Development of QuantiDNA ALU Assay on Luminex Platform for Monitoring Adverse Reactions to Radiation Therapy

Abstract

Radiation treatment is required by 70% of cancer patients, however there is currently no clinical method for determining the therapeutic response or radiation induced toxicity that can be used during a course of radiation therapy to personalize the dose for individual patients. We developed a plate-based QuaniDNA® assay utilizing branched DNA technology (SuperbDNA®) to measure plasma cell free DNA concentration as a marker for monitoring radiation induced toxicity. Utilizing the same branched DNA technology, herein we describe the development of a new QuantiDNA® ALU assay on Luminex platform. The assay employs DNA capture probes coupled to MagPlex™ Microspheres and SuperbDNA® signal amplification technology with biotin-labelled signaling probes which bind to Streptavidin phycoerythrin (SAPE) and the fluorescence signal can be detected by Luminex instruments such as MAGPIX®. The assay can be performed directly on patient plasma samples and can be readily automated. Compared with previous plate-based QuantiDNA® assay, the new assay has a better sensitivity with the lower limit of detection (LOD) being 0.05 ng/ml, which equals 1 pg human DNA given that 20 ul of samples were loaded. The limit of quantitation of the assay is 0.11 ng/mL (equivalent to 2.2 pg human DNA). The inter- and intra-assay coefficients of variance were <20% and <15%, respectively. Bacterial DNA, EDTA (10mM), hemoglobin (500mg/dL) and cholesterol (300mg/mL) do not interfere with the assay. Thirty plasma samples from patients with prostate cancer were tested and the levels of circulating DNA in plasma pre-radiotherapy was 11.6 ∼ 130.6 ng/ml with a median value of 32.2 ng/ml. This assay can be used both for research and clinical testing of plasma samples for patients undergoing radiation therapy for optimization and personalization of treatment.