Lateral Flow Immunoassay Coupled with Copper Enhancement for Rapid and Sensitive SARS-CoV-2 Nucleocapsid Protein Detection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) is still raging all over the world. Hence, the rapid and sensitive screening of the suspected population is in high demand. The nucleocapsid protein (NP) of SARS-CoV-2 has been selected as an ideal marker for viral antigen detection. This study describes a lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles for rapid NP antigen detection, in which sensitivity was improved through copper deposition-induced signal amplification. The detection sensitivity of the developed LFIA for NP antigen detection (using certified reference materials) under the optimized parameters was 0.01 µg/mL and was promoted by three orders of magnitude to 10 pg/mL after copper deposition signal amplification. The LFIA coupled with the copper enhancement technique has many merits such as low cost, high efficiency, and high sensitivity. It provides an effective approach to the rapid screening, diagnosis, and monitoring of the suspected population in the COVID-19 outbreak.

Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19.

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.

Capacitive Aptasensor Coupled with Microfluidic Enrichment for Real-Time Detection of Trace SARS-CoV-2 Nucleocapsid Protein.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for almost 2 years. Stemming its spread has posed severe challenges for clinical virus detection. A long turnaround time, complicated operation, and low accuracy have become bottlenecks in developing detection techniques. Adopting a direct antigen detection strategy, we developed a fast-responding and quantitative capacitive aptasensor for ultratrace nucleocapsid protein detection based on a low-cost microelectrode array (MEA) chip. Employing the solid-liquid interface capacitance with a sensitivity of picofarad level, the tiny change on the MEA surface can be definitively detected. As a result, the limit of detection reaches an ultralow level of femtogram per milliliter in different matrices. Integrated with efficient microfluidic enrichment, the response time of this sensor from the sample to the result is shortened to 15 s, completely meeting the real-time detection demand. Moreover, the wide linear range of the sensor is from 10-5 to 10-2 ng/mL, and a high selectivity of 6369:1 is achieved. After application and evaluation in different environmental and body fluid matrices, this sensor and the detection method have proved to be a label-free, real-time, easy-to-operate, and specific strategy for SARS-CoV-2 screening and diagnosis.

Protein purification and crystallization of HLA-A∗02:01 in complex with SARS-CoV-2 peptides.

Understanding T-cell responses requires identifying viral peptides presented by human leukocyte antigens (HLAs). X-ray crystallography can be used to visualize their presentation. This protocol describes the expression, purification, and crystallization of HLA-A∗02:01, one of the most frequent HLA in the global population in complex with peptides derived from the SARS-CoV-2 nucleocapsid protein. This protocol can be applied to different HLA class I molecules bound to other peptides. For complete details on the use and execution of this protocol, please refer to Szeto et al. (2021).

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.

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.

The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

Improved diagnosis of SARS-CoV-2 by using nucleoprotein and spike protein fragment 2 in quantitative dual ELISA tests.

The novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative enzyme-linked immunosorbent assays (ELISAs) that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive coronavirus disease-2019 (COVID-19) cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients who had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus.

Simultaneous detection of the spike and nucleocapsid proteins from SARS-CoV-2 based on ultrasensitive single molecule assays.

Nucleic acid detection technology based on polymerase chain reaction (PCR) and antibody detection based on immunochromatography still have many problems such as false negatives for the diagnosis of coronavirus disease 2019 (COVID-19). Therefore, it is of great importance to develop new techniques to improve the diagnostic accuracy of COVID-19. We herein developed an ultrasensitive, rapid, and duplex digital enzyme-linked immunosorbent assay (dELISA) for simultaneous detection of spike (S-RBD) and nucleocapsid (N) proteins of SARS-CoV-2 based on a single molecule array. This assay effectively combines magnetic bead encoding technology and the ultrasensitive detection capability of a single molecule array. The detection strategies of S-RBD protein and N-protein exhibited wide response ranges of 0.34-1065 pg/mL and 0.183-338 pg/mL with detection limits of 20.6 fg/mL and 69.8 fg/mL, respectively. It is a highly specific method for the simultaneous detection of S-RBD protein and N-protein and has minimal interference from other blood proteins. Moreover, the spike assay showed a satisfactory and reproducible recovery rate for the detection of S-RBD protein and N-protein in serum samples. Overall, this work provides a highly sensitive method for the simultaneous detection of S-RBD protein and N-protein, which shows ultrasensitivity and high signal-to-noise ratio and contributes to improve the diagnosis accuracy of COVID-19.

Expression and purification of recombinant SARS-CoV-2 nucleocapsid protein in inclusion bodies and its application in serological detection.

The current standard for the diagnosis of COVID-19 is the nucleic acid test of SARS-CoV-2 RNA, however, virus antibody detection has the advantages of convenient sample collection, high throughout, and low cost. When combining detection with nucleic acid detection, antibody detection can effectively compensate for nucleic acid detection. Virus infection always induce high antibody titer against SARS-CoV-2 nucleocapsid protein (N protein), which can be used to detect COVID-19 at both infected and convalescent patients. In this study we reported the expression and purification of N protein in E.coli from inclusion bodies by a combination of two cation exchange chromatography, and the yield of N protein was around 50 mg/L fermentation broth with more than 90% purity. A corresponding colloidal gold detection kit prepared with our purified N protein was used to verify the efficiency and accuracy our N protein in antibody detection method. Of the 58 COVID-19 PCR positive patients' inactivated serum samples, 40 samples were IgM positive (69.0%), and 42 samples were IgG positive (72.4%), and all 95 COVID-19 negative patients' inactivated serum samples were both IgM and IgG negative. Our results indicates that the refolded soluble N protein could be used for the preliminary detection of IgG and IgM antibodies against SARS-CoV- 2.

