Development of Antigen-based Vertical Flow Assay for the Rapid Detection of SARS-CoV-2, Influenza A/B, and RSV

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Project Summary The goal of this Phase I SBIR proposal is to develop a rapid, low-cost, multiplex immunoassay for the detection of common respiratory tract pathogens, including influenza A (Flu A), influenza B (Flu B), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in less than 15 minutes. The development of this antigen-based test will accelerate the rapid diagnosis of SARS-CoV-2 infection, along with influenza A / influenza B and RSV. Influenza A / influenza B and RSV are the other common causative agents of respiratory tract infections other than the ongoing SARS-CoV-2 pandemic. Analyses from the CDC have suggested that there could be 10 times more Coronavirus disease 2019 (COVID-19) positive cases than reported cases. 2 This is primarily due to the slow screening process as it mainly depends on the RT-PCR based testing systems. The rapid identification of SARS-CoV-2 is critical to stop the spread of the infection as it is highly contagious. Apart from SARS-CoV-2, influenza A / influenza B and RSV cause a considerable amount of infections and severe illness. Moreover, influenza A / influenza B and RSV infections show similar symptoms to that of SARS-CoV-2 infection. Differentiating SARS-CoV-2 infections with Flu or RSV is also much needed as Flu and RSV infections have better treatment options if they are diagnosed early. Rapid tests would provide a better opportunity to choose the right treatment strategy and stop the spread of the illness. We propose to develop a rapid, multiplex test for the detection of influenza A / influenza B, RSV, and SARS-CoV-2 without any complex instrumentation. Our rapid test uses immobilized antibodies on the membrane for the capturing and simultaneous qualitative detection of specific antigens for four common respiratory tract pathogens. We will use our innovatively designed sequential injection syringe (SIS) to pre-fill the reagents and disperse them sequentially into the flow-through assay cassette. The components such as the 2nd antibody conjugated with biotin, wash buffers, neutravidin-HRP, and tetramethylbenzidine (TMB) with stable hydrogen peroxide will be sequentially dispersed into the assay cassette. After completing the assay, the colored spots that develop can be viewed without any special instrumentation and can be interpreted easily. If needed, a reader can be used to further increase the sensitivity and ability to record and send test result to the physicians. This multiplex assay can be performed in less than 15 minutes in various point-of-care (POC) and low resource settings without any special training. We will demonstrate that our test can deliver rapid results with the same level of performance as singleplex tests for each pathogen using clinical specimens. We will also validate our assay method at our collaborator's lab at the Medical College of Wisconsin. We plan to achieve 100% specificity and higher sensitivity than current lateral flow assays. If successfully developed, this rapid, multiplex test will be simple to perform and inexpensive enough for all sizes of primary-care physician's offices, nursing homes, pharmacies, and community health clinics to adopt the platform.