Highly Stable Buffer-Based Zinc Oxide/Reduced Graphene Oxide Nanosurface Chemistry for Rapid Immunosensing of SARS-CoV-2 Antigens.

The widespread and long-lasting effect of the COVID-19 pandemic has called attention to the significance of technological advances in the rapid diagnosis of SARS-CoV-2 virus. This study reports the use of a highly stable buffer-based zinc oxide/reduced graphene oxide (bbZnO/rGO) nanocomposite coated on carbon screen-printed electrodes for electrochemical immuno-biosensing of SARS-CoV-2 nuelocapsid (N-) protein antigens in spiked and clinical samples. The incorporation of a salt-based (ionic) matrix for uniform dispersion of the nanomixture eliminates multistep nanomaterial synthesis on the surface of the electrode and enables a stable single-step sensor nanocoating. The immuno-biosensor provides a limit of detection of 21 fg/mL over a linear range of 1-10 000 pg/mL and exhibits a sensitivity of 32.07 ohms·mL/pg·mm2 for detection of N-protein in spiked samples. The N-protein biosensor is successful in discriminating positive and negative clinical samples within 15 min, demonstrating its proof of concept used as a COVID-19 rapid antigen test.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. METHODS: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. RESULTS: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 µg/L, with a precision coefficient of variance below 10 %. CONCLUSION: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.

Construction of bifunctional electrochemical biosensors for the sensitive detection of the SARS-CoV-2 N-gene based on porphyrin porous organic polymers.

In this study, a novel porphyrin-based porous organic polymer (POP) was constructed using 5,10,15,20-tetramine (4-aminophenyl) porphyrin (TAPP) and 5,5'-diformyl-2,2'-bipyridine (DPDD) as organic ligands via a solvothermal method (represented as TAPP-DPDD-POP). Then, it was utilized as a bifunctional scaffold for constructing a sensitive sensing strategy toward the nucleocapsid phosphoprotein (N-gene) of SARS-CoV-2. The obtained TAPP-DPDD-POP is composed of nanospheres with a size of 100-300 nm and possesses a highly conjugated and π-π stacking network. The coexistence of the porphyrin and bipyridine moieties of TAPP-DPDD-POP afforded considerable electrochemical activity and a strong binding interaction toward the SARS-CoV-2 N-gene-targeted antibody and targeted the aptamer strands of the N-gene. The TAPP-DPDD-POP-based aptasensor and immunosensor were manufactured for the sensitive analysis of SARS-CoV-2 N-gene, and exhibited the limit of detection (LOD) of 0.59 fg mL-1 and 0.17 fg mL-1, respectively, within the range of 0.1 fg mL-1 to 1 ng mL-1 of N-gene. The sensing performances of both the TAPP-DPDD-POP-based aptasensor and immunosensor were better than those of existing electrochemical biosensors for analyzing the N-gene, accompanied with excellent stability, high selectivity and reproducibility. The TAPP-DPDD-POP-based aptasensor and immunosensor were then employed to detect the N-gene from various environments, including human serum, river water, and seafoods. This work provides a new method of using an electrochemically active POP to sensitively and selectively analyze SARS-CoV-2 in diverse environments.

Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance.

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.

Paper-based microfluidic chip for rapid detection of SARS-CoV-2 N protein.

This research has developed a method for rapid detection of SARS-CoV-2 N protein on a paper-based microfluidic chip. The chitosan-glutaraldehyde cross-linking method is used to fix the coated antibody, and the sandwich enzyme-linked immunosorbent method is used to achieve the specific detection of the target antigen. The system studied the influence of coating antibody concentration and enzyme-labeled antibody concentration on target antigen detection. According to the average gray value measured under different N protein concentrations, the standard curve of the method was established and the sensitivity was tested, and its linear regression was obtained. The equation is y = 9.8286x+137.6, R2 = 0.9772 > 0.90, which shows a high degree of fit. When the concentration of coating antibody and enzyme-labeled antibody were 1 µg/mL and 2 µg/mL, P > 0.05, the difference was not statistically significant, so the lower concentration of 1 µg/mL was chosen as the coating antibody concentration. The results show that the minimum concentration of N protein that can be detected by this method is 8 µg/mL, and the minimum concentration of coating antibody and enzyme-labeled antibody is 1 µg/mL, which has the characteristics of high sensitivity and good repeatability.

Quantifying SARS-CoV-2 nucleocapsid antigen in oropharyngeal swabs using single molecule array technology.

This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4-98.5%) and specificity of 100% (95% CI 95.1-100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3-98.1%) and a specificity of 100% (95% CI 95.1-100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.

Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue.

BACKGROUND: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. METHODS: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. RESULTS: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. CONCLUSIONS: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

A rapid and reliable liquid chromatography/mass spectrometry method for SARS-CoV-2 analysis from gargle solutions and saliva.