[Rapid SARS-CoV-2 antigenic test: definition, legislation of use, technological principles, analytical and clinical performance comparison]. / Tests rapides antigéniques SARS-CoV-2 : définition, législation d'utilisation, principes technologiques, comparaison des performances analytiques et cliniques.

The SARS-CoV-2 has emerged in China at the end of 2019. In order to meet the growing demand in laboratories for RT-PCR testing for viral genome detection, rapid tests detecting a SARS-CoV-2 protein (antigenic rapid test) have been developed. In this review, we present for different SARS-CoV-2 antigenic rapid tests authorized in France: legislation, technological principle, and analytical and clinical performances. Data bellow are those provided by the manufacturer/distributor. From the list of tests authorized by the French Ministry of Health, we have selected 25 for which the distributors/manufacturers have provided the technical data essential to their comparative analysis. The kits use immunochromatography technology, with detection of the nucleocapsid protein (n = 24) or the spike protein (n = 1). The matrix used is a nasopharyngeal (n = 23), oropharyngeal (n = 9) or nasal (n = 3) swab. According to the test, the reading of the result is done from 15 to 30 minutes after it is performed. The clinical sensitivity, for the more performant tests is conversely linked to the Ct of RT-PCR, ranging from 80.2% to 98.4%, according to the quantity of virus present in the sample. This percentage is inversely proportional to the Ct obtained using RT-PCR. The limit of detection ranges from 31.55 to 7200 TCID50/mL. The clinical specificity, compared to a negative result of RT-PCR, is between 99.2% and 100%. Analytical specificity evaluated on other microorganisms is 100%, except for 3 kits that show cross-reactivities with SARS-CoV-1 (n = 3) and MERS-CoV (n = 1). Positive and negative predictive values range from 96.3% to 100% and 95% to 99.4%, respectively.

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan.

PCR methods are presently the standard for the diagnosis of Coronavirus disease 2019 (COVID-19), but additional methodologies are needed to complement PCR methods, which have some limitations. Here, we validated and investigated the usefulness of measuring serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the iFlash3000 CLIA analyzer. We measured IgM and IgG titers against SARS-CoV-2 in sera collected from 26 PCR-positive COVID-19 patients, 53 COVID-19-suspected but PCR-negative patients, and 20 and 100 randomly selected non-COVID-19 patients who visited our hospital in 2020 and 2017, respectively. The repeatability and within-laboratory precision were obviously good in validations, following to the CLSI document EP15-A3. Linearity was also considered good between 0.6 AU/mL and 112.7 AU/mL for SARS-CoV-2 IgM and between 3.2 AU/mL and 55.3 AU/mL for SARS-CoV-2 IgG, while the linearity curves plateaued above the upper measurement range. We also confirmed that the seroconversion and no-antibody titers were over the cutoff values in all 100 serum samples collected in 2017. These results indicate that this measurement system successfully detects SARS-CoV-2 IgM/IgG. We observed four false-positive cases in the IgM assay and no false-positive cases in the IgG assay when 111 serum samples known to contain autoantibodies were evaluated. The concordance rates of the antibody test with the PCR test were 98.1% for SARS-CoV-2 IgM and 100% for IgG among PCR-negative cases and 30.8% for SARS-CoV-2 IgM and 73.1% for SARS-CoV-2 IgG among PCR-positive cases. In conclusion, the performance of this new automated method for detecting antibody against both N and S proteins of SARS-CoV-2 is sufficient for use in laboratory testing.

Large scale production and characterization of SARS-CoV-2 whole antigen for serological test development.

BACKGROUND: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID-19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS-CoV-2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. METHODS: An enzyme-linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. RESULTS: Preliminary data from 10 sera (5 patients with COVID-19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS-CoV-2-positive individuals. CONCLUSION: This ELISA appears to be a specific and reliable method for detecting COVID-19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus.

Severe Acute Respiratory Syndrome Coronavirus 2 Nucleocapsid Protein in the Ocular Tissues of a Patient Previously Infected With Coronavirus Disease 2019.

Importance: Coronavirus disease 2019 (COVID-19) has been recognized as a pandemic by the World Health Organization. Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can also infect tissues besides the respiratory system, such as the ocular tissues, remains unclear. Objective: To determine whether SARS-CoV-2 exists intracellularly in the ocular tissues of a patient previously infected with COVID-19. Design, Setting, and Participants: This case study analyzed a patient previously infected with COVID-19 who had an acute glaucoma attack during her rehabilitation. Plasma samples and tissue specimens, including ones from the conjunctiva, anterior lens capsular, trabecular meshwork, and iris, were collected during phacoemulsification and trabeculectomy surgery. Specimens from another patient who had glaucoma but not COVID-19 were used as a negative control. Main Outcomes and Measures: Specimens were analyzed using hematoxylin-eosin staining. The nucleocapsid protein antigen of SARS-CoV-2 was measured in the conjunctiva, trabecular meshwork, and iris using immunofluorescence and immunohistochemistry. The expression of angiotensin-converting enzyme 2 receptor on the conjunctiva was measured using immunohistochemistry. Results: The patient with a previous COVID-19 infection was female and 64 years old, and the control patient without a COVID-19 infection history was male and 61 years old. The nucleocapsid protein antigen of SARS-CoV-2 was detected on the cells of the conjunctiva, trabecular, and iris of the patient infected with COVID-19 but not in the control participant, while angiotensin-converting enzyme 2 receptor proteins were detected on the conjunctiva of both patients. Conclusions and Relevance: The nucleocapsid protein antigen of SARS-CoV-2 existed intracellularly in the ocular tissues of a patient previously infected with COVID-19. Thus, SARS-CoV-2 can also infect ocular tissues in addition to the respiratory system.