Journal: PLoS ONE

Article Title: Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

doi: 10.1371/journal.pone.0257089

Figure Lengend Snippet: (A) HEK-cell produced RBD fragment fused to Rabbit IgG Fc fragment (RBD::rFc) binding to immobilized biotinylated recombinant human ACE2 was detected by anti-Rabbit IgG-HRP antibodies and TMB chromogenic reaction. Data shown represent values from one experiment. (B) ACE2 Receptor binding competition assay between a constant concentration of partially purified ER-Golgi Retained Algae-Produced RBD::mClover (~40 nM) and increasing amounts of RBD::rFc or Bovine Serum Albumin showing specific competition. RBD::mClover binding was detected using anti-GFP HRP antibodies. Data points represent mean and error bars represent Standard Error of the Mean of normalized A 490 signal values over three independent experimental repeats.

Article Snippet: Strepavidin coated microtiter plates (Cat#15124, Thermo Fisher) were washed 3X in Receptor Assay Blocking buffer (25 mM TrisHCl, 150mM NaCl, pH 7.4, 0.1% wt/vol Bovine Serum Albumin, 0.05% vol/vol Tween-20) and then were coated with 50 ng per well of biotylated human ACE2 produced in HEK cells lines (Cat#10108-H08H-B Sino Biological) dissolved in 100 μL of PBS for one hour at room temperature with gentle shaking on an orbital table.

Techniques: Produced, Binding Assay, Recombinant, Competitive Binding Assay, Concentration Assay, Purification

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Clone Assay, Plasmid Preparation, Blocking Assay, Binding Assay, Labeling, Sequencing, Expressing, Purification, Activity Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characteristics of potential blocking antibodies

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Expressing, Binding Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Binding Assay, Software, Concentration Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Binding Assay, Software, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Luciferase, Expressing, Incubation, Infection, Inhibition, Concentration Assay

Journal: The EMBO Journal

Article Title: Neutralization of SARS‐CoV‐2 by highly potent, hyperthermostable, and mutation‐tolerant nanobodies

doi: 10.15252/embj.2021107985

Figure Lengend Snippet: HeLa cells were transiently transfected to express the SARS‐CoV‐2 Spike protein. Following fixation, cells were stained for 1 h with fluorophore‐labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated. ACE2 competition experiments with selected VHHs from A. 50 nM RBD was mixed with indicated nanobodies (at 500 nM). Binding to ACE2 (immobilized on the sensors) was monitored by BLI.

Article Snippet: Biotinylated Human ACE2 (10108‐H27B‐B, Sino Biological) was immobilized on biosensors until a 0.5 nm wavelength shift/binding signal was reached.

Techniques: Transfection, Staining, Labeling, Binding Assay, Sequencing

Journal: The EMBO Journal

Article Title: Neutralization of SARS‐CoV‐2 by highly potent, hyperthermostable, and mutation‐tolerant nanobodies

doi: 10.15252/embj.2021107985

Figure Lengend Snippet: Crystal structure of the Re9F06⋅RBD⋅Re5D06 complex (at 1.75 Å resolution; Appendix Table ) as surface (left) or ribbon representation (right). The intramolecular disulfide bonds (shown as yellow sticks) are labeled. Ribbon representation of the Re9F06⋅RBD⋅Re5D06 complex as in A, but with RBD‐bound ACE2 shown as a semitransparent brown surface. Docking of ACE2 is based on the alignment of the RBD with the SARS‐CoV‐2 RBD⋅ACE2 complex (PDB ID 7KMS (Zhou et al , ); RMSD = 0.986 Å). Note the clash of ACE2 with Re5D06 and Re9F06. Cryo‐EM reconstructions of the Spike⋅Re5D06 complex (Appendix Table ). SARS‐CoV‐2 Spike (Hsieh et al , ) was incubated with an excess of Re5D06, purified by size exclusion chromatography, and vitrified immediately for structural characterization by cryo‐EM (Appendix Fig ). The two conformational classes detected were refined to high resolution (2.8 Å global). The sharpened maps are shown at low contour levels. The protomers of the homotrimeric Spike are shown in white, tan or gray, respectively, and are further color‐coded as indicated. See Appendix Fig for details and more information. The SARS‐CoV‐2 RBD from A is shown as a ribbon, colored according to the indicated color gradient, with its N‐terminus in blue and C‐terminus in red. Disulfide bridges are depicted as yellow sticks. Re9F06 has been omitted for clarity. RBD side chains that interact with the fold‐promoting VHH Re9F06 are shown as green "ball‐and‐sticks". See Appendix Fig for a comprehensive analysis of the Re9F06⋅RBD interface. Molecular details of the RBD⋅Re5D06 interaction. Left: overview of the RBD⋅Re5D06 complex, with the RBD shown as a green ribbon overlayed with its semitransparent surface. Re5D06 is shown as a ribbon (in magenta) with orange CDR loops. Right: Details of the RBD⋅Re5D06 interaction interface. RBD and Re5D06 are shown as semitransparent ribbons colored as on the left, with selected interface side chains depicted in green (RBD) or magenta (Re5D06). Blue marks nitrogen, oxygen is shown in red. A water molecule is shown as a yellow sphere. Dashed lines link interacting atoms (distance ≤ 4 Å). Lines pointing onto backbones indicate contacts to carbonyl‐carbons or amide groups. See Appendix Fig for more details. Crystal structure of free Re5D06, solved to 1.25 Å resolution (Appendix Table ). Top: free Re5D06 is shown in magenta, with CDR3 (featuring three solvent‐exposed tyrosine residues) colored in orange. For comparison, Re5D06 in its RBD‐bound conformation is overlayed (RMSD = 0.986 Å) in gray. Bottom: intramolecular interactions that stabilize CDR3. CDR3 side chains are depicted in orange; all other side chains are displayed in magenta. See E for further details. Extensive interactions in free Re5D06 stabilize a CDR3 conformation (with solvent‐exposed Tyr 104, 109, 112) that requires only relatively minor structural changes for strong RBD binding.

Article Snippet: Biotinylated Human ACE2 (10108‐H27B‐B, Sino Biological) was immobilized on biosensors until a 0.5 nm wavelength shift/binding signal was reached.

Techniques: Labeling, Cryo-EM Sample Prep, Incubation, Purification, Size-exclusion Chromatography, Binding Assay