Reverse transcriptase mediated signal amplification in ligand binding assays

A simple method for enhanced detection of biological analytes is presented. It does not involve temperature cycling or temperatures above 37°C, can easily be integrated into existing protocols and requires only standard equipment. The procedure adds an extra amplification step to classical immunoassays. Oligonucleotide primers attached to antibodies are used for signal amplification and analyte detection. The oligonucleotides serve as primers in a reverse transcriptase (RT) reaction using a soluble complementary template and labeled nucleotides. The DNA strand synthesized is detected using the incorporated labels. Factors such as primer length, amount of labeled ligand, amount of RT activity, type of polymerase used, polymerase reaction time and temperature are explored and optimized to maximize the level of signal amplification. The capacity of the method is exemplified by a comparison between a standard capture ELISA for SARS-CoV-2 spike protein, and a polymerase amplified version of the same test. The increase in detection sensitivity achieved was approximately 70 times.