Elongated oligonucleotide-linked immunosorbent assay for sensitive detection of a biomarker in a microwell plate-based platform

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.