(1092-A) Paper-Based Vertical Flow Immunoassay for the Point-of-Care Multiplex Detection of Radiation Dosimetry Genes
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract:
Introduction: In the event of a nuclear or radiological incident, thousands of individuals would be exposed to harmful radiation. First responders must quickly and accurately measure radiation exposure as medical countermeasures are radiation dose-dependent and time-sensitive. Current gold-standard methods to assess absorbed radiation dose include dicentric chromosome, cytokinesis- block micronucleus, and lymphocyte depletion rate assays. However, these methods are not high-throughout and require sample transport off-site as well as expert personnel. Therefore, there is a need to develop a point-of-care (POC) bioassay that can measure quickly and accurately scalable biodosimetry markers. Our lab developed an innovative POC approach based on vertical flow immunoassay (VFI) that possesses superior sensitivity and multiplexing capability than standard lateral flow immunoassays to detect proteins from biothreat pathogens such as Y. Pestis. Our project aims to translate the VFI platform to detect candidate radiation biodosimetry genes (CDKN1A and DDB2). Materials and
Methods: For this study, VFI membranes were prepared using a Nano-plotter 2.1 to dispense capture antibody reagents onto a nitrocellulose substrate in a clean room environment. Samples were prepared by spiking into buffer and incubating with ~ 40 nm gold nanoparticle-labeled detection antibodies for 10 min. The VFI membranes were then assembled into custom housings, and loaded into 3 mL luer-lock syringes and passed through the membrane using a syringe pump at 0.2 mL/min. After passing the samples, membranes were dried and scanned for image analysis. Results and
Discussion: In this study, gold nanoparticle-labeled detection antibodies were used for the first time to detect biodosimetry genes (DDB2 and CDKN1A) in VFI. Based on signal intensities from the performed assays, the target genes could be detected in three different doses (0 Gy, 1Gy, and 2 Gy). All three target genes were shown to be detected with high specificity both in singular and in multiplex. Using both CDKN1A and DDB2 genes, VFI was able to discriminate between non- irradiated and irradiated samples with an AUC of 0.86. Moreover, dose response was also observed with VFI and had similar profiles to the results acquired using qPCR. Also, VFI provided results with excellent reproducibility with intra-assay CV < 10% and inter-assay CV < 20%. Together, these data illustrate the potential of VFI as a POC device in discriminating exposed and non-exposed individuals after a nuclear incident in a short period of time with high accuracy.
Conclusions: VFI has proven the ability to detect multiple genes using gold nanoparticle- labeled detection antibodies. This feature is vital for the prediction, prognostic, diagnostic, or monitoring of a disease condition using biomarkers where their performance is often superior when they are combined, particularly in radiation diagnostic studies.