antibodies (IgGs) isolated from different animals. The binding constants of the four switching-peptides to antibodies were estimated to be in the range of 1.48-3.29 μM. Finally, the feasibility of using switching-peptides for the quantitative one-step immunoassay was demonstrated by human hepatitis B surface antigen (hHBsAg) detection and statistical comparison of the assay results with those of conventional ELISA. The limit of detection for HBsAg was determined to be 56 ng/mL, and the dynamic range was estimated to be 136 ng/mL-33 μg/mL. These results demonstrate the feasibility of the one-step immunoassay for HBsAg.
在此，我们提出了一种用于一步免疫分析的切换肽，无需额外的抗体处理或洗涤步骤即可检测抗原-抗体相互作用。荧光标记的切换肽在抗原与结合口袋结合后不久就从固定化抗体上解离。在这项研究中，我们化学合成了抗体（IgG）框架区域的四个不同部分，并将这些肽作为切换肽结合到固定化抗体上。我们提出了切换肽的设计原理，并使用 Pymol 软件，基于热力学参数的变化，研究了抗体和切换肽之间的相互作用。根据切换肽之间以及从不同动物中分离的切换肽和抗体（IgG）之间的Förster 共振能量转移，分析了切换肽的结合特性。四个切换肽与抗体的结合常数估计在 1.48-3.29 μM 范围内。最后，通过人乙型肝炎表面抗原（hHBsAg）检测和与传统 ELISA 检测结果的统计学比较，证明了使用切换肽进行定量一步免疫分析的可行性。HBsAg 的检测限确定为 56 ng/mL，动态范围估计为 136 ng/mL-33 μg/mL。这些结果证明了一步法免疫分析测定 HBsAg 的可行性。
