Identification of a broad-spectrum high-affinity peptide ligand for the purification of spike proteins.

Since the outbreak of coronavirus disease 2019, the global demand for vaccines has increased rapidly to prevent infection and protect high-risk populations. However, identifying viral mutations poses an additional challenge for chromatographic purification of vaccines and subunit vaccines. In this study, a new affinity peptide model, X1VX2GLNX3WX4RYSK, was established, and a library of 612 peptides was generated for ligand screening. Based on a multistep strategy of ligand screening, 18 candidate peptides were obtained. The top ranking peptide, LP14 (YVYGLNIWLRYSK), and two other representative peptides, LP02 and LP06, with lower rankings were compared via molecular dynamics simulation. The results revealed that peptide binding to the receptor binding domain (RBD) was driven by hydrophobic interactions and the key residues involved in the binding were identified. Surface plasmon resonance analysis further confirmed that LP14 had the highest affinity for the wild RBD (Kd=0.520 µmol/L), and viral mutation had little influence on the affinity of LP14, demonstrating its great potential as a broad-spectrum ligand for RBD purification. Finally, chromatographic performance of LP14-coupled gel-packed column verified that both wild and omicron RBDs could be purified and were eluted by 0.1 mol/L Gly-HCl buffer (pH 3.0). This research identified a broad-spectrum peptide for RBD purification based on rational design and demonstrated its potential application in the purification of RBDs from complex feedstock.

Production of native recombinant proteins using a novel split intein affinity technology.

Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a CC-intein tag is engineered into a protein of interest for binding to a NC-intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the NC-intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the CC-intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems.

A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein.

Recombinant SARS-CoV-2 trimeric spike protein produced by mammalian cell culture is a potential candidate for a COVID-19 vaccine. However, this protein is much larger than most typical biopharmaceutical proteins and its large-scale manufacture is therefore challenging. Particularly, its purification using resin-based chromatography is difficult as the diffusive transport of this protein to and from its binding site within the pores of the stationary phase particles is slow. Therefore, very low flow rates need to be used during binding and elution, and this slows down the purification process. Also, due to its large size, the binding capacity of this protein on resin-based media is low. Membrane chromatography is an efficient and scalable technique for purifying biopharmaceuticals. The predominant mode of solute transport in a membrane is convective and hence it is considered better than resin-based chromatography for purifying large proteins. In this paper, we propose a membrane chromatography-based purification method for fast and scalable manufacture of recombinant SARS-CoV-2 trimeric spike protein. A combination of cation exchange z2 laterally-fed membrane chromatography and size exclusion chromatography was found to be suitable for obtaining a homogeneous spike protein sample from mammalian cell culture supernatant. The proposed method is both fast and scalable and could be explored as a method for manufacturing vaccine grade spike protein.

Assessing the performance of a serological point-of-care test in measuring detectable antibodies against SARS-CoV-2.

This study investigated the performance of a rapid point-of-care antibody test, the BioMedomics COVID-19 IgM/IgG Rapid Test, in comparison with a high-quality, validated, laboratory-based platform, the Roche Elecsys Anti-SARS-CoV-2 assay. Serological testing was conducted on 709 individuals. Concordance metrics were estimated. Logistic regression was used to assess associations with seropositivity. SARS-CoV-2 seroprevalence was 63.5% (450/709; 95% CI 59.8%-67.0%) using the BioMedomics assay and 71.9% (510/709; 95% CI 68.5%-75.2%) using the Elecsys assay. There were 60 discordant results between the two assays, all of which were seropositive in the Elecsys assay, but seronegative in the BioMedomics assay. Overall, positive, and negative percent agreements between the two assays were 91.5% (95% CI 89.2%-93.5%), 88.2% (95% CI 85.1%-90.9%), and 100% (95% CI 98.2%-100%), respectively, with a Cohen's kappa of 0.81 (95% CI 0.78-0.84). Excluding specimens with lower (Elecsys) antibody titers, the agreement improved with overall, positive, and negative percent concordance of 94.4% (95% CI 92.3%-96.1%), 91.8% (95% CI 88.8%-94.3%), and 100% (95% CI 98.2%-100%), respectively, and a Cohen's kappa of 0.88 (95% CI 0.85-0.90). Logistic regression confirmed better agreement with higher antibody titers. The BioMedomics COVID-19 IgM/IgG Rapid Test demonstrated good performance in measuring detectable antibodies against SARS-CoV-2, supporting the utility of such rapid point-of-care serological testing to guide the public health responses and vaccine prioritization.

Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein.

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E, and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii.

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology.

The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q, and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), could be identified. The detection limit for all 9 sites was 1.5 × 103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR cycle threshold (CT) values less than 27 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.

