Journal: Biosensors & Bioelectronics

Article Title: Rapid biosensing SARS-CoV-2 antibodies in vaccinated healthy donors

doi: 10.1016/j.bios.2022.114054

Figure Lengend Snippet: The optical fiber structure and the general principles of optical biosensors for SARS-CoV-2 antibody detection on the fiber. (A) The three-dimensional structure, parameters, and materials of the optical fibers. (B) Principle of the FO-BLI biosensor for rapid detection of BAbs via a sandwich binding format. The BAbs are specific towards RBD, full length protein S-ECD and RBD mutation N501Y. (C) Principle of the FO-BLI biosensor for rapid detection of NAbs via a competitive binding format. In this bioassay, ( i ) when no NAbs exist, the binding between RBD-HRP and hACE2 contributes to high detection signals, while ( ii ) the presence of NAbs blocks HRP-conjugated RBD from binding to immobilized hACE2 protein. In both bioassays, DAB significantly amplified the optical signals.

Article Snippet: The biotinylated SARS-CoV-2 spike RBD-His recombinant protein, SARS-CoV-2 spike RBD(N501Y)-His recombinant protein, SARS-CoV-2 spike ECD (S1+S2)-His recombinant protein, SARS-CoV-2 spike pseudovirus (containing 10 10 virus copies/mL and 860 ng/mL of SARS-CoV-2-S1), human recombinant ACE2 protein (biotinylated), rabbit SARS-CoV-2 spike neutralizing antibody, SARS-CoV-2 spike RBD recombinant protein (HRP labeled, customized), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody Titer Assay Kit, and SARS-CoV-2 (2019-nCoV) Inhibitor Screening ELISA Kit were purchased from Sino Biologicals (Beijing, China).

Techniques: Binding Assay, Mutagenesis, Amplification

Journal: Biosensors & Bioelectronics

Article Title: Rapid biosensing SARS-CoV-2 antibodies in vaccinated healthy donors

doi: 10.1016/j.bios.2022.114054

Figure Lengend Snippet: Assay conditions of the established FO-BLI biosensors for both SARS-CoV-2 BAbs and SARS-CoV-2 NAbs rapid detection in buffer and serum.

Article Snippet: The biotinylated SARS-CoV-2 spike RBD-His recombinant protein, SARS-CoV-2 spike RBD(N501Y)-His recombinant protein, SARS-CoV-2 spike ECD (S1+S2)-His recombinant protein, SARS-CoV-2 spike pseudovirus (containing 10 10 virus copies/mL and 860 ng/mL of SARS-CoV-2-S1), human recombinant ACE2 protein (biotinylated), rabbit SARS-CoV-2 spike neutralizing antibody, SARS-CoV-2 spike RBD recombinant protein (HRP labeled, customized), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody Titer Assay Kit, and SARS-CoV-2 (2019-nCoV) Inhibitor Screening ELISA Kit were purchased from Sino Biologicals (Beijing, China).

Techniques: Serum Assay, Incubation

Journal: Biosensors & Bioelectronics

Article Title: Rapid biosensing SARS-CoV-2 antibodies in vaccinated healthy donors

doi: 10.1016/j.bios.2022.114054

Figure Lengend Snippet: (A) The binding strength trends obtained when the secondary antibody-HRP conjugate, in a series of dilutions from 1000- to 6000-fold, reacts with DAB enhancer for 2 min and 3 min, respectively. (B) Representative non-linear binding profiles of the Anti-RBD BAbs biosensor after signal enhancement, with the assay calibrator MA-RBD S309 ranged from 0 to 500 ng/mL in buffer. (C) Standard binding curves of the FO-BLI biosensors for anti-RBD BAbs, anti-S-ECD and anti-RBD N501Y in 100-fold diluted serum, respectively. (D) Representative non-linear inhibition profiles of the SARS-CoV-2 NAbs biosensor after signal enhancement, with the assay calibrator r-NAb-RBD ranged from 0 to 5000 ng/mL in buffer. (E) Standard inhibition curves of the SARS-CoV-2 NAbs biosensor in buffer and 100-fold diluted sera, with the assay calibrator r-NAb-RBD ranged from 10 to 5000 ng/mL. A range of 10–250 ng/mL r-NAb-RBD in spiked sera produced a linear-regression calibration curve (inserted). Each sample was measured in duplicate.

