A sensitive gold nanoflower-based lateral flow assay coupled with gold staining technique for the detection of SARS-CoV-2 antigen.

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL-1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL-1. The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection.

Utilizing COVID-19 as a Model for Diagnostics Using an Electrochemical Sensor.

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS-1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL-1 to 10.0 µg mL-1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods.

Acoustic ejection mass spectrometry empowers ultra-fast protein biomarker quantification.

The global scientific response to COVID 19 highlighted the urgent need for increased throughput and capacity in bioanalytical laboratories, especially for the precise quantification of proteins that pertain to health and disease. Acoustic ejection mass spectrometry (AEMS) represents a much-needed paradigm shift for ultra-fast biomarker screening. Here, a quantitative AEMS assays is presented, employing peptide immunocapture to enrich (i) 10 acute phase response (APR) protein markers from plasma, and (ii) SARS-CoV-2 NCAP peptides from nasopharyngeal swabs. The APR proteins were quantified in 267 plasma samples, in triplicate in 4.8 h, with %CV from 4.2% to 10.5%. SARS-CoV-2 peptides were quantified in triplicate from 145 viral swabs in 10 min. This assay represents a 15-fold speed improvement over LC-MS, with instrument stability demonstrated across 10,000 peptide measurements. The combination of speed from AEMS and selectivity from peptide immunocapture enables ultra-high throughput, reproducible quantitative biomarker screening in very large cohorts.

Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.

Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 107 fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 µL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 104 fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.

A universal three-dimensional hydrogel electrode for electrochemical detection of SARS-CoV-2 nucleocapsid protein and hydrogen peroxide.

Coronavirus disease 2019 (COVID-19) is a highly contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in a global health crisis. The primary diagnostic method for COVID-19 is quantitative reverse transcription PCR, which is time-consuming and requires expensive instrumentation. Here, we developed an electrochemical biosensor for detecting SARS-CoV-2 biomarkers using a 3D porous polyacrylamide/polyaniline hydrogel (PPG) electrode prepared by UV photopolymerization and in situ polymerization. The electrochemical immunosensor for detecting SARS-CoV-2 N protein via the immune sandwich principle demonstrated a lower detection limit of 42 pg/mL and comparable specificity to a commercial enzyme-linked immunosorbent assay, which was additionally validated in pseudoviruses. The electrochemical sensor for hydrogen peroxide showed a low detection limit of 0.5 µM and excellent selectivity, which was further confirmed in cancer cells under oxidative stress. The biomarkers of SARS-CoV-2 were successfully detected due to the signal amplification capability provided by 3D porous electrodes and the high sensitivity of the antigen-antibody specific binding. This study introduces a novel three-dimensional electrode with great potential for the early detection of SARS-CoV-2.

Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned.

The COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy.

Lateral flow immunoassays (LFIAs) are an essential and widely used point-of-care test for medical diagnoses. However, commercial LFIAs still have low sensitivity and specificity. Therefore, we developed an automatic ultrasensitive dual-color enhanced LFIA (DCE-LFIA) by applying an enzyme-induced tyramide signal amplification method to a double-antibody sandwich LFIA for antigen detection. The DCE-LFIA first specifically captured horseradish peroxidase (HRP)-labeled colored microspheres at the Test line, and then deposited a large amount of tyramide-modified signals under the catalytic action of HRP to achieve the color superposition. A limit of detection (LOD) of 3.9 pg/mL and a naked-eye cut-off limit of 7.8 pg/mL were achieved for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein. Additionally, in the inactivated virus detections, LOD equivalent to chemiluminescence (0.018 TCID50/mL) was obtained, and it had excellent specificity under the interference of other respiratory viruses. High sensitivity has also been achieved for detection of influenza A, influenza B, cardiac troponin I, and human chorionic gonadotrophin using this DCE-LFIA, suggesting the assay is universally applicable. To ensure the convenience and stability in practical applications, we created an automatic device. It provides a new practical option for point-of-care test immunoassays, especially ultra trace detection and at-home testing.

Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

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.

Estimating SARS-CoV-2 Seroprevalence in Canadian Blood Donors, April 2020 to March 2021: Improving Accuracy with Multiple Assays.

We have previously used composite reference standards and latent class analysis (LCA) to evaluate the performance of laboratory assays in the presence of tarnished gold standards. Here, we apply these techniques to repeated, cross-sectional study of Canadian blood donors, whose sera underwent parallel testing with four separate SARS-CoV-2 antibody assays. We designed a repeated cross-sectional design with random cross-sectional sampling of all available retention samples (n = 1500/month) for a 12 -month period from April 2020 until March 2021. Each sample was evaluated for SARS-CoV-2 IgG antibodies using four assays an Abbott Architect assay targeting the nucleocapsid antigen (Abbott-NP, Abbott, Chicago IL) and three in-house IgG ELISAs recognizing distinct recombinant viral antigens: full-length spike glycoprotein (Spike), spike glycoprotein receptor binding domain (RBD) and nucleocapsid (NP). We used two analytic approaches to estimate SAR-CoV-2 seroprevalence: a composite reference standard and LCA. Using LCA to estimate true seropositivity status based on the results of the four antibody tests, we estimated that seroprevalence increased from 0.8% (95% CI: 0.5-1.4%) in April 2020 to 6.3% (95% CI: 5.1-7.6%) in March 2021. Our study provides further support for the use of LCA in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable. IMPORTANCE Here, we describe an approach to estimating seroprevalence in a low prevalence setting when multiple assays are available and yet no known gold standard exists. Because serological studies identify cases through both diagnostic testing and surveillance, and otherwise silent, unrecognized infections, serological data can be used to estimate the true infection fatality ratio of a disease. However, seroprevalence studies rely on assays with imperfect sensitivity and specificity. Seroreversion (loss of antibody response) also occurs over time, and with the advent of vaccination, distinction of antibody response resulting from vaccination as opposed to antibody response due to infection has posed an additional challenge. Our approach indicates that seroprevalence on Canadian blood donors by the end of March 2021was less than 10%. Our study supports the use of latent class analysis in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable.

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.

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.

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.

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.