Hydrogel-Based Slow Release of a Receptor-Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS-CoV-2.

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime-boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

Antisense Peptide Technology for Diagnostic Tests and Bioengineering Research.

Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.

Development of ACE2 autoantibodies after SARS-CoV-2 infection.

BACKGROUND: Activation of the immune system is implicated in the Post-Acute Sequelae after SARS-CoV-2 infection (PASC) but the mechanisms remain unknown. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) resulting in decreased activation of the AT1 receptor and decreased immune system activation. We hypothesized that autoantibodies against ACE2 may develop after SARS-CoV-2 infection, as anti-idiotypic antibodies to anti-spike protein antibodies. METHODS AND FINDINGS: We tested plasma or serum for ACE2 antibodies in 67 patients with known SARS-CoV-2 infection and 13 with no history of infection. None of the 13 patients without history of SARS-CoV-2 infection and 1 of the 20 outpatients that had a positive PCR test for SARS-CoV-2 had levels of ACE2 antibodies above the cutoff threshold. In contrast, 26/32 (81%) in the convalescent group and 14/15 (93%) of patients acutely hospitalized had detectable ACE2 antibodies. Plasma from patients with antibodies against ACE2 had less soluble ACE2 activity in plasma but similar amounts of ACE2 protein compared to patients without ACE2 antibodies. We measured the capacity of the samples to inhibit ACE2 enzyme activity. Addition of plasma from patients with ACE2 antibodies led to decreased activity of an exogenous preparation of ACE2 compared to patients that did not have antibodies. CONCLUSIONS: Many patients with a history of SARS-CoV-2 infection have antibodies specific for ACE2. Patients with ACE2 antibodies have lower activity of soluble ACE2 in plasma. Plasma from these patients also inhibits exogenous ACE2 activity. These findings are consistent with the hypothesis that ACE2 antibodies develop after SARS-CoV-2 infection and decrease ACE2 activity. This could lead to an increase in the abundance of Ang II, which causes a proinflammatory state that triggers symptoms of PASC.

Sensitive Immunodetection of Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern 501Y.V2 and 501Y.V1.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may influence the effectiveness of existing laboratory diagnostics. In the current study we determined whether the British (20I/501Y.V1) and South African (20H/501Y.V2) SARS-CoV-2 variants of concern are detected with an in-house S1-based antigen detection assay, analyzing spiked pools of quantitative reverse-transcription polymerase chain reaction-negative nasopharyngeal swab specimens. The assay, combining 4 monoclonal antibodies, allowed sensitive detection of both the wild type and the variants of concern, despite accumulation of several mutations in the variants' S1 region-results suggesting that this combination, targeting distinct epitopes, enables both specificity and the universality.

Emerging SARS-CoV-2 variants of concern and potential intervention approaches.

The major variant of concerns (VOCs) have shared mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins, mostly on the S1 unit and resulted in higher transmissibility rate and affect viral virulence and clinical outcome. The spike protein mutations and other non-structural protein mutations in the VOCs may lead to escape approved vaccinations in certain extend. We will discuss these VOC mutations and discuss the need for combination therapeutic strategies targeting viral cycle and immune host responses.

Porous Au@Pt nanoparticles with superior peroxidase-like activity for colorimetric detection of spike protein of SARS-CoV-2.

The development of colorimetric assays for rapid and accurate diagnosis of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is of practical importance for point-of-care (POC) testing. Here we report the colorimetric detection of spike (S1) protein of SARS-CoV-2 based on excellent peroxidase-like activity of Au@Pt nanoparticles, with merits of rapidness, easy operation, and high sensitivity. The Au@Pt NPs were fabricated by a facile seed-mediated growth approach, in which spherical Au NPs were premade as seeds, followed by the Pt growth on Au seeds, producing uniform, monodispersed and porous Au@Pt core-shell NPs. The as-obtained Au@Pt NPs showed a remarkable enhancement in the peroxidase-mimic catalysis, which well abided by the typical Michaelis-Menten theory. The enhanced catalysis of Au@Pt NPs was ascribed to the porous nanostructure and formed electron-rich Pt shells, which enabled the catalytic pathway to switch from hydroxyl radical generation to electron transfer process. On a basis of these findings, a colorimetric assay of spike (S1) protein of SARS-CoV-2 was established, with a linear detection range of 10-100 ng mL-1 of protein concentration and a low limit of detection (LOD) of 11 ng mL-1. The work presents a novel strategy for diagnosis of COVID-19 based on metallic nanozyme-catalysis.

Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies.

In December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

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.

COVID-19 serological survey using micro blood sampling.

During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for more than 9 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.

[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.