Wyatt Blackson

In the event of a nuclear or radiological incident, thousands of individuals would be exposed to harmful radiation. Since medical countermeasures are dose-dependent, it is critical to evaluate the absorbed dose to organize efficient triage. Current gold-standard methods to assess absorbed radiation dose are time-consuming, not adapted for high-throughput, and require heavy equipment as well as expert personnel. Therefore, there is a need to develop a point-of-care (POC) bioassay that can measure biodosimetry markers both quickly and accurately. In this project, a vertical flow immunoassay (VFI) platform previously developed by our lab to detect proteins from biothreat pathogens is translated to detect radiation biodosimetry genes (CDKN1A and DDB2) as well as proteins (BAX and DDB2). In order to detect genes, our amplification method used specific labeled primers. The forward primer was conjugated with biotin for CDKN1A, digoxin (DIG) for DDB2, and 2,4-Dinitrophenol (DNP) for MRPS5 (housekeeping gene control). The reverse primer was conjugated with fluorescein isothiocyanate (FITC) for all three genes, thus enabling colorimetric detection when combined with a unique gold nanoparticle anti-FITC antibody. Capture antibodies for each gene were dispensed on a nitrocellulose membrane to specifically bind the forward primers. Target genes were detected by the VFI with high specificity both in singular and in multiplex; we discriminated between non-irradiated and irradiated samples based on an AUC of 0.95. A dose response between radiation exposure and signal intensity was observed using the VFI, with similar profiles to the results acquired using qPCR. In order to detect protein biodosimetry markers, ELISA was first used to determine optimal antibody pairs. Pairs for DDB2 and β-Tubulin (housekeeping protein) were successfully screened and, therefore, integrated onto the VFI platform. Results showed that only β-Tubulin could be detected on the VFI, demonstrating that ELISA translation into VFI will require further optimization. Overall, this work presents the initial development of a POC diagnostic tool that could help quickly determine an individual’s absorbed radiation dose and save thousands in response to nuclear or radiological incidents.