Journal: ACS Infectious Diseases

Article Title: Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2

doi: 10.1021/acsinfecdis.1c00486

Figure Lengend Snippet: Microfluidic antibody affinity profiling (MAAP) of nine convalescent COVID-19 patients and three pre-pandemic sera. (A) Probability density plots of MAAP against fluorescently labeled SARS-CoV-2 RBD, spike S1, and spike S2. The graphs show the affinity ( K D ) and the molar concentration of antibody binding sites for each convalescent serum sample. No binding could be detected in the three pre-pandemic sera. Points correspond to the maximum a posteriori values in the two-dimensional posterior probability distribution, and shaded regions correspond to the probability density. Gray shaded regions indicate the area of nonbinding for samples with [antibody binding sites] < K D . (B) ELISA −log (EC 50 ) values for the same nine convalescent COVID-19 patient samples and three pre-pandemic sera ( 1 : NL63+, 229E+; 2 : NL63+, 229E+, OC43+, HKU1+; 3 : 229E+). A SARS-CoV-2 seronegative sample and a SARS-CoV-2 seropositive sample served as negative and positive controls, respectively. Immobilized antigens are SARS-CoV-2 spike ectodomain, spike S1, and the RBD. Detection was performed with fluorescently labeled anti-human IgG antibodies. (A, B) Red boxes/circles indicate convalescent sera 4 and 5 , which have the strongest immune response to all SARS-CoV-2 spike subunits.

Article Snippet: HCoV-NL63 Spike RBD (10605-CV) and HCoV-HKU1 Spike RBD (10600-CV) were obtained from R&D Systems.

Techniques: Labeling, Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: ACS Infectious Diseases

Article Title: Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2

doi: 10.1021/acsinfecdis.1c00486

Figure Lengend Snippet: Microfluidic antibody affinity profiling against HCoV-NL63 spike S1 and RBD to establish cross-reactivity of antibodies in convalescent COVID-19 serum. (A, B) Equilibrium binding curves of a neutralizing SARS-CoV-2 antibody against 10 nM fluorescently labeled spike S1 from (A) HCoV-HKU1 or (B) HCoV-NL63, respectively. The hydrodynamic radii ( R h ) of the free labeled spike proteins did not increase upon addition of the antibody, indicating the absence of binding. (C) Equilibrium binding curve of an anti-HKU1 antibody against 10 nM fluorescently labeled spike S1 of HCoV-HKU1 showed very tight binding with a K D below 0.1 nM. The K D was determined by nonlinear least-squares fitting using eq . (D) Probability density plots of MAAP against fluorescently labeled HCoV-NL63 spike S1 and HCoV-NL63 RBD. The graphs show the affinity ( K D ) and the molar concentration of antibody binding sites for each of the convalescent COVID-19 (red) and the pre-pandemic sera (blue), whereby pre-pandemic sera and 2 were found to be seropositive for HCoV-NL63. Points correspond to the maximum a posteriori values in the two-dimensional posterior probability distribution, and shaded regions correspond to the probability density.

Article Snippet: HCoV-NL63 Spike RBD (10605-CV) and HCoV-HKU1 Spike RBD (10600-CV) were obtained from R&D Systems.

Techniques: Binding Assay, Labeling, Concentration Assay

Journal: ACS Infectious Diseases

Article Title: Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2

doi: 10.1021/acsinfecdis.1c00486

Figure Lengend Snippet: (A) Schematic of SARS-CoV-2–HCoV-NL63 cross-reactivity competition assay. Typically, 10 nM fluorescently labeled HCoV-NL63 RBD was mixed either with buffer or with patient serum, incubated for 1 h, and subjected to microfluidic diffusional sizing to determine the size of the free labeled RBD ( R h,free ) and the size of the immune-complex ( R h,complex ). For binding competition, 250 nM unlabeled SARS-CoV-2 RBD was added to the mixture of 10 nM HCoV-NL63 RBD and patient serum and incubated for 1 h before measuring the hydrodynamic radius ( R h ). If the serum contains cross-reactive antibodies, a size decrease is observed, as the antibodies will bind to the excess of unlabeled RBD (left box). If the antibodies in the serum are not cross-reactive, they remain bound to the labeled HCoV-NL63 RBD, and the R h of the immuno-complex will stay constant (right box). (B) SARS-CoV-2–HCoV-NL63 cross-reactivity competition assay in convalescent COVID-19 sera and pre-pandemic sera. Filled circles (●) indicate the size of the HCoV-NL63 RBD–antibody immune complex. Empty circles (○) indicate the size of HCoV-NL63 RBD after addition of excess unlabeled SARS-CoV-2 RBD. (Red) Convalescent COVID-19 sera, (Blue) pre-pandemic sera. No decrease of R h upon addition of the unlabeled SARS-CoV-2 RBD could be observed in any of the sera, indicating no cross-reactivity of anti-NL63 antibodies with SARS-CoV-2 RBD.