We describe a rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the direct detection and quantitation of SARS-CoV-2 nucleoprotein in gargle solutions and saliva. The method is based on a multiple-reaction monitoring (MRM) mass spectrometry approach with a total cycle time of 5 min per analysis and allows the detection and accurate quantitation of SARS-CoV-2 nucleoprotein as low as 500 amol/µL. We improved the sample preparation protocol of our recent piloting SARS-CoV-2 LC-MS study regarding sensitivity, reproducibility, and compatibility with a complementary reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of the same sample. The aim of this work is to promote diagnostic tools that allow identifying and monitoring SARS-CoV-2 infections by LC-MS/MS methods in a routine clinical environment.

Ultrasensitive and Simultaneous Detection of Two Specific SARS-CoV-2 Antigens in Human Specimens Using Direct/Enrichment Dual-Mode Fluorescence Lateral Flow Immunoassay.

Sensitive point-of-care methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens are urgently needed to achieve rapid screening of viral infection. We developed a magnetic quantum dot-based dual-mode lateral flow immunoassay (LFIA) biosensor for the high-sensitivity simultaneous detection of SARS-CoV-2 spike (S) and nucleocapsid protein (NP) antigens, which is beneficial for improving the detection accuracy and efficiency of SARS-CoV-2 infection in the point-of-care testing area. A high-performance magnetic quantum dot with a triple-QD shell (MagTQD) nanotag was first fabricated and integrated into the LFIA system to provide superior fluorescence signals, enrichment ability, and detectability for S/NP antigen testing. Two detection modes were provided by the proposed MagTQD-LFIA. The direct mode was used for rapid screening or urgent detection of suspected samples within 10 min, and the enrichment mode was used for the highly sensitive and quantitative analysis of SARS-CoV-2 antigens in biological samples without the interference of the "hook effect." The simultaneous detection of SARS-CoV-2 S/NP antigens was conducted in one LFIA strip, and the detection limits for two antigens under direct and enrichment modes were 1 and 0.5 pg/mL, respectively. The MagTQD-LFIA showed high accuracy, specificity, and stability in saliva and nasal swab samples and is an efficient tool with flexibility to meet the testing requirements for SARS-CoV-2 antigens in various situations.

SARS-CoV-2 RNA and antibody detection in breast milk from a prospective multicentre study in Spain.

OBJECTIVES: To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies. DESIGN AND PATIENTS: This is a prospective, multicentre longitudinal study (April-December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included. SETTING: Seven health centres from different provinces in Spain. MAIN OUTCOME MEASURES: Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)-IgA, IgG and IgM-in breast milk samples from patients with COVID-19. RESULTS: All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%-87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation). CONCLUSIONS: Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence. Trial registration number NCT04768244.

Proteomic Signature of Host Response to SARS-CoV-2 Infection in the Nasopharynx.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a global health pandemic. COVID-19 severity ranges from an asymptomatic infection to a severe multiorgan disease. Although the inflammatory response has been implicated in the pathogenesis of COVID-19, the exact nature of dysregulation in signaling pathways has not yet been elucidated, underscoring the need for further molecular characterization of SARS-CoV-2 infection in humans. Here, we characterize the host response directly at the point of viral entry through analysis of nasopharyngeal swabs. Multiplexed high-resolution MS-based proteomic analysis of confirmed COVID-19 cases and negative controls identified 7582 proteins and revealed significant upregulation of interferon-mediated antiviral signaling in addition to multiple other proteins that are not encoded by interferon-stimulated genes or well characterized during viral infections. Downregulation of several proteasomal subunits, E3 ubiquitin ligases, and components of protein synthesis machinery was significant upon SARS-CoV-2 infection. Targeted proteomics to measure abundance levels of MX1, ISG15, STAT1, RIG-I, and CXCL10 detected proteomic signatures of interferon-mediated antiviral signaling that differentiated COVID-19-positive from COVID-19-negative cases. Phosphoproteomic analysis revealed increased phosphorylation of several proteins with known antiviral properties as well as several proteins involved in ciliary function (CEP131 and CFAP57) that have not previously been implicated in the context of coronavirus infections. In addition, decreased phosphorylation levels of AKT and PKC, which have been shown to play varying roles in different viral infections, were observed in infected individuals relative to controls. These data provide novel insights that add depth to our understanding of SARS-CoV-2 infection in the upper airway and establish a proteomic signature for this viral infection.

Exponential Amplification Using Photoredox Autocatalysis.

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here, we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a nonfluorescent eosin Y derivative (EYH3-) under green light. The deactivated photocatalyst is stable and rapidly activated under low-intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH3- is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e-/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH3- degradation, we successfully improved EYH3--to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photoinduced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19.

Analysis of SARS-CoV-2 nucleocapsid phosphoprotein N variations in the binding site to human 14-3-3 proteins.

