Yi, Joon-Yeop Ryu, Jaewon Jeong, Yujin Cho, Yoeseph Kim, Minyoung Jeon, Mijin Park, Hee Ho Hwang, Nathaniel S. Jeong, Hee-Jin Sung, Changmin 2024-10-26T16:30:30Z 2024-10-26T16:30:30Z 2024-10-25 2024-08 0003-2670 https://pubs.kist.re.kr/handle/201004/150899 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-C6-labeled 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-1-1 for buffer samples, 1.0 ng mL-1-1 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. English Elsevier BV One-step detection of procollagen type III N-terminal peptide as a fibrosis biomarker using fluorescent immunosensor (quenchbody) Article 10.1016/j.aca.2024.342887 1 Analytica Chimica Acta, v.1317 Analytica Chimica Acta 1317 Y scie scopus 001333524400001 2-s2.0-85196410777 Chemistry, Analytical Chemistry Article SERUM CANCER BIOSENSOR MARKERS LIVER Quenchbody Immunosensor fluorescence Fibrosis biomarker