Article Snippet: HCoV-NL63 Spike RBD (10605-CV) and HCoV-HKU1 Spike RBD (10600-CV) were obtained from R&D Systems.

Techniques: Competitive Binding Assay, Labeling, Incubation, Binding Assay

Journal: Cell

Article Title: Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody

doi: 10.1016/j.cell.2022.03.009

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 Spike RBD-His (Alpha) , , Sino Biological Inc. , Cat# 40592-V08H18.

Techniques: Produced, Virus, Recombinant, Saline, Modification, Luciferase, Antibody Labeling, Clone Assay, Expressing, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2

doi: 10.1038/s41467-022-30878-4

Figure Lengend Snippet: a Illustration of mRNA vaccine construct expressing SARS-CoV-2 WT and Omicron spike genes. The spike open reading frame were flanked by 5′ untranslated region (UTR), 3′ UTR, and polyA tail. The Omicron mutations (red) and HexaPro mutations (black) were numbered based on WA-1 spike residue number. b Distribution of Omicron spike mutations (magenta) were displayed in one protomer of spike trimer of which N-terminal domain (NTD), receptor-binding domain (RBD), hinge region and S2 were colored in purple, blue, green, and orange respectively (PDB: 7SBL). The HexaPro mutations in S2 were colored in cyan. c Schematics illustrating the formulation and biophysical characterization of lipid nanoparticle (LNP)-mRNA. Created with BioRender.com. d Dynamic light scattering derived histogram depicting the particle radius distribution of Omicron spike LNP-mRNA. e Omicron LNP-mRNA image collected on transmission electron microscope. f Human ACE2 receptor binding of LNP-mRNA encoding Omicron spike expressed in 293T cells as detected by human ACE2-Fc fusion protein and PE-anti-human Fc antibody on Flow cytometry.

Article Snippet: The antigen panel used in the ELISA includes RBDs of SARS RBD (AcroBiosystems, SPD-S52H6), MERS RBD (AcroBiosystems, SPD-M52H6), 2019-nCoV WA-1 (Sino Biological 40592-V08B), Delta variant B.1.617.2 (Sino Biological 40592-V08H90), Beta variant B.1.351 (Sino Biological 40592-V08H85) and Omicron variant B.1.1.529 (Sino Biological 40592-V08H121).

Techniques: Construct, Expressing, Binding Assay, Derivative Assay, Transmission Assay, Microscopy, Flow Cytometry

Journal: Nature Communications

Article Title: Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2

doi: 10.1038/s41467-022-30878-4

Figure Lengend Snippet: a Schematics showing the immunization and blood sampling schedule of mice administered with 1 µg WT LNP-mRNA prime (WT × 1) and boost (WT × 2) as well as 10 µg WT or Omicron-specific LNP-mRNA booster shots. The data was collected and combined from two independent experiments shown in Supplementary Fig. and . Created with BioRender.com. b Bar graph comparing binding antibody titers of mice administered with PBS or WT and Omicron LNP-mRNA against Omicron, Delta, and WA-1 RBD (ELISA antigens). The antibody titers were quantified as Log 10 AUC based on titration curves in Supplementary Fig. S1a. PBS sub-groups ( n = 6 each) collected from different matched time points showed no statistical differences between each other, and were combined as one group ( n = 18). c Pseudovirus neutralizing antibody titers in the form of log 10 -transformed reciprocal IC50 calculated from fitting the titration curve with a logistic regression model ( n = 12 mice before booster, n = 5 in WT × 3, n = 7 in WT × 2 + Omicron). d Infectious virus neutralization titer comparisons between mice before and after vaccination with WT or Omicron boosters ( n = 9 mice before booster, n = 5 in WT × 3, n = 4 in WT × 2 + Omicron). Titer ratios were indicated in each graph and fold change described in manuscript is calculated from (ratio − 1). Data on dot-bar plots are shown as mean ± s.e.m. with individual data points in plots. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. Statistical significance labels: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Non-significant comparisons are not shown, unless otherwise noted as n.s., not significant. Sample number is designated as n from biologically independent samples.

Article Snippet: The antigen panel used in the ELISA includes RBDs of SARS RBD (AcroBiosystems, SPD-S52H6), MERS RBD (AcroBiosystems, SPD-M52H6), 2019-nCoV WA-1 (Sino Biological 40592-V08B), Delta variant B.1.617.2 (Sino Biological 40592-V08H90), Beta variant B.1.351 (Sino Biological 40592-V08H85) and Omicron variant B.1.1.529 (Sino Biological 40592-V08H121).

