title: Elongated oligonucleotide-linked immunosorbent assay for sensitive detection of a biomarker in a microwell plate-based platform.
abstract: The immunoassay is a representative method for detecting disease biomarkers and pathogenic biological agents. While the conventional enzyme-linked immunosorbent assay (ELISA) has been routinely used for the analysis of biological samples, methods with higher sensitivity are still in demand because the detection of low-level biomarkers is important for early diagnosis of lethal diseases. In this study, we developed a sensitive immunoassay called elongated oligonucleotide-linked immunoassay (EOLISA), employing long DNA oligonucleotides (80-mer), a fluorogenic RNA probe and RNase H for signal amplification. The elongated DNA oligonucleotides led to a highly amplified fluorescence signal via iterative cycles of DNA-RNA duplexation and subsequent degradation of the RNA in the duplex by RNase H. The immunoassay was evaluated for sensitive detection of fatty acid binding protein (FABP) in the 0-1 ng mL(-1) range. When compared with ELISA, EOLISA showed about 10-fold improved detection sensitivity. With its simple procedure and reliable detection performance in the conventional platform, the proposed immunoassay is expected to have potential applications in clinical diagnostics.
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea. Electronic address: biolord@kist.re.kr.
authors.forename: Ki-Cheol
authors.initials: KC
authors.lastname: Han
authors.forename: Eun Gyeong
authors.initials: EG
authors.lastname: Yang
authors.forename: Dae-Ro
authors.initials: DR
authors.lastname: Ahn
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D050556:Fatty Acid-Binding Proteins / Q000097:blood*; D005453:Fluorescence; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D015347:RNA Probes / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D050556:Fatty Acid-Binding Proteins; D009841:Oligonucleotides; D015347:RNA Probes; D004247:DNA; D016914:Ribonuclease H
keywords: Elongated oligonucleotide-linked immunosorbent assay; Fatty acid binding protein; Fluorescence intensity; Immunoassay