Article Snippet: The biotinylated SARS-CoV-2 spike RBD-His recombinant protein, SARS-CoV-2 spike RBD(N501Y)-His recombinant protein, SARS-CoV-2 spike ECD (S1+S2)-His recombinant protein, SARS-CoV-2 spike pseudovirus (containing 10 10 virus copies/mL and 860 ng/mL of SARS-CoV-2-S1), human recombinant ACE2 protein (biotinylated), rabbit SARS-CoV-2 spike neutralizing antibody, SARS-CoV-2 spike RBD recombinant protein (HRP labeled, customized), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody Titer Assay Kit, and SARS-CoV-2 (2019-nCoV) Inhibitor Screening ELISA Kit were purchased from Sino Biologicals (Beijing, China).

Techniques: Binding Assay, Inhibition, Produced

Journal: Biosensors & Bioelectronics

Article Title: Rapid biosensing SARS-CoV-2 antibodies in vaccinated healthy donors

doi: 10.1016/j.bios.2022.114054

Figure Lengend Snippet: Inhibition of three monoclonal antibodies and two aptamers against SARS-CoV-2 RBD as measured using both the FO-BLI NAb biosensor (blue lines) and a commercial inhibitor screening ELISA kit (black lines). (A) The NAb assay calibrator r-NAb-RBD was determined to have an IC50 of 0.87 nM, close to the IC50 of 0.59 nM measured by the kit as reported in the product's official document. (B) Inhibition of MA-S2, a non-neutralizing antibody against S2; dashed line indicates a theoretical 0% neutralizing capacity. (C) Inhibition of MA-RBD S309, the SARS-CoV-2 BAb biosensors calibrator; both assays revealed limited inhibition for this antibody. (D) Inhibition of the literature-reported RBD-specific aptamer CoV-2-RBD-1C; both assays reported non-neutralization for it. (E) Inhibition of the literature-reported RBD-specific aptamer CoV-2-RBD-4C; both assays reported limited neutralization for it. (F) Correlation and agreement between inhibition values obtained from both methods; data generally aligned with each other.

Article Snippet: The biotinylated SARS-CoV-2 spike RBD-His recombinant protein, SARS-CoV-2 spike RBD(N501Y)-His recombinant protein, SARS-CoV-2 spike ECD (S1+S2)-His recombinant protein, SARS-CoV-2 spike pseudovirus (containing 10 10 virus copies/mL and 860 ng/mL of SARS-CoV-2-S1), human recombinant ACE2 protein (biotinylated), rabbit SARS-CoV-2 spike neutralizing antibody, SARS-CoV-2 spike RBD recombinant protein (HRP labeled, customized), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody Titer Assay Kit, and SARS-CoV-2 (2019-nCoV) Inhibitor Screening ELISA Kit were purchased from Sino Biologicals (Beijing, China).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Biosensors & Bioelectronics

Article Title: Rapid biosensing SARS-CoV-2 antibodies in vaccinated healthy donors

doi: 10.1016/j.bios.2022.114054

Figure Lengend Snippet: Detection of anti-RBD, anti-S-ECD BAbs and anti-SARS-CoV-2 NAbs in sera of 16 individual vaccine-injected healthy donors (V-HD1 to V-HD16; V-HD9-2 is the second sample from V-HD9). (A) Prevalence of the anti-RBD antibodies in the 16 donors measured using a commercial RBD antibody titer assay kit. Dashed line indicates the kit's cut-off for detection at 0.19 in absorbance. (B i ) Measurements of anti-RBD antibodies in the 16 donors using the Anti-RBD BAb biosensor. Statistical comparison between the anti-RBD BAb positive and negative samples is listed on the right side. (B ii ) Measurements of anti-S-ECD antibodies in the 16 donors using the Anti-S-ECD BAb biosensor. Statistical comparison between the anti-S-ECD BAb positive and negative samples is listed on the right side. (B iii ) Measurements of anti-SARS-CoV-2 NAbs in the 16 donors using the Anti-SARS-CoV-2 NAb biosensor. Statistical comparison between the NAbs positive and negative samples is listed on the right side. Dashed lines represent the Cut-off D (Cut-off for Detection) and Cut-off Q (Cut-off for Quantification) of the corresponding assay, all listed in . Error bars were from duplicate measurements. (C) Inhibition of hACE2−RBD binding by the two screened high-quality NAb serum antibodies: ( i ) sample of V-HD9 and ( ii ) sample of V-HD9-2. Inhibition was calculated in the presence of increasing concentrations of S-ECD (0, 4, 10 and 20 μg/mL) and spike pseudovirus (10 × and stock) and assessed using the SARS-CoV-2 NAb biosensor (n = 1).