Techniques: Sampling, Binding Assay, Enzyme-linked Immunosorbent Assay, Titration, Transformation Assay, Neutralization

Journal: medRxiv

Article Title: Immune response to SARS-CoV-2 variants of concern in vaccinated individuals

doi: 10.1101/2021.03.08.21252958

Figure Lengend Snippet: UK ( a ) and South African (SA) RBD mutants ( b ) have differing effects upon antibody binding. RBD mutant antigens were generated and added to MULTICOV-AB to measure the immune response towards them in sera from vaccinated pre-second dose (light blue) (n=25), post-second dose (dark blue) (n=20) and infected (red) (n=35) individuals, compared to the wild-type (wt) RBD. A linear curve (y=x) is shown as a dashed grey-line to indicate identical response between wild-type and mutant. Kendall’s tau was calculated to measure ordinal association between the mutant and wild-type.

Article Snippet: All RBD mutants except the Mink variant (#40592-V08H80, Sino Biological) were produced in-house.

Techniques: Binding Assay, Mutagenesis, Generated, Infection

Journal: medRxiv

Article Title: Immune response to SARS-CoV-2 variants of concern in vaccinated individuals

doi: 10.1101/2021.03.08.21252958

Figure Lengend Snippet: When compared to wild-type (wt), RBD mutants for both the Mink ( a ) and LA ( b ) variants of concern resulted in similar response. RBD mutant antigens were generated (LA) or purchased (Mink) and added to MULTICOV-AB to measure the immune response towards them from vaccinated (N=45) and infected (N=35) sera, compared to the wild-type RBD. A linear curve (y=x) is shown as a dashed grey-line to indicate identical response between wild-type and mutant. Kendall’s tau was calculated to measure ordinal association between the mutant and wild-type.

Article Snippet: All RBD mutants except the Mink variant (#40592-V08H80, Sino Biological) were produced in-house.

Techniques: Mutagenesis, Generated, Infection

Journal: medRxiv

Article Title: Immune response to SARS-CoV-2 variants of concern in vaccinated individuals

doi: 10.1101/2021.03.08.21252958

Figure Lengend Snippet: To determine the effect variants of concern had upon neutralization potential, an ACE2 competition assay was developed. RBD mutants for all variants of concern included within this manuscript (UK ( a ), Mink ( b ), LA ( c )) were examined as well as the wild-type (wt) variant on sera from infected (red, N=35) and vaccinated (pre second vaccination (light blue, N=25), post second vaccination (dark blue, N=20)) individuals. Linear regression (y=x) was calculated for each panel, with the R 2 value shown. Linear regressions had the following equations for the different figure panels: ( a ) y = −0.026 + 0.820x ( b ) y = −0.036 + 0.840x ( c ) y = −0.03 + 0.835x

Article Snippet: All RBD mutants except the Mink variant (#40592-V08H80, Sino Biological) were produced in-house.

Techniques: Neutralization, Competitive Binding Assay, Variant Assay, Infection

Journal: Mikrochimica Acta

Article Title: Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification

doi: 10.1007/s00604-023-05747-6

Figure Lengend Snippet: Evaluation of specificity of the RCA-enabled fluorometric ( a ) and colorimetric assay ( b ). The signal intensity generated from S protein RBD is compared with that from N protein, MERS protein RBD, SARS-Cov-2 B.1.617.2 spike protein RBD, and a mixture of the three proteins. The total protein concentration in each sample was 50 ng/mL

Article Snippet: Recombinant SARS-CoV-2 receptor-binding domain (RBD, source: HEK293-derived SARS-CoV-2 Spike RBD protein (Arg319-Phe541), with a 6x-His tag at C-terminal), recombinant MERS-CoV Spike RBD (Chinese Hamster Ovary-derived MERS-CoV Spike RBD protein (Glu367-Tyr606), with a 6x-His tag at C-terminal), recombinant SARS-CoV-2 B.1.617.2 RBD (source: HEK293-derived SARS-CoV-2 Spike RBD protein (Arg319-Phe541 (Leu452Arg, Thr478Lys), with a 6x-His tag at C-terminal), and recombinant SARS-CoV-2 nucleocapsid (source: Spodoptera frugiperda , Sf 21 (baculovirus)-derived SARS-CoV-2 nucleocapsid (Met1-Ala419), with a 6x-His tag at C-terminal) were purchased from Bio-Techne (Minneapolis, USA).

Techniques: Colorimetric Assay, Generated, Protein Concentration