“…The mAb was purified using a HiTrap Protein G column and subjected to surface plasmon resonance analysis to determine the dissociation constant ( K d value) to the epitope peptide (the GTH peptide). Interestingly, the K d value was 1.1 × 10 –10 M, which was a relatively low K d value compared with that of previously reported antibodies 31 , 32 . Figure 3 Hybridomas producing antibodies against GTH peptide, virus (WT), and recombinant S1 + S2 spike protein.…”
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prolonged the duration of the pandemic because of the continuous emergence of new variant strains. The emergence of these mutant strains makes it difficult to detect the virus with the existing antibodies; thus, the development of novel antibodies that can target both the variants as well as the original strain is necessary. In this study, we generated a high-affinity monoclonal antibody (5G2) against the highly conserved region of the SARS-CoV-2 spike protein to detect the protein variants. Moreover, we generated its single-chain variable antibody fragment (sc5G2). The sc5G2 expressed in mammalian and bacterial cells detected the spike protein of the original SARS-CoV-2 and variant strains. The resulting sc5G2 will be a useful tool to detect the original SARS-CoV-2 and variant strains.
“…The mAb was purified using a HiTrap Protein G column and subjected to surface plasmon resonance analysis to determine the dissociation constant ( K d value) to the epitope peptide (the GTH peptide). Interestingly, the K d value was 1.1 × 10 –10 M, which was a relatively low K d value compared with that of previously reported antibodies 31 , 32 . Figure 3 Hybridomas producing antibodies against GTH peptide, virus (WT), and recombinant S1 + S2 spike protein.…”
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prolonged the duration of the pandemic because of the continuous emergence of new variant strains. The emergence of these mutant strains makes it difficult to detect the virus with the existing antibodies; thus, the development of novel antibodies that can target both the variants as well as the original strain is necessary. In this study, we generated a high-affinity monoclonal antibody (5G2) against the highly conserved region of the SARS-CoV-2 spike protein to detect the protein variants. Moreover, we generated its single-chain variable antibody fragment (sc5G2). The sc5G2 expressed in mammalian and bacterial cells detected the spike protein of the original SARS-CoV-2 and variant strains. The resulting sc5G2 will be a useful tool to detect the original SARS-CoV-2 and variant strains.
“…Human serum samples included pre-pandemic samples collected before 2020 and samples from individuals who were only vaccinated, infected and vaccinated, or only infected. These samples have been described and evaluated in previous studies [23][24][25]. Tiger sera were collected in 2021 by the Mexico-United States Commission for the Prevention of Foot-and-Mouth Disease and Other Exotic Animal Diseases (CPA) of the National Service for Disease Control, Safety, and Agri-food Quality (SENASICA) in Mexico City as part of the passive surveillance of SARS-CoV-2 (Case number: CPA-06880-21).…”
SARS-CoV-2 infects humans and a broad spectrum of animal species, such as pets, zoo animals, and nondomestic animals. Monitoring infection in animals is important in terms of the risk of interspecies transmission and the emergence of new viral variants. Economical, fast, efficient, and sensitive diagnostic tests are needed to analyze animal infection. Double-antigen sandwich ELISA has the advantage of being multispecies and can be used for detecting infections caused by pathogens that infect several animal hosts. This study aimed to develop a double-antigen sandwich ELISA using two SARS-CoV-2 proteins, N and RBD. We compared its performance, when using these proteins separately, with an indirect ELISA and with a surrogate virus neutralization test. Positive and negative controls from a cat population (n = 31) were evaluated to compare all of the tests. After confirming that double-antigen sandwich ELISA with both RBD and N proteins had the best performance (AUC= 88%), the cutoff was adjusted using positive and negative samples from cats, humans (n = 32) and guinea pigs (n = 3). The use of samples from tigers (n = 2) and rats (n = 51) showed good agreement with the results previously obtained using the microneutralization test. Additionally, a cohort of samples from dogs with unknown infection status was evaluated. These results show that using two SARS-CoV-2 proteins in the double-antigen sandwich ELISA increases its performance and turns it into a valuable assay with which to monitor previous infection caused by SARS-CoV-2 in different animal species.
Here, we performed single‐cell RNA sequencing of S1 and receptor binding domain protein‐specific B cells from convalescent COVID‐19 patients with different clinical manifestations. This study aimed to evaluate the role and developmental pathway of atypical memory B cells (MBCs) in response to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. The results revealed a proinflammatory signature across B cell subsets associated with disease severity, as evidenced by the upregulation of genes such as GADD45B, MAP3K8, and NFKBIA in critical and severe individuals. Furthermore, the analysis of atypical MBCs suggested a developmental pathway similar to that of conventional MBCs through germinal centers, as indicated by the expression of several genes involved in germinal center processes, including CXCR4, CXCR5, BCL2, and MYC. Additionally, the upregulation of genes characteristic of the immune response in COVID‐19, such as ZFP36 and DUSP1, suggested that the differentiation and activation of atypical MBCs may be influenced by exposure to SARS‐CoV‐2 and that these genes may contribute to the immune response for COVID‐19 recovery. Our study contributes to a better understanding of atypical MBCs in COVID‐19 and the role of other B cell subsets across different clinical manifestations.
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