Article Snippet: The biotinylated SARS-CoV-2 spike RBD-His recombinant protein, SARS-CoV-2 spike RBD(N501Y)-His recombinant protein, SARS-CoV-2 spike ECD (S1+S2)-His recombinant protein, SARS-CoV-2 spike pseudovirus (containing 10 10 virus copies/mL and 860 ng/mL of SARS-CoV-2-S1), human recombinant ACE2 protein (biotinylated), rabbit SARS-CoV-2 spike neutralizing antibody, SARS-CoV-2 spike RBD recombinant protein (HRP labeled, customized), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody Titer Assay Kit, and SARS-CoV-2 (2019-nCoV) Inhibitor Screening ELISA Kit were purchased from Sino Biologicals (Beijing, China).

Techniques: Injection, Titer Assay, Inhibition, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Mutations on the spike glycoprotein of SARS-CoV-2 Omicron subvariants. Residues that are not identical among Omicron subvariants are colored red. b , Workflow to generate cryo-EM structure of BA.2, BA.3, BA.2.13, BA.2.12.1, BA.4/5 spike glycoprotein trimer with S6P and R683A, R685A substitutions. c , Binding affinities of Omicron variants spike trimers to hACE2 measured by SPR. SPR analyses were conducted in biological duplicates. d , MD simulated interactions between hACE2 and RBD of Omicron variants. Structures of the RBD from Omicron variants and hACE2 are shown as ribbons.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Surface representation of S-trimers of BA.1, BA.2, BA.3, BA.2.13, BA.2.12.1 and BA.4/BA.5 (BA.4/5) variants. b , Structural interpretation and functional verification of the stability of the spike protein of BA.1, BA.2, BA.3, BA.2.13, BA.2.12.1 and BA.4/BA.5 variants. Left, superimposed structures of spike protein and the S2 domains of BA.1 (purple), BA.2 (red) and BA.4/BA.5 (blue). The binding surface areas between S2 subunits of the variants are calculated in the table on the right. c , Thermoflour analysis for these Omicron variants. Analyses were performed as biological duplicates. d , Binding affinities of RBDs of Omicron variants for hACE2 measured by SPR. Analyses were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Functional Assay, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a – d , Neutralizing titres against SARS-CoV-2 D614G, Omicron subvariants and SARS-CoV-1 pseudoviruses in plasma from vaccinated and convalescent individuals. a , Individuals who had received 3 doses of CoronaVac ( n = 40). b , Individuals who had received 2 doses of CoronaVac and 1 dose of ZF2001 ( n = 38). c , Individuals who, after receiving 3 doses of CoronaVac, had been infected with BA.1 and recovered ( n = 50). d , People who had recovered from SARS and received 2 doses of CoronaVac and 1 dose of ZF2001 ( n = 28). P -values were calculated using two-tailed Wilcoxon signed-rank tests of paired samples. The geometric mean titre is shown above each group of points. e , Neutralizing activity against SARS-CoV-2 variants and sarbecoviruses by therapeutic NAbs. Green, half-maximal inhibitory concentration (IC 50 ) ≤ 30 ng ml −1 ; white, 30 ng ml −1 < IC 50 < 1,000 ng ml −1 ; red, IC 50 ≥ 1,000 ng ml −1 ; *, IC 50 ≥ 10,000 ng ml −1 . All neutralization assays were performed as biological duplicates. * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant ( P > 0.05).

