Journal: Molecules

Article Title: Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model

doi: 10.3390/molecules29112676

Figure Lengend Snippet: SARS-CoV-2 Omicron BA.5 RBD expression and purification in E. coli . ( a ) Schematics of the sequence location of RBD in the SARS-CoV-2 spike protein. ( b ) Ribbon model of SARS-CoV-2 RBD with disulfide bond pairing. ( c ) SARS-CoV-2 Omicron BA.5 RBD expression and purification protocol in E. coli . ( d ) Binding of the SARS-CoV-2 Omicron BA.5 RBD to the hACE2 using an Octet-N1 Bio-Layer Interferometer. RBD was immobilized on a Ni-NTA sensor chip, and hACE2 was in the mobile phase.

Article Snippet: SARS-CoV-2 RBD (5 μg/mL) was immobilized on the Ni-NTA biosensor for 180 s, followed by an association phase with recombinant hACE2 (purity > 90% SDS-PAGE) (Bioworld Tech, St. Louis Park, MN, USA) diluted in kinetics buffer for 300 s. Dissociation was then carried out in the kinetics buffer for 300 s. Binding affinities were calculated using a 1:1 Langmuir binding model.

Techniques: Expressing, Purification, Sequencing, Binding Assay

Journal: Molecules

Article Title: Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model

doi: 10.3390/molecules29112676

Figure Lengend Snippet: hACE2 inhibition assay. ( a ) Steps of the hACE2 binding inhibition assay using bio-layer interferometry (BLI). RBD was immobilized on the biosensor chip, followed by antisera binding. hACE2 was loaded for the association and dissociation steps assessed in the kinetic buffer. ( b ) Enlarged figure of hACE2 association and dissociation step. ( c ) Inhibition of RBD binding to hACE2 by E. coli -expressed RBD-immunized antisera of one mouse from each group; “M” indicates the identity of the mouse.

Article Snippet: SARS-CoV-2 RBD (5 μg/mL) was immobilized on the Ni-NTA biosensor for 180 s, followed by an association phase with recombinant hACE2 (purity > 90% SDS-PAGE) (Bioworld Tech, St. Louis Park, MN, USA) diluted in kinetics buffer for 300 s. Dissociation was then carried out in the kinetics buffer for 300 s. Binding affinities were calculated using a 1:1 Langmuir binding model.

Techniques: Inhibition, Binding Assay

Journal: Vaccines

Article Title: Surrogate Virus Neutralisation Test Based on Nanoluciferase-Tagged Antigens to Quantify Inhibitory Antibodies against SARS-CoV-2 and Characterise Omicron-Specific Reactivity in a Vaccination Cohort

doi: 10.3390/vaccines11121832

Figure Lengend Snippet: Detection of inhibitory antibodies by sVNT. ( A ) In-house sVNT employing the secNLuc-RBD construct was used to determine antibodies inhibiting binding of the RBD to hACE2 in vaccinee sera at time points t0–t5. The cut-off determined by ROC curve analysis (25% inhibition) is indicated by the dashed line. Horizontal bars delineate the variable median. ( B ) Analysis of vaccinee sera by the cPass sVNT at time points t0–t5. The cut-off value according to the manufacturer’s manual is indicated by the dashed line (30% inhibition), and horizontal bars delineate the variable median. ( C ) Correlation of in-house sVNT and cPass sVNT values < 0% were set to 0%, and the correlation coefficient was calculated according to Spearman. Ih: in-house.

Article Snippet: Black high-binding 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with 200 ng/well of recombinant hACE2 (Sino Biological Inc., Beijing, China, #10108-H08H) diluted in 100 µL carbonate buffer at 4 °C for 16 h. The coated plates were washed four times using 300 µL/well TRIS-buffered saline (TBS)/0.1% Tween (TBS-T), blocked with 300 µL/well TBS-T/2% BSA ( w/v ) for 1.5 h at 37 °C, and washed four times using TBS-T.

Techniques: Construct, Binding Assay, Inhibition