ace2 buffer  (Sino Biological)

Journal: eLife

Article Title: Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein

doi: 10.7554/eLife.88387

Figure Lengend Snippet: ( A ) Characterization of S1-specific monoclonal antibodies obtained from No. 1 mouse. The heatmap represents the relative intensity of antibody binding to receptor binding domains (RBDs) and blocking of the RBD-angiotensin-converting enzyme-2 (ACE2) interaction. Blue (0–25%), green (25–50%), orange (50–75%), and red (>75%). N-terminal domain (NTD) binding was considered positive (+) when the OD at 405 nm was >0.3 after the background was subtracted. Neutralizing activity was considered positive (+) when the antibody suppressed Wuhan pseudotyped virus infection by 50%. The figure reports values from a single experiment. UN, antibody type not determined. ND, antibody activity not determined. ( B ) Maximum-likelihood phylogenetic tree of the VH and VL chains of the S1-specific antibodies. Different colored fonts indicate antibodies obtained from the nose (red), spleen (blue), and lung (green). Bands on the outer ring indicate antibody groups. The color of the ring indicates antibody types: Type 1 (red), Type 2 (orange), Type 3 (green), Type 4 (blue), and Type 5 (gray). The antibody group is defined as clones using the same V-(D)-J usage and having an overall sequence identity of at least 95% from the signal peptide to framework 4 (FR4). The prefixes N, S, and L in the antibody clone numbers refer to antibodies derived from the nose, spleen, and lung, respectively. The suffixes A, G, and K in the antibody clone numbers refer to alpha, gamma, and kappa chains, respectively. ( C ) Nucleotide sequence alignment of VH and VL genes in the G2 and G3 antibodies from No. 1 mouse. The VH and VL sequences from the beginning of the signal peptide through the end of FR4 are shown as horizontal lines. Nucleotide changes relative to S632A and N109A are depicted as vertical bars across the horizontal lines. Different colored fonts indicate antibodies derived from the nose (red) and spleen (blue). Antibody phylogenetic trees based on VH/VK paired sequences are depicted. Gray circles represent the hypothetical germline configuration. White circles represent hypothetical ancestors. Colors indicate nasal (red) and splenic (blue) antibodies. Circles and squares indicate IgA and IgG, respectively.

Article Snippet: Recombinant protein , Biotinylated Human ACE2 , Sino Biological , 10108-H08H-B , .

Techniques: Bioprocessing, Binding Assay, Blocking Assay, Activity Assay, Virus, Infection, Clone Assay, Sequencing, Derivative Assay

Journal: eLife

Article Title: Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein

doi: 10.7554/eLife.88387

Figure Lengend Snippet: The VH and VL sequences from the beginning of the signal peptide through the end of FR4 are shown as horizontal lines. Nucleotide changes relative to N220A are depicted as vertical bars across the horizontal lines. Different colored fonts indicate antibodies derived from the nose (red) and spleen (blue). Antibody phylogenetic trees based on VH/VK paired sequences are depicted. Gray circles represent the hypothetical germline configuration. White circles represent hypothetical ancestors. Colors indicate nasal (red), splenic (blue), and lung (green) antibodies. Circles and squares indicate IgA and IgG, respectively. The antibodies in the G1-G4 clusters were categorized as Type 1. The G1 antibody appeared only once. This clone displayed potent binding to the Wuhan, Kappa, and Delta RBD, and moderately to the Beta RBD. It also blocked ACE2 binding to all RBDs except the Beta RBD. The antibodies in G2-4 clusters bound to Wuhan and Delta RBD but not Beta and Kappa RBD. They were able to block ACE2 binding to the RBD of Wuhan and Delta but not to the RBD of Beta and Kappa. The antibodies in G5-G9 clusters were categorized as Type 2, bound to all four RBD variants, and uniformly blocked ACE2 binding to the RBDs of all four strains. G10 antibodies were classified as Type 3, accounting for 38% of the nasal IgA repertoire. They bound to the RBDs of all four strains but did not prevent ACE2 binding to the RBDs of any of the strains. The G11 antibody, classified as Type 4, was an anti-NTD antibody that did not prevent ACE2 binding to the RBDs of any strains.

Article Snippet: Recombinant protein , Biotinylated Human ACE2 , Sino Biological , 10108-H08H-B , .

Techniques: Derivative Assay, Binding Assay, Blocking Assay

Journal: eLife

Article Title: Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein

doi: 10.7554/eLife.88387

Figure Lengend Snippet: ( A ) Graphs of the competitive enzyme-linked immunosorbent assay (ELISA) results showing the binding of biotinylated angiotensin-converting enzyme-2 (ACE2) to the immobilized Wuhan, Delta, or Omicron receptor binding domain (RBD) in the presence of antibodies. The results are expressed as the mean ± SD of three technical replicates. The figure reports values from a single experiment. The IC 50 values of the indicated antibodies that inhibit the RBD-ACE2 interaction are shown in the diagrams. ( B ) Comparison of neutralization activity between M-IgA and secretory IgA (S-IgA) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudotyped viruses. Neutralization curves of the indicated antibody against pseudotyped viruses bearing spike proteins of Wuhan, Delta, or Omicron are shown. Pseudotyped viruses preincubated with antibodies at the indicated concentrations were used to infect VeroE6 cells, and luciferase activities in cell lysates were measured at 20 hr post-transduction to calculate infection (%) relative to nonantibody-treated controls. The results are expressed as the mean ± SD of three technical replicates. The NT 50 values of the indicated antibodies are shown in the diagrams. Antibodies that did not reach >70% inhibition at the highest concentration tested were listed as data not determined (ND). ( C ) Comparison of neutralization potential between M-IgA and S-IgA against authentic SARS-CoV-2 BA.1. The neutralizing potential of the antibody was determined using a reverse transcription polymerase chain reaction (RT-PCR)-based SARS-CoV-2 neutralization assay. VeroE6 cells preincubated with authentic SARS-CoV-2 BA.1 virus were incubated with the indicated antibodies at various concentrations. The virus in the cell culture medium was measured at 48 hr post-transduction to calculate infection (%) relative to non-antibody-treated controls. The results are expressed as the mean ± SD of three technical replicates. The NT 50 values of the indicated antibodies are shown in the diagrams. Antibodies that did not reach >50% inhibition at the highest concentration tested are listed as ND. **p<0.01.

Article Snippet: Recombinant protein , Biotinylated Human ACE2 , Sino Biological , 10108-H08H-B , .

Techniques: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Neutralization, Activity Assay, Luciferase, Transduction, Infection, Inhibition, Concentration Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Virus, Incubation, Cell Culture

Journal: eLife

Article Title: Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein

doi: 10.7554/eLife.88387

Figure Lengend Snippet: The affinity, angiotensin-converting enzyme-2 (ACE2) inhibitory activity, and the in vitro neutralizing activity of the indicated antibodies are illustrated in a 3D scatter plot.

Article Snippet: Recombinant protein , Biotinylated Human ACE2 , Sino Biological , 10108-H08H-B , .

Techniques: Activity Assay, In Vitro