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 oligonucleolide-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 R Nase 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 alpha-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.