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Infection, Two Tailed Test, Activity Assay, Concentration Assay, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: NT50 against SARS-CoV-2, SARS-CoV-1 D614G and Omicron subvariants spike-pseudotyped VSV by plasma samples from a , individuals who received 3 doses CoronaVac with (n = 50) or without (n = 40) BA.1 breakthrough infection; b , individuals who received 2 doses CoronaVac and ZF2001 booster with (n = 28) or without (n = 38) previous SARS-CoV-1 infection; c , individuals who received 3 doses CoronaVac (n = 40) or 2 doses CoronaVac with ZF2001 booster (n = 38). P-values were calculated using two-tailed Wilcoxon rank-sum tests and labeled above the bars. n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. Geometric means are labeled. Error bars refer to geometric standard deviations.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Infection, Two Tailed Test, Labeling, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , FACS analysis of pooled memory B cells (IgM − CD27 + ) from plasma of individuals who have recovered from BA.1 breakthrough infection after vaccination, vaccinated individuals and unvaccinated individuals who have recovered from BA.1 breakthrough infection. The percentage of cells recognizing WT or BA.1 RBD are shown. b , The heavy chain V domain somatic hypermutation (SHM) rate of BA.1-specific ( n = 968) and BA.1–WT cross-reactive ( n = 4,782) BCRs obtained from 10X scVDJ-seq from individuals who have recovered from BA.1 breakthrough infection after vaccination. Two-tailed Wilcoxon rank-sum test. Boxes show 25th percentile, median and 75th percentile, and violin plots show kernel density estimation curves of the distribution. c , t -SNE and unsupervised clustering of antibodies that bind WT SARS-CoV-2 RBD. Twelve epitope groups were identified on the basis of DMS of 1,538 antibodies. d , e , Epitope distribution and projection of antibodies from plasma of individuals who had recovered from infection with the WT virus, individuals who have recovered from BA.1 breakthrough infection after vaccination, and vaccinated individuals who had recovered from SARS. f , ACE2 competition level determined by competition ELISA ( n = 1,286) were projected onto the t -SNE. g , Neutralizing activity against SARS-CoV-2 D614G ( n = 1,509) and SARS-CoV-1 (HKU-39849; n = 1,457). h , Average mutational escape score projection of each epitope group on SARS-CoV-2 RBD (Protein Data Bank (PDB): 6M0J). All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Infection, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: Neutralizing activity against SARS-CoV-2 D614G and Omicron subvariants pseudovirus by antibodies of each epitope group from BA.1 convalescents (BA.1-stimulated. A, n = 30; B, n = 41; C, n = 20; D1, n = 49; D2, n = 17; E1, n = 11; E2.1, n = 64; E2.2, n = 122; E3, n = 57; F1, n = 80; F2, n = 13; F3, n = 2), and from wildtype convalescents or vaccinees (WT-stimulated. A, n = 98; B, n = 55; C, n = 88; D1, n = 46; D2, n = 36; E1, n = 59; E2.1, n = 26; E2.2, n = 39; E3, n = 68; F1, n = 97; F2, n = 158; F3, n = 67). Geometric mean titers (GMT) are annotated above each group of points, and error bars indicate geometric standard deviation. P-values were calculated using two-tailed Wilcoxon rank-sum tests and labeled above the bars. n.s., not significant, p > 0.05. NAbs in the boxed epitope groups showed substantial neutralization potency changes against BA.2.12.1 or BA.4/5 compared to BA.1. All neutralization assays were conducted in biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Standard Deviation, Two Tailed Test, Labeling, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: Heavy chain V-J genes combination of a , WT-stimulated antibodies. b , BA.1-stimulated antibodies or each epitope group. The number of NAbs is annotated above the chord plot. IGHV genes are annotated only if the corresponding number of antibodies is greater than one.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques:

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Neutralizing activity against SARS-CoV-2 D614G and Omicron subvariants by BA.1-stimulated (A, n = 30; B, n = 41; C, n = 20) and WT-stimulated (A, n = 98; B, n = 55; C, n = 88) antibodies in Group A, B and C. Geometric mean of IC50 fold changes compared to IC50 against BA.2 are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, in comparison to IC50 against BA.2. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. b , Averaged escape maps at escape hotspots of BA.1-stimulated and WT-stimulated antibodies in group A, B and C, and corresponding MSA of various sarbecovirus RBDs. Height of each amino acid in the escape maps represents its mutation escape score. Mutated sites in Omicron variants are marked in bold.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Neutralization, Mutagenesis

