(B) ELISA using hybridoma supernatants from the spleen of mouse EB024 infected with SARS-CoV-2 S1. Hybridoma supernatants were tested with S1 from SARS-CoV-2, SARS-CoV-1, MERS, NL63, 229E, HKU1, and OC43 to evaluate cross-reactivity. (C) ELISA using hybridoma supernatants Tenatoprazole from the lymph nodes of mouse EB025 infected with SARS-CoV-2 N. Hybridoma supernatants were tested with S1 from SARS-CoV-2 and SARS-CoV-1 to evaluate cross-reactivity.(TIF) pntd.0010311.s001.tif (187K) GUID:?934425CF-6215-4FC6-B055-E37E73406E99 S2 Fig: Flow cytometric analysis of SARS-CoV-2 mAbs. Hybridoma supernatants were used to stain permeabilized Vero cells infected with SARS-CoV-2, the human coronaviruses 229E or OC43, or non-infected cells as a control (Mock). FACS analysis was performed using (A) SARS-CoV-2 S1 mAbs derived from mouse EB017 lymph nodes, (B) SARS-CoV-2 S1 mAbs derived from mouse EB024 lymph nodes, (C) SARS-CoV-2 N mAbs derived from mouse EB025 lymph nodes. Fluorescence is normalized as Fold Above Background.(TIF) pntd.0010311.s002.tif (150K) GUID:?3A44C4F4-B631-468E-8AB7-D14B87DDBF52 S3 Fig: Combinatorial Dipstick Analysis. (A) SARS-CoV-2 S1 mAbs and (B) SARS-CoV-2 N mAbs were tested in a matrix for interaction with SARS-CoV-2 S1 or N and without added antigen as a control (). mAbs Tenatoprazole were either conjugated to the nanoparticle or placed onto the nitrocellulose paper. SARS-CoV-2 S1 or SARS-CoV-2 N, at final concentrations of 125 ng/ml, were allowed to react with the rapid antigen tests. The pairwise immunochromatography signal intensities are scored as low binding (1, >201 normalized grey scale pixel intensity), medium binding (2, 141C200 normalized grey scale pixel intensity), or high binding (3, <140 normalized grey scale pixel intensity), with a majority of the combinations presenting with no signal (white). Grey boxes represent combinations not tested.(TIF) pntd.0010311.s003.tif (467K) Tenatoprazole GUID:?83EB3AB6-56A6-45F5-90AC-2AE482EB0CD3 S4 Fig: Western blot analysis using SARS-CoV-2 S1 mAbs. Immunoblots were performed using SARS-CoV-2 spike protein immunostained with (A) mAb 46, (B) mAb 124, (C) mAb 349, and (D) mAb 474 to elucidate mAb binding to spike subunit 1 (S1), spike subunit 2 (S2), or the receptor binding domain (RBD). kDa, kilodalton.(TIF) pntd.0010311.s004.tif (241K) GUID:?A6AE61B7-F263-4558-8A09-489AD81036B2 S1 Table: SARS-CoV-2 rapid antigen tests with emergency use authorizations (EUA). (DOCX) pntd.0010311.s005.docx (18K) GUID:?99112095-6F3F-4CAB-9CF3-76630CFE8E18 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Background The focus on laboratory-based diagnosis of coronavirus disease 2019 (COVID-19) warrants alternative public health Tenatoprazole tools such as rapid antigen tests. While there are a number of commercially available antigen tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), all cross-react with the genetically similar SARS-CoV-1 or require an instrument for results interpretation. Methodology/Principal findings We developed and validated rapid antigen tests that use pairs of murine-derived monoclonal antibodies (mAbs), along with gold nanoparticles, to detect SARS-CoV-2 with or without cross-reaction to SARS-CoV-1 and other coronaviruses. In this development, we demonstrate a robust antibody screening methodology for the selection of mAb TMSB4X pairs that can recognize SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. Linear epitope mapping of the mAbs helped elucidate SARS-CoV-2 S and N interactions in lateral flow chromatography. A candidate rapid antigen test for SARS-CoV-2 N was validated using nasal swab specimens that were confirmed positive or negative by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Test results were image-captured using a mobile phone and normalized signal pixel intensities were calculated; signal intensities were inversely correlated to RT-PCR cycle threshold (Ct) value. Conclusion/Significance Overall, our results suggest that the rapid antigen test is optimized to detect SARS-CoV-2 N during the acute phase of COVID-19. The rapid antigen tests developed in this study are alternative tools for wide scale public health surveillance of COVID-19. Author summary The delays in diagnostic testing and lack of proper surveillance during the COVID-19 pandemic have contributed, in part, to the unprecedented death toll and impediment to wellbeing. As asymptomatic individuals have contributed to a large portion of disease spread, improved public health tools for widespread screening are necessary to maintain low transmission levels. We developed new rapid antigen tests that can be administered in less than 15 minutes without instrumentation, offering potential for frequent asymptomatic screening at-home and for point-of-care use in high-traffic areas such as schools, hospitals, and airports. The SARS-CoV-2 monoclonal antibody screening methodology resulted in pairs with or without cross-reaction to SARS-CoV-1, offering new opportunities for public health tools such as wide scale surveillance. This paper provides important insights for nanoparticle-based immunochromatographic assays. Introduction The coronavirus disease 2019 (COVID-19) pandemic has resulted in an unprecedented public health crisis. As of October 2021, there have been approximately 245 million confirmed cases globally dealing devastating effects to economies and livelihood [1C3]. Individuals experiencing asymptomatic infection are a main source for.