Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Serological survey of SARS-CoV-2 in companion animals in China.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can be transmitted from human to companion animals. The national wide serological surveillance against SARS-CoV-2 was conducted among pet animals, mainly in cats and dogs, 1 year after the first outbreak of COVID-19 in China. All sera were tested for SARS-CoV-2 IgG antibodies using an indirect enzyme linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of spike protein. This late survey takes advantage of the short duration of the serological response in these animals to track recent episode of transmission. A total of 20,592 blood samples were obtained from 25 provinces across 7 geographical regions. The overall seroprevalence of SARS-CoV-2 infections in cats was 0.015% (2/13397; 95% confidence intervals (CI): 0.0, 0.1). The virus infections in cats were only detected in Central (Hubei, 0.375%) and Eastern China (Zhejiang, 0.087%) with a seroprevalence estimated at 0.090 and 0.020%, respectively. In dogs, the seroprevalence of SARS-CoV-2 infections was 0.014% (1/7159; 95% CI: 0.0, 0.1) in the entire nation, seropositive samples were limited to Beijing (0.070%) of Northern China with a prevalence of 0.054%. No seropositive cases were discovered in other geographic regions, nor in other companion animals analyzed in this study. These data reveal the circulation of SARS-CoV-2 in companion animals, although transmission of the virus to domestic cats and dogs is low in China, continuous monitoring is helpful for the better understand of the virus transmission status and the effect on animals.

Spy&IAC enables specific capture of SpyTagged proteins for rapid assembly of plug-and-display nanoparticle vaccines.

From modular vaccine production to protein assembly on nanoparticles, the SpyCatcher/SpyTag system provides a convenient plug-and-display procedure. Here, we established a general-purpose immunoaffinity chromatography (IAC) method for SpyTagged proteins (Spy&IAC). SpyTags are displayed on the surface of nanoparticles to induce high-affinity monoclonal antibodies, allowing the specific capture of the target protein. Taking the key core antigenic regions of two coronaviruses that are currently more threatened in the field of human and animal diseases, the nucleocapsid (N) protein of SARS-CoV-2 and the COE protein of porcine epidemic diarrhea virus (PEDV) as model proteins, a purification model with SpyTag at the N-terminal or C-terminal expressed in E. coli or mammalian cells was constructed. After the efficient elution of Spy&IAC, the final yield of several proteins is about 3.5-15 mg/L culture, and the protein purity is above 90 %. Purification also preserves the assembly function and immunogenicity of the protein to support subsequent modular assembly and immunization programs. This strategy provides a general tool for the efficient purification of SpyTagged proteins from different expression sources and different tag positions, enabling the production of modular vaccines at lower cost and in a shorter time, which will prepare the public health field for potential pandemic threats.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL−1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL−1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Identification of three conserved linear B cell epitopes on the SARS-CoV-2 spike protein.

Spike (S) glycoprotein is the most significant structural protein of SARS-CoV-2 and a key target for neutralizing antibodies. In light of the on-going SARS-CoV-2 pandemic, identification and screening of epitopes of spike glycoproteins will provide vital progress in the development of sensitive and specific diagnostic tools. In the present study, NTD, RBD, and S2 genes were inserted into the pcDNA3.1(+) vector and designed with N-terminal 6× His-tag for fusion expression in HEK293F cells by transient transfection. Six monoclonal antibodies (4G, 9E, 4B, 7D, 8F, and 3D) were prepared using the expressed proteins by cell fusion technique. The characterization of mAbs was performed by indirect -ELISA, western blot, and IFA. We designed 49 overlapping synthesized peptides that cover the extracellular region of S protein in which 6 amino acid residues were offset between adjacent (S1-S49). Peptides S12, S19, and S49 were identified as the immunodominant epitope regions by the mAbs. These regions were further truncated and the peptides S12.2 286TDAVDCALDPLS297, S19.2 464FERDISTEIYQA475, and S49.4 1202ELGKYEQYIKWP1213 were identified as B- cell linear epitopes for the first time. Alanine scans showed that the D467, I468, E471, Q474, and A475 of the epitope S19.2 and K1205, Q1208, and Y1209 of the epitope S49.4 were the core sites involved in the mAbs binding. The multiple sequence alignment analysis showed that these three epitopes were highly conserved among the variants of concern (VOCs) and variants of interest (VOIs). Taken together, the findings provide a potential material for rapid diagnosis methods of COVID-19.

Differential diagnosis of the infection caused by wild-type or CD2v-deleted ASFV strains by quantum dots-based immunochromatographic assay.

African swine fever (ASF), a highly contagious and lethal disease, poses a tremendous threat and burden to the swine industry worldwide. Lack of available vaccines or treatments leaves rapid diagnosis as the key tool to control the disease. Quantum dots (QDs) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In this study, we developed a quantum dots-based fluorescent immunochromatographic assay (QDs-FICA) using CD2v as the diagnosis antigen to detect ASFV antibodies. The titre of the test strip was 1 : 5·12 × 105 . In addition, the strip was highly specific to anti-ASFV serum and had no cross-reaction with CSFV, PPV, PRRSV, PCV-2, PRV and FMDV. Moreover, a comparative test of 71 clinical samples showed that the coincidence rate was 85·92% between the test strip and the commercial ELISA kit (coated with p30, p62 and p72). The QDs-FICA can be used to detect ASFV antibodies, which is meaningful for the surveillance, control and purification of ASF.

A Universal Fluorescent Immunochromatography Assay Based on Quantum Dot Nanoparticles for the Rapid Detection of Specific Antibodies against SARS-CoV-2 Nucleocapsid Protein.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogenic agent leading to COVID-19. Due to high speed of transmission and mutation rates, universal diagnosis and appropriate prevention are still urgently needed. The nucleocapsid protein of SARS-CoV-2 is considered more conserved than spike proteins and is abundant during the virus' life cycle, making it suitable for diagnostic applications. Here, we designed and developed a fluorescent immunochromatography assay (FICA) for the rapid detection of SARS-CoV-2-specific antibodies using ZnCdSe/ZnS QDs-conjugated nucleocapsid (N) proteins as probes. The nucleocapsid protein was expressed in E.coli and purified via Ni-NTA affinity chromatography with considerable concentration (0.762 mg/mL) and a purity of more than 90%, which could bind to specific antibodies and the complex could be captured by Staphylococcal protein A (SPA) with fluorescence displayed. After the optimization of coupling and detecting conditions, the limit of detection was determined to be 1:1.024 × 105 with an IgG concentration of 48.84 ng/mL with good specificity shown to antibodies against other zoonotic coronaviruses and respiratory infection-related viruses (n = 5). The universal fluorescent immunochromatography assay simplified operation processes in one step, which could be used for the point of care detection of SARS-CoV-2-specific antibodies. Moreover, it was also considered as an efficient tool for the serological screening of potential susceptible animals and for monitoring the expansion of virus host ranges.

Screening and identification of B cell epitope of the nucleocapsid protein in SARS-CoV-2 using the monoclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease (COVID-19). It is confirmed that nucleocapsid (N) protein is closely related to viral pathogenesis, modulation of host immune response, RNA transcription, and replication and virus packaging. Therefore, the N protein is a preponderant antigen target for virus detection. The codon-optimized N gene was designed according to the encoding characteristics of insect cells and inserted into pFastBacTM1 vector with 6 × His-tag-fused N protein for expression in insect sf21 cells. Six anti-N mAbs (4G3, 5B3, 12B6, 18C7-A2, 21H10-A3, 21H10-E9) were prepared by recombinant N protein. The mAbs showed high titers, antibody affinity, and reactivity with the SARS-CoV-2 N protein. Then, fourteen overlapped peptides that covered the intact N protein were synthesized (N1-N14). Peptide N14 was identified as the main linear B-cell epitope region via peptide-ELISA and dot-blot assay, and this region was truncated gradually until mapping the peptide 401-DFSKQLQQ-408. Simultaneously, compared with the sequence of variants of concern (VOCs) and variants of interest (VOIs) strains among the several countries, epitope 401-DFSKQLQQ-408 is very conservative among them. The findings provide new guidance for the design and detection of COVID-19 targets. KEY POINTS: • The N protein was optimized according to the insect cell codon preference and was highly expressed. • The monoclonal antibodies prepared in this study were shown high antibody titers and high affinity. • Monoclonal antibodies were used to map the epitope 401-408 amino acids of N protein for the first time in this study.

Epitope Profiling Reveals the Critical Antigenic Determinants in SARS-CoV-2 RBD-Based Antigen.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is a huge public health crisis for the globe. The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in viral infection and serves as a major target for developing neutralizing antibodies. In this study, the antibody response to the RBD of SARS-CoV-2 S protein was analyzed by a panel of sera from animals immunized with RBD-based antigens and four linear B-cell epitope peptides (R345, R405, R450 and R465) were revealed. The immunogenicity of three immunodominant peptides (R345, R405, R465) was further accessed by peptide immunization in mice, and all of them could induced potent antibody response to SARS-CoV-2 S protein, indicating that the three determinants in the RBD were immunogenic. We further generated and characterized monoclonal antibodies (15G9, 12C10 and 10D2) binding to these epitope peptides, and finely mapped the three immunodominant epitopes using the corresponding antibodies. Neutralization assays showed that all three monoclonal antibodies had neutralization activity. Results from IFA and western blotting showed that 12C10 was a cross-reactive antibody against both of SARS-CoV-2 and SARS-CoV. Results from conservative and structural analysis showed that 350VYAWN354 was a highly conserved epitope and exposed on the surface of SARS-CoV-2 S trimer, whereas 473YQAGSTP479 located in the receptor binding motif (RBM) was variable among different SARS-CoV-2 strains. 407VRQIAP412 was a highly conserved, but cryptic epitope shared between SARS-CoV-2 and SARS-CoV. These findings provide important information for understanding the humoral antibody response to the RBD of SARS-CoV-2 S protein and may facilitate further efforts to design SARS-CoV-2 vaccines and the target of COVID-19 diagnostic.

Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein.

The outbreak and worldwide pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have a significant impact on global economy and human health. In order to reduce the disease spread, 16 monoclonal antibodies (McAbs) again SARS-CoV-2 were generated by immunized mice with the spike protein receptor binding domain (RBD), which was expressed in Chinese hamster ovary cell (CHO). A colloidal gold-based immunochromatographic strip was developed with two McAbs to detect SARS-CoV-2 spike protein, which can play a potential role in monitoring vaccine quality. The strip is highly specific, detecting only SARS-CoV-2 spike protein, and does not show any non-specific reactions with syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and other coronavirus and influenza viruses. The strip detected subunit vaccine in our laboratory with a detection limit of spike protein of 62.5 ng/mL. This strip provides an effective method in monitoring vaccine quality by detecting the antigen content of spike protein.