The SARS-CoV-2 N protein binds several cell host proteins including 14-3-3γ, a well-characterized regulatory protein. However, the biological function of this interaction is not completely understood. We analyzed the variability of ∼90 000 sequences of the SARS-CoV-2 N protein, particularly, its mutations in disordered regions containing binding motifs for 14-3-3 proteins. We studied how these mutations affect the binding energy to 14-3-3γ and found that changes positively affecting the predicted interaction with 14-3-3γ are the most successfully spread, with the highest prevalence in the phylogenetic tree. Although most residues are highly conserved within the 14-3-3 binding site, compensatory mutations to maintain the interaction energy of N-14-3-3γ were found, including half of the current variants of concern and interest. Our results suggest that binding of N to 14-3-3γ is beneficial for the virus, thus targeting this viral-host protein-protein interaction seems an attractive approach to explore antiviral strategies.

Immunohistochemistry for protein detection in PFA-fixed paraffin-embedded SARS-CoV-2-infected COPD airway epithelium.

Patients with chronic lung disease are vulnerable to getting severe diseases associated with SARS-CoV-2 infection. Here, we describe protocols for subculturing and differentiating primary normal human bronchial epithelial (NHBE) cells of patients with chronic obstructive lung disease. The differentiation of NHBE cells in air-liquid interface mimics an in vivo airway and provides an in vitro model for studying SARS-CoV-2 infection. We also describe a protocol for detecting proteins in the sectioned epithelium for detailing SARS-CoV-2 infection-induced pathobiology with a vertical view.

On-field evaluation of a ultra-rapid fluorescence immunoassay as a frontline test for SARS-CoV-2 diagnostic.

BACKGROUND: Viral RNA amplification by real-time RT-PCR still represents the gold standard for the detection of SARS-CoV-2, but the development of rapid, reliable and easy-to-perform diagnostic methods is crucial for public health, because of the need of shortening the time of result-reporting with a cost-efficient approach. OBJECTIVES: The aim of our research was to assess the performance of FREND™ COVID-19 Ag assay (NanoEntek, South Korea) as a ultra-rapid frontline test for SARS-CoV-2 identification, in comparison with RT-PCR and another COVID-19 antigen fluorescence immunoassay (FIA). STUDY DESIGN: The qualitative FIA FREND™ test, designed to detect within 3 min the Nucleocapsid protein of SARS-CoV-2, was evaluated using nasopharyngeal swabs in Universal Transport Medium (UTM™, Copan Diagnostics Inc, US) from suspected COVID-19 cases who accessed the Emergency Room of the Ospedale Policlinico San Martino, Genoa, Liguria, Northwest Italy. Diagnostic accuracy was determined in comparison with SARS-CoV-2 RT-PCR and STANDARD F™ COVID-19 Ag FIA test (SD BIOSENSOR Inc., Republic of Korea). RESULTS: In November 2020, 110 nasopharyngeal samples were collected consecutively; 60 resulted RT-PCR positive. With respect to RT-PCR results, sensitivity and specificity of FREND™ COVID-19 Ag test were 93.3 % (95 % CI: 83.8-98.2) and 100 % (95 % CI: 92.9-100), respectively. FREND™and STANDARD F™ COVID-19 Ag FIA assays showed a concordance of 96.4 % (Cohen's k = 0.93, 95 % CI: 0.86-0.99). CONCLUSIONS: FREND™ FIA test showed high sensitivity and specificity in nasopharyngeal swabs. The assay has the potential to become an important tool for an ultra-rapid identification of SARS-CoV-2 infection, particularly in situations with limited access to molecular diagnostics.

Voltammetric-based immunosensor for the detection of SARS-CoV-2 nucleocapsid antigen.

Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL-1 to 100 ng.mL-1 and a limit of detection of 0.4 pg.mL-1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.

A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT ] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an "in-house" test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing.IMPORTANCE PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best. Antigen tests directly detect parts of the infectious agent, which may enable faster diagnosis but often at lower sensitivity and specificity. Here, we describe a technique for rapid antigen detection and demonstrate the test format's potential using SARS-CoV-2 as the model pathogen. The 10-min test, performed in a buffer that readily inactivates SARS-CoV-2, from nasopharyngeal samples identified 97.4% (37/38) of the samples from which we could isolate the virus. This suggests that the test performs well in identifying patients potentially shedding the virus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of concept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases.

Protein-based lateral flow assays for COVID-19 detection.

To combat the enduring and dangerous spread of COVID-19, many innovations to rapid diagnostics have been developed based on proteinprotein interactions of the SARS-CoV-2 spike and nucleocapsid proteins to increase testing accessibility. These antigen tests have most prominently been developed using the lateral flow assay (LFA) test platform which has the benefit of administration at point-of-care, delivering quick results, lower cost, and does not require skilled personnel. However, they have gained criticism for an inferior sensitivity. In the last year, much attention has been given to creating a rapid LFA test for detection of COVID-19 antigens that can address its high limit of detection while retaining the advantages of rapid antibodyantigen interaction. In this review, a summary of these proteinprotein interactions as well as the challenges, benefits, and recent improvements to protein based LFA for detection of COVID-19 are discussed.

Spike vs nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens.

Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many "antigen" detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false positive results. In this work, we developed a novel alternative spike-based antigen assay, comprising four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results. Schematic representation of sample collection and analysis. The figure was created using BioRender.com.