title: An approach to multiplexing an immunosorbent assay with antibody-oligonucleotide conjugates.
abstract: Early detection of cancer biomarkers provides clinically valuable information. While the conventional enzyme-linked immunosorbent assay (ELISA) has been routinely used for individual cancer markers, methods for simultaneous determination of multiple markers within a single sample are still in demand. Here, we present a novel oligonucleotide-linked immunosorbent assay (OLISA) with a multiplexing capability on the same microwell plate-based system as in ELISA. Employing a DNA oligonucleotide that is covalently conjugated to the detection antibody and a complementary RNA oligonucleotide which is appended with a fluorophore and a quencher, degradation of the RNA in the DNA-RNA duplex by RNase H is exploited for fluorescent signal generation. Iterative cycles of DNA-RNA duplexation and subsequent degradation of the RNA in the duplex by RNase H further lead to amplification of the detection signal in OLISA. Moreover, the use of antibody-oligonucleotide conjugates enables multiplexing of OLISA, which is successfully demonstrated by tethering DNA molecules to detection antibodies and by performing assays for three common cancer markers including α-fetoprotein, prostate-specific antigen, and carcinoembryonic antigen. With the simple procedure and reliable detection performance, the developed multiplex OLISA has a wide potential for use in analysis of a panel of biomarkers in clinical diagnostics.
authors.affiliation: Life Sciences Research Division, Korea Institute of Science and Technology, Cheongryang, Seoul 130-650, Republic of Korea.
authors.forename: Ki-Cheol
authors.initials: KC
authors.lastname: Han
authors.forename: Dae-Ro
authors.initials: DR
authors.lastname: Ahn
authors.forename: Eun Gyeong
authors.initials: EG
authors.lastname: Yang
mesh_terms: D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000032:analysis; D015415:Biomarkers / Q000032:analysis*; D002272:Carcinoembryonic Antigen / Q000032:analysis; D003110:Colonic Neoplasms / Q000175:diagnosis; D004247:DNA / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D018796:Immunoconjugates / Q000032:analysis* / Q000737:chemistry; D007163:Immunosorbent Techniques; D008113:Liver Neoplasms / Q000175:diagnosis; D008297:Male; D009841:Oligonucleotides / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000032:analysis; D011471:Prostatic Neoplasms / Q000175:diagnosis; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000032:analysis
chemical_list: D000906:Antibodies; D000941:Antigens; D015415:Biomarkers; D002272:Carcinoembryonic Antigen; D005456:Fluorescent Dyes; D018796:Immunoconjugates; D009841:Oligonucleotides; D000509:alpha-Fetoproteins; D012313:RNA; D004247:DNA; D016914:Ribonuclease H; D017430:Prostate-Specific Antigen