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African swine fever (ASF) is a highly contagious and often lethal infectious disease of swine. Given the absence of treatment and vaccines, rapid and accurate ASF diagnostics is crucial to border inspection, surveillance, and outbreak containment. Currently, there is still a lack of low-cost yet accurate antibody or antigen tests that can be deployed at the time, simultaneously in the same device to pen sides for surveillance purposes. The Wang lab at ASU and the INIA-CISA/CSIC in Spain propose a multidisciplinary research plan to address the fundamental challenges in low-cost, portable, fast, simple, and quantitative ASF assay format. In this project, our major goal is to design and validate a new metal nanoparticle (MNP)-based assay platform, namely nanoparticle-supported rapid, electronic detection (NaSRED), and quantify its performance in ASF diagnostics. As shown in the management plan, this project has four important tasks.We have four tasks/objectives in this project.In Task 1, we will innovate on a spherical metal nanoparticle (MNP) based NaSRED assay for the detection of ASF antigen based on the p72 ASFV protein and Anti-p72 as antibodies. Expected outcome: we expect a dynamic range of > 3 logs, e.g. from >100 nM to <100 pM in antigen detection. We will also evaluate the NaSRED specificity by using two negative controls (NC), one as PBS buffer and another as a different ASF protein, such as p30. Later we will test at ASU and INIA-CISA/CSIC semipurified soluble ASFV antigen (SAg) /and or P72 antigen and other closely clinically related Classical swine fever virus (CSFV). We should expect minimal signal response for the negative control samples and antigens coming from CSFV. Further, we will combine experimental analysis with physical interpretations and a theoretical model to comprehensively study the mechanisms of MNP-based multivalent analyte-binding.In Task 2, we will design two sets of heterogeneous MNPs, e.g. gold NPs and nanorods (NRs), to display distinct plasmonic wavelengths. Such MNPs will be conjugated with specific ligands towards ASF (SAg) or protein (p72) and antibody (anti-p54, anti-p30) markers, respectively. The simultaneous spectrometric readout of two or more markers at separated wavelengths allows multiplexed profiling of ASF protein markers. The assay performance will be assessed in LoD, cross-reactivity, assay time, etc. Expected outcome: we will mix very low-concentration (e.g. 1 to 100 pM) anti-p30 and high-concentration p72 (e.g. 10 to 100 nM) as the ASF sample to mimic early stage ASF detection, during which antibodies are not released yet by the immune systems. We expect only the SAg/p72 proteins are detectable, triggering AuNR precipitation. Then we will spike both the antigens and antibodies at high concentration (e.g. 10 to 100 nM). This mimics the stage from about two weeks ASF infection and up to at least 1.5 month, during which both antigens and antibodies are expected present in swine serum. We expect the mixing to cause precipitation of both MNPs. Here the exact color would depend on the ratio between the remaining NPs and NRs. Lastly, for very late-stage ASF infection or survivors, only antibodies should be detectable. We will mix low-concentration (e.g. 1 to 100 pM) p72 and high-concentration anti-p30 (e.g. 10 to 100 nM) as the ASF sample. This will cause AuNPs to precipitate. In this task, this multiplexed diagnosis will prove the feasibility of ASF detection across different infection stages at ASU.Towards faster detection for mass screening, particularly considering the need of future pen-side testing, in Task 3 we will develop a rapid detection system to complete the test within a few minutes, assisted by a simple centrifugation followed by mixing. The target ASF protein markers will be quantified in centrifuge tubes with portable electronic readout system, including an integrated circuit board comprising two LEDs for the two selected MNPs, photodetectors, battery connectors, and signal transmitters. Expected outcome: We expect to demonstrate rapid and accurate detection of ASF p72 proteins and anti-p54 (or anti-p30) antibodies. Here we will use p72 as antigen and anti-p72-bound AuNPs as sensor to evaluate different spinning speed from 300 rpm to 2000 rpm, and optimize the centrifugation and incubation processes. The assay limit of detection, dynamic range and linearity will be evaluated using the electronic readout, and compared against the spectroscopic readout. We estimate the total detection time can be reduced to within 5 minutes even including all the sample handling, such as pipetting, centrifugation, vortex, and readout (if by bare eye), or within 30 min if using a 20-min incubation. In addition, we will demonstrate the feasibility of portable readout by integrating low-cost electronic elements on PCB boards with 3D printed low-cost tube holders. This will eliminate the need to extract the assay supernatant to PDMS well plate and also simplify the readout.In Task 4, the NaSRED assay will be evaluated at INIA-CISA/CSIC for antigen detection, antibody detection, and multiplexed detection. Using ASF reference samples, we will evaluate the analytical limit of detection (LoD) and specificity against closely clinically related swine viruses, and estimate diagnostic sensitivity and specificity. During NaSRED validation, we will determine the diagnostic sensitivity and diagnostic specificity. We will further investigate the cross-reactivity with other swine virus diseases, and determine the analytical speci?city of NaSRED by testing experimental samples from closely clinically related Classical swine fever virus (CSFV) as well as and clinical material (EDTA-blood and serum) obtained from a non-infected donor pig. The repeatability will be measured by running 10 replicates of 10 positive pig samples previously experimentally infected with ASFV and 8 replicates of 8 negative samples from non-infected pigs. Lastly, to evaluate the feasibility of deploying the NaSRED assay to the pen-side during future outbreaks, both teams at ASU and INIA-CISA/CSIC will load the NaSRED device, together with needed accessories (pipettes, centrifuge tubes, low-cost centrifuges and vortex mixers, biosamples, batteries, and laptop) on a car, and perform ASF NaSRED assay in the field. We will record the data and compare to the test results obtained in a lab setting. The outside humidity, temperature and the duration of the tests will be recorded as well.

Wang, C.; Gallardo, CA, .
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