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Epitope of representative antibodies in group D1 (C110; PDB: 7K8V), D2 (LY-CoV1404; PDB: 7MMO), E2.1 (BD-744; PDB: 7EY0) and E2.2 (FC08; PDB: 7DX4). Residues highlighted in red indicate sites that are mutated in Omicron variants. b , Neutralizing activity of NAbs in group D1 ( n = 95), D2 ( n = 53), E2.1 ( n = 90) and E2.2 ( n = 161) against spike-pseudotyped SARS-CoV-2 variants. The geometric mean of the fold change in IC 50 relative to BA.2 is shown above each plot. Two-tailed Wilcoxon signed-rank test of paired samples, in comparison to IC 50 values versus BA.2. c , Neutralizing activity of representative potent NAbs in group D1 ( n = 24), D2 ( n = 12), E2.1 ( n = 23) and E2.2 ( n = 23) against SARS-CoV-2 spike L452 mutants. Geometric mean of the fold change in IC 50 relative to D614G is shown above each plot. Two-tailed Wilcoxon signed-rank test of paired samples. d , Average escape maps at escape hotspots of antibodies in epitope groups D1, D2, E2.1 and E2.2, and the corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron subvariants are marked in bold. All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Neutralizing activity against SARS-CoV-2 D614G and Omicron subvariants by antibodies in group E3 (n = 125) and F1 (n = 177). Geometric mean of IC50 fold changes compared to BA.2 are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, in comparison to IC50 against BA.2. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. b , Epitope of representative antibodies in group E3 (S2H97, PDB: 7M7W) and F1 (S304, PDB: 7JW0). Residues highlighted in red indicate mutated sites in Omicron variants. c , Averaged escape maps at escape hotspots of antibodies in group E3 and F1, and corresponding MSA of various sarbecovirus RBDs. Height of each amino acid in the escape maps represents its mutation escape score. Mutated sites in Omicron variants are marked in bold.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Neutralization, Mutagenesis

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a – c , Neutralizing activity against SARS-CoV-1 and SARS-CoV-2 subvariants by NAbs in group E1 ( a ; n = 70), F2 ( b ; n = 171) and F3 ( c ; n = 69). The geometric mean of the fold change in IC 50 relative to BA.2 is shown above each plot. P -values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, compared with the IC 50 for BA.2. d , The epitope of Group E1 antibody BD55-3152 on the BA.1 RBD. e , Overlay of BD55-5840 in the complex with BA.1 or BA.2 RBD. f , g , The epitope and interactions on the binding interface of BD55-1239 (group F2) ( f ) and BD55-3372 (group F3) ( g ). Antibody residues are shown in blue, and RBD residues are in black or red. Residues highlighted in red indicate sites that are mutated in Omicron variants. h , Average escape maps of antibodies in epitope groups E1, F2 and F3, and the corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron subvariants are marked in bold. All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Binding Assay, Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Cartoon models of Cryo-EM structures of BD55-3152 in complex of BA.1 RBD, BD55-1239 in complex of BA.1 RBD, and BD55-3372 in complex of Delta RBD. b , Workflow to generate refined structural model of BD55-3152 and BD55-1239 in complex of BA.1 RBD, BD55-3372 in complex of Delta RBD, and BD55-5840 in complex of BA.2 RBD. c , Neutralizing activity of representative NAbs in group E1 (n = 68), F2 (n = 139) and F3 (n = 61) against SARS-CoV-2 D614G, in addition to D614G+D405N and D614G+R408S. Geometric mean of IC50 fold changes compared to IC50 against D614G are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. d , Conformational comparison between BA.1 and BA.2 RBD regarding the 366-377 hairpin. e , Biolayer interferometry analysis of Group E1 antibodies S309 and BD55-5840 binding to Omicron BA.1 and BA.2 Spike trimer. Biolayer interferometry analyses were conducted in biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Cryo-EM Sample Prep, Activity Assay, Two Tailed Test, Neutralization, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Four epitope groups were identified among 102 BA.1-specific NAbs via k -means clustering and t -SNE of BA.1 RBD-based DMS profiles. b , c , Distribution of ACE2 competition level ( b ) and neutralizing activities ( c ) against BA.1. d , Neutralizing activities of BA.1-specific antibodies against pseudovirus with SARS-CoV-1 and SARS-CoV-2 spike variants (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. e , Average mutational escape score projection of each BA.1-specific epitope group on SARS-CoV-2 RBD (PDB: 7WPB). f , Averaged escape maps at escape hotspots of the 102 NAbs in the four epitope groups, and corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron variants are marked in bold. WT-related escaping mutations are highlighted. g , Neutralizing activities of BA.1-specific NAbs against BA.1- or BA.2-based pseudoviruses carrying single substitutions (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. Wilcoxon signed-rank test of paired samples, compared with IC 50 for BA.1. All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: Heavy chain V-J gene combination of BA.1-specific neutralizing antibodies in BA.1-specific epitope groups A Omi , B Omi , D Omi and F3 Omi .

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: