Antibody detection of SARS-CoV spike and nucleocapsid protein (original) (raw)
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Clinical and Vaccine Immunology, 2006
To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any crossreactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.
Scientific Reports
SARS-CoV-2 contains four structural proteins, two of which, the spike and nucleocapsid, are commonly used for the standardization of novel methods for antibody detection; however, some limitations in their use have been observed due to the homology of this virus with other phylogenetically-related viruses. We performed in silico analysis to search for novel immunogenic and antigenic peptides. A total of twenty-five peptides were preliminarily selected, located in the 3D structure of both proteins. Finally, eight peptides were selected: one located in the N protein and seven in the S1 domain of the spike protein. Additionally, the localization of selected peptides in 2D structures and possible changes in the sequences of these peptides in SARS-CoV-2 variants of concern were analyzed. All peptides were synthetized in MAP8 format, and recombinant S (trimer and RBD) and N proteins were used as antigens to search for antibodies in serum samples derived from COVID-19 patients, and for ant...
Pathogens and Disease, 2022
Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor-binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Our results showed that while reactivity of patients' sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (P < 0.001), no significant differences were observed for reactivity of patients' sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassi...
Brazilian Journal of Microbiology
Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.
European Journal of Immunology, 2021
Monoclonal antibodies (mAbs) are used clinically in the diagnosis and therapy of many diseases. There is a need for the use of high‐affinity antibodies specific to SARS‐CoV‐2 in the rapid and accurate diagnosis of the global COVID‐19 outbreak. In this study, mouse mAbs against the receptor‐binding domain (RBD) and S1 subunit of the spike protein which are structural proteins of SARS‐CoV‐2, were developed using hybridoma technology. BALB/c mice were immunized with the recombinant S1 and RBD proteins. Spleen cells of the immunized mice which have high antibody response were fused with mouse myeloma cells (F0; ATTC CRL‐1646) in the presence of polyethylene glycol to obtain hybrid cells. Selection and subcloning processes were performed for the hybridoma cells which have high‐level antibody response, after that, the specificity of the developed mAbs to SARS‐CoV‐2 proteins was demonstrated by enzyme‐linked immunosorbent assay (ELISA) and western blotting methods. Therewithal, the isotype of the developed antibodies was determined as IgG1 and the productivity of purification processes was compared by using Protein A and Protein G columns. As a conclusion 2 mAbs were obtained, one of them was only specific to S1 protein, but the other one could detect RBD protein at the same level as S1 protein even was immunized with S1 protein. Thanks to its high response to RBD, a neutralizing antibody candidate was obtained for potential drug development. At the same time, purified mAbs began to be used in the development of antibody‐based detection systems for SARS‐CoV‐2 diagnosis.
Journal of Immunological Methods, 2008
Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402-622) and the N4 fragment (aa 220-336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS-CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS-CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS-CoV, these assays will be a useful tool to trace the origin and transmission of SARS-CoV and to minimise the risk of animal-to-human transmission.
Recombinant protein-based ELISA and immuno-cytochemical assay for the diagnosis of SARS
Journal of Medical Virology, 2005
A new Coronavirus (SARS-CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in-house ELISA based on SARS-CoV recombinant antigens was developed. The SARS-CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine-tagged proteins. Six reference sera from SARS patients were used to detect virus-specific IgG in an ELISA using each recombinant protein as coating antigen. High-titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non-SARS, low-tract respiratory infections. Specific hyper-immune sera to SARS-CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS-CoV infected and uninfected cells.
Nucleocapsid protein of SARS‐CoV‐2 is a potential target for developing new generation of vaccine
Journal of Clinical Laboratory Analysis
BackgroundSARS‐CoV‐2 has spread worldwide causing more than 400 million people with virus infections since early 2020. Currently, the existing vaccines targeting the spike glycoprotein (S protein) of SARS‐CoV‐2 are facing great challenge from the infection of SARS‐CoV‐2 virus and its multiple S protein variants. Thus, we need to develop a new generation of vaccines to prevent infection of the SARS‐CoV‐2 variants. Compared with the S protein, the nucleocapsid protein (N protein) of SARS‐CoV‐2 is more conservative and less mutations, which also plays a vital role in viral infection. Therefore, the N protein may have the great potential for developing new vaccines.MethodsThe N protein of SARS‐CoV‐2 was recombinantly expressed and purified in Escherichia coli. Western Blot and ELISA assays were used to demonstrate the immunoreactivity of the recombinant N protein with the serum of 22 COVID‐19 patients. We investigated further the response of the specific serum antibodies and cytokine pr...
2021
Seroconversion to SARS-CoV-2 has been widely studied to evaluate infection spreading for epidemiological purpose, or even in studies of protective immunity in convalescent or vaccinated individuals. The viral particle has an envelope harboring the spike glycoprotein, which can be used as an antigen for assay development, to detect antiviral antibodies to SARS-CoV-2. Since several vaccines encode a spike subunit, the full length spike-based immunoassay should be a universal tool to evaluate seroconversion. In this manuscript, we propose a low-cost ELISA that can be used to detect antiviral IgG to SARS-CoV-2 in human serum.