One-step detection of procollagen type III N-terminal peptide as a fibrosis biomarker using fluorescent immunosensor (quenchbody).

Autor: Yi JY; Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea; Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea., Ryu J; Department of Biological and Environmental Science, Dongguk University, Goyang-si, Gyeonggi-do, 10326, Republic of Korea., Jeong Y; Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea; Department of Bioengineering, Hanyang University, Seoul, 04763, Republic of Korea., Cho Y; Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea., Kim M; Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea; Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea., Jeon M; Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea., Park HH; Department of Bioengineering, Hanyang University, Seoul, 04763, Republic of Korea., Hwang NS; Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea; School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea; Institute of Engineering Research, Seoul National University, Seoul, 08826, Republic of Korea; Bio-Max/N-Bio Institute, Seoul National University, Seoul, 08826, Republic of Korea., Jeong HJ; Department of Biological and Chemical Engineering, Hongik University, Sejong, 30016, Republic of Korea., Sung C; Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea. Electronic address: scm@kist.re.kr.
Jazyk: angličtina
Zdroj: Analytica chimica acta [Anal Chim Acta] 2024 Aug 15; Vol. 1317, pp. 342887. Date of Electronic Publication: 2024 Jun 18.
DOI: 10.1016/j.aca.2024.342887
Abstrakt: Background: Procollagen type III N-terminal peptide (P-III-NP) is a fibrosis biomarker associated with liver and cardiac fibrosis. Despite the value of P-III-NP as a biomarker, its analysis currently relies on enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), which require more than 3 h. To facilitate early diagnosis and treatment through rapid biomarker testing, we developed a one-step immunoassay for P-III-NP using a quenchbody, which is a fluorescence-labeled immunosensor for immediate signal generation.
Results: To create quenchbodies, the total mRNA of P-III-NP antibodies was extracted from early-developed hybridoma cells, and genes of variable regions were obtained through cDNA synthesis, inverse PCR, and sequencing. A single-chain variable fragment (scFv) with an N-terminal Cys-tag was expressed in E. coli Shuffle T7, resulting in a final yield of 9.8 mg L -1 . The fluorescent dye was labeled on the Cys-tag of the anti-P-III-NP scFv using maleimide-thiol click chemistry, and the spacer arm lengths between the maleimide-fluorescent dyes were compared. Consequently, a TAMRA-C 6 -labeled quenchbody exhibited antigen-dependent fluorescence signals and demonstrated its ability to detect P-III-NP at concentrations as low as 0.46 ng mL -1 for buffer samples, 1.0 ng mL -1 for 2 % human serum samples.
Significance: This one-step P-III-NP detection method provides both qualitative and quantitative outcomes within a concise 5-min timeframe. Furthermore, its application can be expanded using a 96-well platform and human serum, making it a high-throughput and sensitive method for testing fibrotic biomarkers.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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