Combining a Nanosensor and ELISA for Measurement of Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Activity and Protein Amount in Cell and Tissue Extract

Abstract

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) was initially discovered by its ability to remove topoisomerase I (TOPI) covalently trapped to DNA. However, it has since been found to be involved in the repair of a number of DNA lesions other than TOPI–DNA complexes e.g., damages caused by ionizing radiation and free radical-based genotoxins. TDP1 has been reported to be posttranslational regulated, and in nonsmall cell lung cancer (NSCLC) it has been reported that the enzyme activity of TDP1 can be upregulated without detectable upregulation in protein amount. Activity and protein analyses are normally performed sequentially using radioactively labeled DNA substrates analyzed using gel-electrophoresis for the activity measurement and western blot or ELISA for protein measurement. We demonstrate here that our previously developed TDP1 nanosensor due to the optical real-time readout can be combined with ELISA measurement into one assay facilitating measurement of both the enzymatic activity and the protein amount of TDP1. We show that the combined assay can be used for measurements both in cell line-based studies and for measurement in clinical tissue samples. Due to all measurements being performed in the exact same aliquot of cell or tissue extract, the combined assay allows the use of minimal amount of cells or tissue and without additional incubation steps compared to the normal ELISA procedure. Measuring protein amount and activity within the same portion of extract also minimizes the risk of variation being introduced when comparing amount to activity. Since the assay only uses small amounts of extract, it requires no advanced equipment besides a plate reader, and can work in whole-cell or tissue extract. We expect that it could be useful for analysis of posttranslational modifications influencing enzymatic activities both in basic research and in clinical studies.